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  1. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

    International Nuclear Information System (INIS)

    Robinson, Lisa J.; Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L.; Blair, Harry C.

    2009-01-01

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.

  2. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-{alpha} with BCAR1 and Traf6

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Lisa J., E-mail: robinsonlj@msx.upmc.edu [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Blair, Harry C. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Veteran' s Affairs Medical Center, Pittsburgh, PA 15243 (United States)

    2009-04-15

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at {approx} 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-{beta}-estradiol. Estrogen receptor-{alpha} (ER{alpha}) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ER{alpha}. However, ER{alpha} was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ER{alpha} in the presence of estrogen, was abundant. Immunoprecipitation showed rapid ({approx} 5 min) estrogen-dependent formation of ER{alpha}-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-{kappa}B activity, precipitated with this complex. Reduction of NF-{kappa}B nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of I{kappa}B in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ER{alpha}.

  3. Expression of RANKL/OPG during bone remodeling in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, H., E-mail: tnk@ymghp.jp [Department of Orthopedic Surgery, Yamaguchi Grand Medical Center, 77 Ohsaki, Hofu, Yamaguchi 747-8511 (Japan); Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224 (United States); Mine, T. [Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Ogasa, H. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224 (United States); Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Taguchi, T. [Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Liang, C.T. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224 (United States); National Health Research Institutes, Taipei 115, Taiwan (China)

    2011-08-12

    Highlights: {yields} This is the first study to determine the relationship between osteogenic differentiation and RANKL/OPG expression during bone remodeling in vivo. {yields} The OPG expression peak occurred during the bone formation phase, whereas the marked elevation of RANKL expression was observed during the bone resorption phase. {yields} Histological analysis showed that RANKL/OPG immunoreactivity was predominantly associated with bone marrow cells in the marrow cavity. {yields} The present study confirmed that RANKL/OPG are key factors linking bone formation to resorption during the bone remodeling process. -- Abstract: The interaction between receptor activator of nuclear factor {kappa}B ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation. Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen {alpha}1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The

  4. Expression of RANKL/OPG during bone remodeling in vivo

    International Nuclear Information System (INIS)

    Tanaka, H.; Mine, T.; Ogasa, H.; Taguchi, T.; Liang, C.T.

    2011-01-01

    Highlights: → This is the first study to determine the relationship between osteogenic differentiation and RANKL/OPG expression during bone remodeling in vivo. → The OPG expression peak occurred during the bone formation phase, whereas the marked elevation of RANKL expression was observed during the bone resorption phase. → Histological analysis showed that RANKL/OPG immunoreactivity was predominantly associated with bone marrow cells in the marrow cavity. → The present study confirmed that RANKL/OPG are key factors linking bone formation to resorption during the bone remodeling process. -- Abstract: The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation. Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation

  5. Toll-Like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL

    Science.gov (United States)

    Kassem, Ali; Lindholm, Catharina; Lerner, Ulf H

    2016-01-01

    Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. We, therefore, investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption. S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. Bone resorption induced by S. aureus was abolished by OPG. S. aureus increased the expression of osteoclastogenic cytokines and prostaglandins in the parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption. In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. Our study indicates the importance of using different in vitro approaches for studies of how S

  6. Regulation of vacuolar H+-ATPase in microglia by RANKL

    International Nuclear Information System (INIS)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

    2009-01-01

    Vacuolar H + -ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor κB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  7. Glechoma hederacea Suppresses RANKL-mediated Osteoclastogenesis.

    Science.gov (United States)

    Hwang, J K; Erkhembaatar, M; Gu, D R; Lee, S H; Lee, C H; Shin, D M; Lee, Y R; Kim, M S

    2014-07-01

    Glechoma hederacea (GH), commonly known as ground-ivy or gill-over-the-ground, has been extensively used in folk remedies for relieving symptoms of inflammatory disorders. However, the molecular mechanisms underlying the therapeutic action of GH are poorly understood. Here, we demonstrate that GH constituents inhibit osteoclastogenesis by abrogating receptor activator of nuclear κ-B ligand (RANKL)-induced free cytosolic Ca(2+) ([Ca(2+)]i) oscillations. To evaluate the effect of GH on osteoclastogenesis, we assessed the formation of multi-nucleated cells (MNCs), enzymatic activity of tartrate-resistant acidic phosphatase (TRAP), expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), and [Ca(2+)]i alterations in response to treatment with GH ethanol extract (GHE) in primarily cultured bone marrow-derived macrophages (BMMs). Treatment of RANKL-stimulated or non-stimulated BMMs with GHE markedly suppressed MNC formation, TRAP activity, and NFATc1 expression in a dose-dependent manner. Additionally, GHE treatment induced a large transient elevation in [Ca(2+)]i while suppressing RANKL-induced [Ca(2+)]i oscillations, which are essential for NFATc1 activation. GHE-evoked increase in [Ca(2+)]i was dependent on extracellular Ca(2+) and was inhibited by 1,4-dihydropyridine (DHP), inhibitor of voltage-gated Ca(2+) channels (VGCCs), but was independent of store-operated Ca(2+) channels. Notably, after transient [Ca(2+)] elevation, treatment with GHE desensitized the VGCCs, resulting in an abrogation of RANKL-induced [Ca(2+)]i oscillations and MNC formation. These findings demonstrate that treatment of BMMs with GHE suppresses RANKL-mediated osteoclastogenesis by activating and then desensitizing DHP-sensitive VGCCs, suggesting potential applications of GH in the treatment of bone disorders, such as periodontitis, osteoporosis, and rheumatoid arthritis. © International & American Associations for Dental Research.

  8. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

    2009-11-06

    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  9. Imbalanced expression of RANKL and osteoprotegerin mRNA in pannus tissue of rheumatoid arthritis.

    Science.gov (United States)

    Ainola, M; Mandelin, J; Liljeström, M; Konttinen, Y T; Salo, J

    2008-01-01

    To test if the pannus tissue is characterized by a high receptor activator of nuclear factor kappaB ligand to osteoprotegerin (RANKL:OPG) ratio, which could explain local osteoclastogenesis and formation of bony erosions. Messenger RNA and protein expressions of RANKL and OPG in rheumatoid and osteoarthritic tissue samples were measured using quantitative real-time RT-PCR and Western blot/densitometry. Pannus and synovitis fibroblasts explanted from tissue samples were cultured in vitro without and with TNF-alpha, IL-1Beta or IL-17 and analyzed quantitatively for RANKL expression. The ability of pannus fibroblasts to induce formation of multinuclear osteoclast-like cells from human monocytes, with macrophage-colony stimulating factor (M-CSF) but without RANKL added, was tested. Histochemical staining was used to assess the eventual presence of RANKL and tartrate resistant acid phosphatase positive osteoclast-like cells at the pannus-bone interface. RANKL:OPG ratios of messenger RNA (ppannus (2.06+/-0.73 and 2.2+/-0.65) compared to rheumatoid (0.62+/-0.13 and 1.31+/-0.69) and osteoarthritis (0.62+/-0.32 and 0.52+/-0.16) synovial membranes. Resting and stimulated (p dependent on the cytokine used) pannus fibroblasts produced RANKL in excess (p=0.0005) and unstimulated pannus fibroblasts also effectively induced osteoclast-like cell formation from monocytes in vitro without any exogenous RANKL added. Compatible with these findings, multinuclear osteoclasts-like cells were frequent in the fibroblast- and macrophage-rich pannus tissue at the soft tissue-to-bone interface. The high RANKL:OPG ratio, together with close fibroblast-to-monocyte contacts in pannus tissue, probably favor local generation of bone resorbing osteoclasts at the site of erosion in rheumatoid arthritis.

  10. IL-33 inhibits RANKL-induced osteoclast formation through the regulation of Blimp-1 and IRF-8 expression

    Energy Technology Data Exchange (ETDEWEB)

    Kiyomiya, Hiroyasu [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Ariyoshi, Wataru; Okinaga, Toshinori [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Kaneuji, Takeshi [Division of Oral Medicine, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Mitsugi, Sho [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Sakurai, Takuma [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Habu, Manabu [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Yoshioka, Izumi [Division of Oral Medicine, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Tominaga, Kazuhiro [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); and others

    2015-05-01

    Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, including Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression. - Highlights: • IL-33 inhibits RANKL-induced osteoclast formation. • IL-33 has inhibitory effect on the RANKL-induced NFATc1 expression. • IL-33-induced NFATc1 suppression depends on the regulation of Blimp-1 and IRF-8.

  11. IL-33 inhibits RANKL-induced osteoclast formation through the regulation of Blimp-1 and IRF-8 expression

    International Nuclear Information System (INIS)

    Kiyomiya, Hiroyasu; Ariyoshi, Wataru; Okinaga, Toshinori; Kaneuji, Takeshi; Mitsugi, Sho; Sakurai, Takuma; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro

    2015-01-01

    Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, including Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression. - Highlights: • IL-33 inhibits RANKL-induced osteoclast formation. • IL-33 has inhibitory effect on the RANKL-induced NFATc1 expression. • IL-33-induced NFATc1 suppression depends on the regulation of Blimp-1 and IRF-8

  12. Circulating RANKL and RANKL/OPG and Breast Cancer Risk by ER and PR Subtype: Results from the EPIC Cohort.

    Science.gov (United States)

    Sarink, Danja; Schock, Helena; Johnson, Theron; Overvad, Kim; Holm, Marianne; Tjønneland, Anne; Boutron-Ruault, Marie-Christine; His, Mathilde; Kvaskoff, Marina; Boeing, Heiner; Lagiou, Pagona; Papatesta, Eleni-Maria; Trichopoulou, Antonia; Palli, Domenico; Pala, Valeria; Mattiello, Amalia; Tumino, Rosario; Sacerdote, Carlotta; Bueno-de-Mesquita, H B As; van Gils, Carla H; Peeters, Petra H; Weiderpass, Elisabete; Agudo, Antonio; Sánchez, Maria-José; Chirlaque, Maria-Dolores; Ardanaz, Eva; Amiano, Pilar; Khaw, Kay Tee; Travis, Ruth; Dossus, Laure; Gunter, Mark; Rinaldi, Sabina; Merritt, Melissa; Riboli, Elio; Kaaks, Rudolf; Fortner, Renée T

    2017-09-01

    Receptor activator of nuclear factor-kappa B (RANK)-RANK ligand (RANKL) signaling promotes mammary tumor development in experimental models. Circulating concentrations of soluble RANKL (sRANKL) may influence breast cancer risk via activation of RANK signaling; this may be modulated by osteoprotegerin (OPG), the decoy receptor for RANKL. sRANKL and breast cancer risk by hormone receptor subtype has not previously been investigated. A case-control study was nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. This study included 1,976 incident invasive breast cancer cases [estrogen receptor positive (ER+), n = 1,598], matched 1:1 to controls. Women were pre- or postmenopausal at blood collection. Serum sRANKL was quantified using an ELISA, serum OPG using an electrochemiluminescent assay. Risk ratios (RR) and 95% confidence intervals (95% CI) were calculated using conditional logistic regression. Associations between sRANKL and breast cancer risk differed by tumor hormone receptor status ( P het = 0.05). Higher concentrations of sRANKL were positively associated with risk of ER+ breast cancer [5th vs. 1st quintile RR 1.28 (95% CI, 1.01-1.63); P trend = 0.20], but not ER- disease. For both ER+ and estrogen and progesterone receptor positive (ER+PR+) breast cancer, results considering the sRANKL/OPG ratio were similar to those for sRANKL; we observed a suggestive inverse association between the ratio and ER-PR- disease [5th vs. 1st quintile RR = 0.60 (0.31-1.14); P trend = 0.03]. This study provides the first large-scale prospective data on circulating sRANKL and breast cancer. We observed limited evidence for an association between sRANKL and breast cancer risk. Cancer Prev Res; 10(9); 525-34. ©2017 AACR . ©2017 American Association for Cancer Research.

  13. Isoproterenol Increases RANKL Expression in a ATF4/NFATc1-Dependent Manner in Mouse Osteoblastic Cells

    Directory of Open Access Journals (Sweden)

    Kyunghwa Baek

    2017-10-01

    Full Text Available Sympathetic nervous system stimulation-induced β-adrenergic signal transduction is known to induce bone loss and increase of osteoclast activity. Although isoproterenol, a nonspecific β-adrenergic receptor agonist, has been shown to increase receptor activator of NF-κB ligand (RANKL, the details of the regulatory mechanisms remain unclear. In the present study, we investigated the role of the nuclear factor of activated T-cells (NFAT in isoproterenol-induced RANKL expression in C2C12 and in primary cultured mouse calvarial cells. Isoproterenol increased nuclear factor of activated T-cells cytoplasmic 1 (NFATc1 and RANKL expressions at both mRNA and protein levels and increased NFAT reporter activity. NFATc1 knockdown blocked isoproterenol-mediated RANKL expression. Isoproterenol also promoted cAMP response element-binding protein 1 (CREB1 and activating transcription factor 4 (ATF4 phosphorylation. Isoproterenol-mediated transcriptional activation of NFAT was blocked by protein kinase A (PKA inhibitor H89. Isoproterenol-induced CREB1, ATF4, NFATc1, and RANKL expressions were suppressed by H89. Mutations in cAMP response element-like or NFAT-binding element suppressed isoproterenol-induced RANKL promoter activity. Chromatin immunoprecipitation analysis demonstrated that isoproterenol increased NFAT-binding and ATF4-binding activities on the mouse RANKL promoter, but did not increase CREB1-binding activity. Association of NFATc1 and ATF4 was not observed in a co-immunoprecipitation study. ATF4 knockdown suppressed isoproterenol-induced NFAT binding to the RANKL promoter, whereas NFATc1 knockdown did not suppress isoproterenol-induced ATF4 binding to the RANKL promoter. ATF4 knockdown suppressed isoproterenol-induced expressions of NFATc1 and RANKL. These results suggest that isoproterenol increases RANKL expression in an ATF4/NFATc1-dependent manner.

  14. Oxidized lipids enhance RANKL production by T lymphocytes: implications for lipid-induced bone loss.

    Science.gov (United States)

    Graham, Lucia S; Parhami, Farhad; Tintut, Yin; Kitchen, Christina M R; Demer, Linda L; Effros, Rita B

    2009-11-01

    Osteoporosis is a systemic disease that is associated with increased morbidity, mortality and health care costs. Whereas osteoclasts and osteoblasts are the main regulators of bone homeostasis, recent studies underscore a key role for the immune system, particularly via activation-induced T lymphocyte production of receptor activator of NFkappaB ligand (RANKL). Well-documented as a mediator of T lymphocyte/dendritic cell interactions, RANKL also stimulates the maturation and activation of bone-resorbing osteoclasts. Given that lipid oxidation products mediate inflammatory and metabolic disorders such as osteoporosis and atherosclerosis, and since oxidized lipids affect several T lymphocyte functions, we hypothesized that RANKL production might also be subject to modulation by oxidized lipids. Here, we show that short term exposure of both unstimulated and activated human T lymphocytes to minimally oxidized low density lipoprotein (LDL), but not native LDL, significantly enhances RANKL production and promotes expression of the lectin-like oxidized LDL receptor-1 (LOX-1). The effect, which is also observed with 8-iso-Prostaglandin E2, an inflammatory isoprostane produced by lipid peroxidation, is mediated via the NFkappaB pathway, and involves increased RANKL mRNA expression. The link between oxidized lipids and T lymphocytes is further reinforced by analysis of hyperlipidemic mice, in which bone loss is associated with increased RANKL mRNA in T lymphocytes and elevated RANKL serum levels. Our results suggest a novel pathway by which T lymphocytes contribute to bone changes, namely, via oxidized lipid enhancement of RANKL production. These findings may help elucidate clinical associations between cardiovascular disease and decreased bone mass, and may also lead to new immune-based approaches to osteoporosis.

  15. Attenuated RANKL-induced cytotoxicity by Portulaca oleracea ethanol extract enhances RANKL-mediated osteoclastogenesis.

    Science.gov (United States)

    Erkhembaatar, Munkhsoyol; Choi, Eun-Joo; Lee, Hak-Yong; Lee, Choong Hun; Lee, Young-Rae; Kim, Min Seuk

    2015-07-14

    Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive. In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca(2+) mobilization, nuclear factor of activated T-cell c1 (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity. Our results demonstrated that POEE suppressed RANKL-induced Ca(2+) oscillations by inhibition of Ca(2+) release from internal Ca(2+) stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5'-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs. These results present in vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis.

  16. Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

    Science.gov (United States)

    Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo

    2012-05-05

    Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

    Science.gov (United States)

    Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

    2014-03-01

    Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. RANK und RANKL - Vom Knochen zum Mammakarzinom

    Directory of Open Access Journals (Sweden)

    Sigl V

    2012-01-01

    Full Text Available RANK („Receptor Activator of NF-κB“ und sein Ligand RANKL sind Schlüsselmoleküle im Knochenmetabolismus und spielen eine essenzielle Rolle in der Entstehung von pathologischen Knochenveränderungen. Die Deregulation des RANK/RANKL-Systems ist zum Beispiel ein Hauptgrund für das Auftreten von postmenopausaler Osteoporose bei Frauen. Eine weitere wesentliche Funktion von RANK und RANKL liegt in der Entwicklung von milchsekretierenden Drüsen während der Schwangerschaft. Dabei regulieren Sexualhormone, wie zum Beispiel Progesteron, die Expression von RANKL und induzieren dadurch die Proliferation von epithelialen Zellen der Brust. Seit Längerem war schon bekannt, dass RANK und RANKL in der Metastasenbildung von Brustkrebszellen im Knochengewebe beteiligt sind. Wir konnten nun das RANK/RANKLSystem auch als essenziellen Mechanismus in der Entstehung von hormonellem Brustkrebs identifizieren. In diesem Beitrag werden wir daher den neuesten Erkenntnissen besondere Aufmerksamkeit schenken und diese kritisch in Bezug auf Brustkrebsentwicklung betrachten.

  19. Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts*

    Science.gov (United States)

    Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

    2016-01-01

    Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. PMID:26670608

  20. OPG/RANKL/RANK cytokine system in renal osteodystrophy

    Directory of Open Access Journals (Sweden)

    Ivica Avberšek-Lužnik

    2007-11-01

    Full Text Available Background: Renal osteodystrophy is one of the most common complications affecting patients with endstage renal disease treated with hemodialysis (HD. The action of calciotropic hormones in renal osteodystrophy is regulated by the OPG/RANKL/RANK system. Its function is modulated by interleukines, calcitriol and parathyroid hormone (PTH.The aim of our study was to confirm that this system is involved in the pathogenesis of renal osteodystrophy and supports the mechanism of PTH action on bone.Methods: 106 HD patients (mean age 60 years and 50 healthy volunteers (mean age 64 years were enrolled in the study. In serum samples of patients and controls we determined concentrations of OPG, RANKL, tartarat resistant acid phosphatase 5b (TRAP 5b, serum Cterminal telopeptide cross-links of type I collagen (CTx, bone specific alkaline phosphatase (BALP, osteocalcin (OC and parathyroid hormone (PTH. We compared serum measurements of HD patients and controls and assessed the correlation of OPG and RANKL with bone markers. The most frequent OPG promotor gene polymorphisms were also determined. SPSS 12.1 for Windows was used for statistical analysis.Results: Median OPG concentrations were approximately three times higher in HD patients (0.804 µg/l than in healthy volunteers (0.272 µg/l. Mean serum RANKL concentrations were 1.66- fold higher in HD patients (1.36 pmol/l than in controls (0.82 pmol/l. Serum RANKL levels significantly differed between patients with and without calcitriol therapy (p = 0.001. After dividing HD patients into tertiles according to PTH, we observed significantly higher OPG values in the lower and RANKL in the upper tertile (p < 0.001. OPG did not correlate with bone resorption markers. Only weak correlation of bone formation markers with osteocalcin was noted. In contrast to OPG, RANKL correlated well with PTH, OC and CTX. OPG promoter gene polymorphisms (149 T → C, 163 A → G, 950 T → C do not influence OPG expression and

  1. Constitutive expression of TNF-related activation-induced cytokine (TRANCE/receptor activating NF-κB ligand (RANK-L by rat plasmacytoid dendritic cells.

    Directory of Open Access Journals (Sweden)

    Thomas Anjubault

    Full Text Available Plasmacytoid dendritic cells (pDCs are a subset of DCs whose major function relies on their capacity to produce large amount of type I IFN upon stimulation via TLR 7 and 9. This function is evolutionary conserved and place pDC in critical position in the innate immune response to virus. Here we show that rat pDC constitutively express TNF-related activation-induced cytokine (TRANCE also known as Receptor-activating NF-κB ligand (RANKL. TRANCE/RANKL is a member of the TNF superfamily which plays a central role in osteoclastogenesis through its interaction with its receptor RANK. TRANCE/RANK interaction are also involved in lymphoid organogenesis as well as T cell/DC cross talk. Unlike conventional DC, rat CD4(high pDC were shown to constitutively express TRANCE/RANKL both at the mRNA and the surface protein level. TRANCE/RANKL was also induced on the CD4(low subsets of pDC following activation by CpG. The secreted form of TRANCE/RANKL was also produced by rat pDC. Of note, levels of mRNA, surface and secreted TRANCE/RANKL expression were similar to that observed for activated T cells. TRANCE/RANKL expression was found on pDC in all lymphoid organs as well blood and BM with a maximum expression in mesenteric lymph nodes. Despite this TRANCE/RANKL expression, we were unable to demonstrate in vitro osteoclastogenesis activity for rat pDC. Taken together, these data identifies pDC as novel source of TRANCE/RANKL in the immune system.

  2. RANKL signaling and osteoclastogenesis is negatively regulated by cardamonin.

    Directory of Open Access Journals (Sweden)

    Bokyung Sung

    Full Text Available Bone loss/resorption or osteoporosis is a disease that is accelerated with aging and age-associated chronic diseases such as cancer. Bone loss has been linked with human multiple myeloma, breast cancer, and prostate cancer and is usually treated with bisphosphonates, and recently approved denosumab, an antibody against receptor activator of NF-κB ligand (RANKL. Because of the numerous side effects of the currently available drugs, the search continues for safe and effective therapies for bone loss. RANKL, a member of the TNF superfamily, has emerged as a major mediator of bone loss via activation of osteoclastogenesis. We have identified cardamonin, a chalcone isolated from Alpinia katsumadai Hayata that can affect osteoclastogenesis through modulation of RANKL. We found that treatment of monocytes with cardamonin suppressed RANKL-induced NF-κB activation and this suppression correlated with inhibition of IκBα kinase and of phosphorylation and degradation of IκBα, an inhibitor of NF-κB. Furthermore, cardamonin also downregulated RANKL-induced phosphorylation of MAPK including ERK and p38 MAPK. Cardamonin suppressed the RANKL-induced differentiation of monocytes to osteoclasts in a dose-dependent and time-dependent manner. We also found that an inhibitor of NF-κB essential modulator (NEMO blocked RANKL-induced osteoclastogenesis, indicating a direct link with NF-κB. Finally, osteoclastogenesis induced by human breast cancer cells or human multiple myeloma cells were completely suppressed by cardamonin. Collectively, our results indicate that cardamonin suppresses osteoclastogenesis induced by RANKL and tumor cells by suppressing activation of the NF-κB and MAPK pathway.

  3. RANKL release from self-assembling nanofiber hydrogels for inducing osteoclastogenesis in vitro.

    Science.gov (United States)

    Xing, James Z; Lu, Lei; Unsworth, Larry D; Major, Paul W; Doschak, Michael R; Kaipatur, Neelambar R

    2017-02-01

    To develop a nanofiber hydrogel (NF-hydrogel) for sustained and controlled release of the recombinant receptor activator of NF-kB ligand; (RANKL) and to characterize the release kinetics and bioactivity of the released RANKL. Various concentrations of fluorescently-labelled RANKL protein were added to NF-hydrogels, composed of Acetyl-(Arg-Ala-Asp-Ala) 4 -CONH 2 [(RADA) 4 ] of different concentrations, to investigate the resulting in vitro release rates. The nano-structures of NF-hydrogel, with and without RANKL, were determined using atomic force microscopy (AFM). Released RANKL was further analyzed for changes in secondary and tertiary structure using CD spectroscopy and fluorescent emission spectroscopy, respectively. Bioactivity of released RANKL protein was determined using NFATc1 gene expression and tartrate resistant acid phosphatase (TRAP) activity of osteoclast cells as biomarkers. NF-hydrogel concentration dependent sustained release of RANKL protein was measured at concentrations between 0.5 and 2%(w/v). NF-hydrogel at 2%(w/v) concentration exhibited a sustained and slow-release of RANKL protein up to 48h. Secondary and tertiary structure analyses confirmed no changes to the RANKL protein released from NF-hydrogel in comparison to native RANKL. The results of NFATc1 gene mRNA expression and TRAP activities of osteoclast, showed that the release process did not affect the bioactivity of released RANKL. This novel study is the first of its kind to attempt in vitro characterization of NF-hydrogel based delivery of RANKL protein to induce osteoclastogenesis. We have shown the self-assembling NF-hydrogel peptide system is amenable to the sustained and controlled release of RANKL locally; that could in turn increase local concentration of RANKL to induce osteoclastogenesis, for application to the controlled mobilization of tooth movement in orthodontic procedures. Orthodontic tooth movement (OTM) occurs through controlled application of light forces to teeth

  4. Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

    Science.gov (United States)

    Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

    2016-02-12

    Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages.

    Science.gov (United States)

    Choe, Jung-Yoon; Kim, Seong-Kyu

    2017-03-01

    Melittin is a major toxic component of bee venom (Apis mellifera). It is not known whether melittin is involved in bone metabolism and osteoclastogenesis. The aim of this study was to determine the role of melittin in the regulation of osteoclastogenesis. In vitro osteoclastogenesis assays were performed using mouse RAW 264.7 cells and bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Morphologic and functional analyses for osteoclast-like multinucleated cells (MNCs) were performed by tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and pit formation methods. The gene expression of TRAP, cathepsin K, matrix metalloproteinase-9 (MMP-9) and carbonic anhydrase II was measured by reverse transcription-quantitative PCR. The protein expression levels of mitogen-activated protein kinases (MAPKs), the p65 subunit of nuclear factor-κB (NF-κB), c-Fos, c-Jun, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TNF receptor-associated factor-6 (TRAF6), and interleukin-1β (IL-1β) were assessed by western blot analysis. Melittin inhibited the mRNA expression of TRAP, cathepsin K, MMP-9 and carbonic anhydrase II in RANKL-stimulated RAW 264.7 cells. The increased protein expression of TRAF6, p-extracellular signal-regulated kinase (ERK), p-JNK, p-p65, p-c-Fos and NFATc1 induced by RANKL was significantly suppressed in the RAW 264.7 cells treated with melittin. A synergistic effect of IL-1β on the formation of RANKL-induced osteoclast-like MNCs was found in two experimental cells. The increased expression of IL-1β following the stimulation of RAW 264.7 cells with RANKL activated TRAF6, p-ERK, p-JNK, p-p65, p-c-Fos and NFATc1. These effects were attenuated by the downregulation of IL-1β using siRNA against IL-1β, and also by treatment with melittin. On the whole, the findings of this study demonstrate that melittin

  6. Irreversible inhibition of RANK expression as a possible mechanism for IL-3 inhibition of RANKL-induced osteoclastogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Khapli, Shruti M.; Tomar, Geetanjali B.; Barhanpurkar, Amruta P.; Gupta, Navita; Yogesha, S.D.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

    2010-09-03

    Research highlights: {yields} IL-3 inhibits receptor activator of NF-{kappa}B ligand (RANKL)-induced osteoclastogenesis. {yields} IL-3 inhibits RANKL-induced JNK activation. {yields} IL-3 down-regulates expression of c-Fos and NFATc1 transcription factors. {yields} IL-3 down-regulates RANK expression posttranscriptionally and irreversibly. {yields} IL-3 inhibits in vivo RANK expression. -- Abstract: IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-{kappa}B (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.

  7. Irreversible inhibition of RANK expression as a possible mechanism for IL-3 inhibition of RANKL-induced osteoclastogenesis

    International Nuclear Information System (INIS)

    Khapli, Shruti M.; Tomar, Geetanjali B.; Barhanpurkar, Amruta P.; Gupta, Navita; Yogesha, S.D.; Pote, Satish T.; Wani, Mohan R.

    2010-01-01

    Research highlights: → IL-3 inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. → IL-3 inhibits RANKL-induced JNK activation. → IL-3 down-regulates expression of c-Fos and NFATc1 transcription factors. → IL-3 down-regulates RANK expression posttranscriptionally and irreversibly. → IL-3 inhibits in vivo RANK expression. -- Abstract: IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-κB (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.

  8. Administration of RANKL boosts thymic regeneration upon bone marrow transplantation.

    Science.gov (United States)

    Lopes, Noella; Vachon, Hortense; Marie, Julien; Irla, Magali

    2017-06-01

    Cytoablative treatments lead to severe damages on thymic epithelial cells (TECs), which result in delayed de novo thymopoiesis and a prolonged period of T-cell immunodeficiency. Understanding the mechanisms that govern thymic regeneration is of paramount interest for the recovery of a functional immune system notably after bone marrow transplantation (BMT). Here, we show that RANK ligand (RANKL) is upregulated in CD4 + thymocytes and lymphoid tissue inducer (LTi) cells during the early phase of thymic regeneration. Importantly, whereas RANKL neutralization alters TEC recovery after irradiation, ex vivo RANKL administration during BMT boosts the regeneration of TEC subsets including thymic epithelial progenitor-enriched cells, thymus homing of lymphoid progenitors, and de novo thymopoiesis. RANKL increases specifically in LTi cells, lymphotoxin α, which is critical for thymic regeneration. RANKL treatment, dependent on lymphotoxin α, is beneficial upon BMT in young and aged individuals. This study thus indicates that RANKL may be clinically useful to improve T-cell function recovery after BMT by controlling multiple facets of thymic regeneration. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  9. Effects of interleukin-7/interleukin-7 receptor on RANKL-mediated osteoclast differentiation and ovariectomy-induced bone loss by regulating c-Fos/c-Jun pathway.

    Science.gov (United States)

    Zhao, Ji-Jun; Wu, Zhao-Feng; Yu, Ying-Hao; Wang, Ling; Cheng, Li

    2018-09-01

    To explore the effects of IL-7/IL-7R on the RANKL-mediated osteoclast differentiation in vitro and OVX-induced bone loss in vivo. BMMs and RAW264.7 were transfected with IL-7, IL-7R siRNA, c-Fos siRNA, and c-jun siRNA and later stimulated by RANKL. TRAP and toluidine blue staining were used to observe osteoclast formation and bone resorption, respectively. HE and TRAP staining were used to detect trabecular bone microstructure and osteoclasts of mice, respectively. qRT-PCR and Western blot analysis were used to examine expression. IL-7 unregulated the expression of CTSK, NFATc1, MMP9, and the phosphorylation of p38 and Akt by activating the c-Fos/c-Jun pathway, which increased osteoclast numbers and bone resorption in RANKL-stimulated macrophages. While IL-7R siRNA and c-Fos siRNA decreased the expression, as well as and the phosphorylation of p38 and Akt.IL-7 decreased the BMD and OPG expression in OVX-induced mice and increased the TRAP positive cells, the mRNA expression of c-fos, c-jun, and RANKL, which was contradictory to IL-7R siRNA, and c-Fos siRNA. Furthermore, IL-7R siRNA and c-Fos siRNA caused thicker trabeculae, increased trabecular number, and decreased osteolysis in OVX mice. IL-7/IL-7R can promote RANKL-mediated osteoclast formation and bone resorption by activating the c-Fos/c-Jun pathway, as well as inducing bone loss in OVX mice. © 2018 Wiley Periodicals, Inc.

  10. RANK, RANKL and osteoprotegerin in arthritic bone loss

    Directory of Open Access Journals (Sweden)

    M.C. Bezerra

    2005-02-01

    Full Text Available Rheumatoid arthritis is characterized by the presence of inflammatory synovitis and destruction of joint cartilage and bone. Tissue proteinases released by synovia, chondrocytes and pannus can cause cartilage destruction and cytokine-activated osteoclasts have been implicated in bone erosions. Rheumatoid arthritis synovial tissues produce a variety of cytokines and growth factors that induce monocyte differentiation to osteoclasts and their proliferation, activation and longer survival in tissues. More recently, a major role in bone erosion has been attributed to the receptor activator of nuclear factor kappa B ligand (RANKL released by activated lymphocytes and osteoblasts. In fact, osteoclasts are markedly activated after RANKL binding to the cognate RANK expressed on the surface of these cells. RANKL expression can be upregulated by bone-resorbing factors such as glucocorticoids, vitamin D3, interleukin 1 (IL-1, IL-6, IL-11, IL-17, tumor necrosis factor-alpha, prostaglandin E2, or parathyroid hormone-related peptide. Supporting this idea, inhibition of RANKL by osteoprotegerin, a natural soluble RANKL receptor, prevents bone loss in experimental models. Tumor growth factor-ß released from bone during active bone resorption has been suggested as one feedback mechanism for upregulating osteoprotegerin and estrogen can increase its production on osteoblasts. Modulation of these systems provides the opportunity to inhibit bone loss and deformity in chronic arthritis.

  11. RANKL/RANK: from bone loss to the prevention of breast cancer.

    Science.gov (United States)

    Sigl, Verena; Jones, Laundette P; Penninger, Josef M

    2016-11-01

    RANK and RANKL, a receptor ligand pair belonging to the tumour necrosis factor family, are the critical regulators of osteoclast development and bone metabolism. Besides their essential function in bone, RANK and RANKL have also been identified as the key factors for the formation of a lactating mammary gland in pregnancy. Mechanistically, RANK and RANKL link the sex hormone progesterone with stem cell expansion and proliferation of mammary epithelial cells. Based on their normal physiology, RANKL/RANK control the onset of hormone-induced breast cancer through the expansion of mammary progenitor cells. Recently, we and others were able to show that RANK and RANKL are also critical regulators of BRCA1-mutation-driven breast cancer. Currently, the preventive strategy for BRCA1-mutation carriers includes preventive mastectomy, associated with wide-ranging risks and psychosocial effects. The search for an alternative non-invasive prevention strategy is therefore of paramount importance. As our work strongly implicates RANK and RANKL as key molecules involved in the initiation of BRCA1-associated breast cancer, we propose that anti-RANKL therapy could be a feasible preventive strategy for women carrying BRCA1 mutations, and by extension to other women with high risk of breast cancer. © 2016 The Authors.

  12. Porphyromonas endodontalis lipopolysaccharides induce RANKL by mouse osteoblast in a way different from that of Escherichia coli lipopolysaccharide.

    Science.gov (United States)

    Tang, Yin; Sun, Feifei; Li, Xiaoting; Zhou, Yuan; Yin, Shihai; Zhou, Xuedong

    2011-12-01

    Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P endodontalis LPS; only anti-TLR2 antibody had a significant (P endodontalis LPS-infected osteoblasts (P endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4-JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS). Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  13. Comparison of low-intensity pulsed ultrasound and pulsed electromagnetic field treatments on OPG and RANKL expression in human osteoblast-like cells

    NARCIS (Netherlands)

    Borsje, Manon A.; Ren, Yijin; de Haan-Visser, H. Willy; Kuijer, Roel

    OBJECTIVE: To compare two clinically applied treatments to stimulate bone healing-low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF)-for their effects on RANKL and OPG expression in osteoblast-like cells in vitro. MATERIALS AND METHODS: LIPUS or PEMF was applied to

  14. Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis

    International Nuclear Information System (INIS)

    Takeshita, Harunori; Kitano, Masayasu; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto; Miyazawa, Keiji; Hla, Timothy; Sano, Hajime

    2012-01-01

    Highlights: ► MH7A cells and CD4 + T cells expressed S1P1 and RANKL. ► S1P increased RANKL expression in MH7A cells and CD4 + T cells. ► The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4 + T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4 + T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4 + T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4 + T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4 + T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

  15. Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Takeshita, Harunori [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Kitano, Masayasu, E-mail: mkitano6@hyo-med.ac.jp [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi [Department of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima Kobe, Hyogo 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Miyazawa, Keiji [Discovery Research III, Research and Development, Kissei Pharmaceutical Company, 4365-1 Hodakakashiwara, Azumino, Nagano 399-8304 (Japan); Hla, Timothy [Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 1300 York Avenue, Box 69, NY 10065 (United States); Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

  16. Interleukin-4 inhibits RANKL-induced expression of NFATc1 and c-Fos: A possible mechanism for downregulation of osteoclastogenesis

    International Nuclear Information System (INIS)

    Kamel Mohamed, Saad Gad; Sugiyama, Eiji; Shinoda, Kouichiro; Hounoki, Hiroyuki; Taki, Hirofumi; Maruyama, Muneharu; Miyahara, Tatsuro; Kobayashi, Masashi

    2005-01-01

    Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been shown to inhibit osteoclast differentiation. Therefore, this cytokine is considered to be a promising therapeutic applicant for bone-resorbing diseases such as rheumatoid arthritis (RA). Recently NFATc1, a transcription factor, has been shown to play critical roles in osteoclastogenesis. The aim of this study was to clarify the role of IL-4 on the intracellular signaling of NFATc1. A RAW264.7 monocyte/macrophage cell line and murine bone marrow precursors were differentiated into osteoclasts in the presence of receptor activator of nuclear factor κB ligand (RANKL) and/or macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine were used for the identification of activated osteoclasts. The protein expression of IL-4 receptor, NFATc1, and c-Fos was determined by Western blot analysis. In addition, the gene expression of NFATc1 and c-Fos was determined by reverse transcription and polymerase chain reaction. The IL-4 receptor was constitutively expressed in RAW264.7 cells. RANKL induced osteoclast generation, as determined by TRAP staining and pit assay. IL-4 inhibited RANKL-induced osteoclastogenesis at low concentrations of 10 ng/ml and more. Interestingly, IL-4 potently inhibited RANKL-induced expression of NFATc1 at mRNA level. Furthermore, IL-4 inhibited c-Fos expression, which is shown to be responsible for NFATc1 expression, in time- and dose-dependent manners. In addition, IL-4 inhibited the RANKL-induced expression of NFATc1 and c-Fos in murine bone marrow cells. Thus, we suggest that IL-4 may downregulate osteoclastogenesis in part through inhibition of the expression of transcription factors, NFATc1 and c-Fos. These findings provide new insight into development of new medication for osteoporosis and RA

  17.  Dehydroepiandrosterone sulfate, osteoprotegerin and its soluble ligand sRANKL and bone metabolism in girls with anorexia nervosa

    Directory of Open Access Journals (Sweden)

    Zofia Ostrowska

    2012-09-01

    Full Text Available  Background:Only scarce data exist concerning the relationship between dehydroepiandrosterone (DHEA and/or its sulfate form DHEAS and bone status in adolescents with anorexia nervosa (AN.Aim:We investigated whether a relationship existed between DHEAS and bone metabolism (as assessed based on serum osteocalcin [OC], and collagen type I cross-linked carboxy-terminal telopeptide [CTx]. We also aimed to establish whether the above mentioned relationship might be affected by osteoprotegerin (OPG and its soluble ligand sRANKL.Material/Methods:Fifty-six female patients with AN and 21 healthy female subjects aged 13 to 16 years participated in the study. Serum DHEAS, OC, CTx, OPG and sRANKL were measured by ELISA.Results:Our female patients with AN demonstrated significant suppression of DHEAS and bone markers, an increase in OPG and sRANKL levels, and a reduction of the OPG/sRANKL ratio. DHEAS, CTx and the OPG/sRANKL ratio correlated positively with BMI. A significant positive correlation was also observed between DHEAS and the OPG/sRANKL ratio, OC and the OPG/sRANKL ratio, and CTx and sRANKL. The correlation was negative in the case of DHEAS and CTx, DHEAS and sRANKL, CTx and the OPG/sRANKL ratio, and sRANKL and the OPG/sRANKL ratio.Discussion/DHEAS suppression in girls with anorexia nervosa was associated with a decrease in the levels of bone markers, an increase in OPG and sRANKL concentrations and a significant decrease in the OPG/sRANKL ratio. DHEAS suppression in girls with anorexia nervosa might have a harmful effect on their bone tissue, probably via a shift in the OPG/RANKL ratio toward a functional excess of sRANKL.

  18. Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease.

    Science.gov (United States)

    Kajiya, Mikihito; Giro, Gabriela; Taubman, Martin A; Han, Xiaozhe; Mayer, Marcia P A; Kawai, Toshihisa

    2010-11-08

    Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider (1) the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and (2) the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria.

  19. The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. (Alton Ochsner Medical Foundation, New Orleans, LA (USA))

    1991-01-01

    In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

  20. The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-{kappa}B and MAPK pathways

    Energy Technology Data Exchange (ETDEWEB)

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit [Graduate Program in Industrial Microbiology, Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand); Pudhom, Khanitha [Department of Chemistry, Faculty of Science and Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand); Palaga, Tanapat, E-mail: tanapat.p@chula.ac.th [Graduate Program in Industrial Microbiology, Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. Black-Right-Pointing-Pointer Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. Black-Right-Pointing-Pointer The mode of action of this limonoid is by inhibiting activation of the NF-{kappa}B and MAPK pathways which are activated by RANKL. -- Abstract: Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-{kappa}B (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 {mu}M. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-{kappa}B p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

  1. The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-κB and MAPK pathways

    International Nuclear Information System (INIS)

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit; Pudhom, Khanitha; Palaga, Tanapat

    2011-01-01

    Highlights: ► A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. ► Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. ► The mode of action of this limonoid is by inhibiting activation of the NF-κB and MAPK pathways which are activated by RANKL. -- Abstract: Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

  2. Generation mechanism of RANKL(+) effector memory B cells: relevance to the pathogenesis of rheumatoid arthritis.

    Science.gov (United States)

    Ota, Yuri; Niiro, Hiroaki; Ota, Shun-Ichiro; Ueki, Naoko; Tsuzuki, Hirofumi; Nakayama, Tsuyoshi; Mishima, Koji; Higashioka, Kazuhiko; Jabbarzadeh-Tabrizi, Siamak; Mitoma, Hiroki; Akahoshi, Mitsuteru; Arinobu, Yojiro; Kukita, Akiko; Yamada, Hisakata; Tsukamoto, Hiroshi; Akashi, Koichi

    2016-03-16

    The efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T-B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL(+) effector B cells and their impacts on osteoclast differentiation. Peripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells. RANKL expression was accentuated in CD80(+)CD86(+) B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21. Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3(+)RANKL(+) effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL(+) effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro. Our current findings have shed light on the generation mechanism of pathogenic RANKL(+) effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future.

  3. RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Changhong; Zhao, Jinxia; Sun, Lin; Yao, Zhongqiang; Liu, Rui [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China); Huang, Jiansheng [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO (United States); Liu, Xiangyuan, E-mail: liu-xiangyuan@263.net [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China)

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer CXCR6 is down-regulated during RANKL-induced osteoclastogenesis in RAW264.7 cells. Black-Right-Pointing-Pointer CXCR6 reduction was nearly reversed by inhibition of JAK2/STAT3 signaling pathway. Black-Right-Pointing-Pointer CXCL16 alone does not positively regulate osteoclastogenesis. -- Abstract: The receptor activator of nuclear factor-{kappa}B ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.

  4. Evaluation of the osteoclastogenic process associated with RANK / RANK-L / OPG in odontogenic myxomas

    Science.gov (United States)

    González-Galván, María del Carmen; Mosqueda-Taylor, Adalberto; Bologna-Molina, Ronell; Setien-Olarra, Amaia; Marichalar-Mendia, Xabier; Aguirre-Urizar, José-Manuel

    2018-01-01

    Background Odontogenic myxoma (OM) is a benign intraosseous neoplasm that exhibits local aggressiveness and high recurrence rates. Osteoclastogenesis is an important phenomenon in the tumor growth of maxillary neoplasms. RANK (Receptor Activator of Nuclear Factor κappa B) is the signaling receptor of RANK-L (Receptor activator of nuclear factor kappa-Β ligand) that activates the osteoclasts. OPG (osteoprotegerin) is a decoy receptor for RANK-L that inhibits pro-osteoclastogenesis. The RANK / RANKL / OPG system participates in the regulation of osteolytic activity under normal conditions, and its alteration has been associated with greater bone destruction, and also with tumor growth. Objectives To analyze the immunohistochemical expression of OPG, RANK and RANK-L proteins in odontogenic myxomas (OMs) and their relationship with the tumor size. Material and Methods Eighteen OMs, 4 small ( 3cm) and 18 dental follicles (DF) that were included as control were studied by means of standard immunohistochemical procedure with RANK, RANKL and OPG antibodies. For the evaluation, 5 fields (40x) of representative areas of OM and DF were selected where the expression of each antibody was determined. Descriptive and comparative statistical analyses were performed with the obtained data. Results There are significant differences in the expression of RANK in OM samples as compared to DF (p = 0.022) and among the OMSs and OMLs (p = 0.032). Also a strong association is recognized in the expression of RANK-L and OPG in OM samples. Conclusions Activation of the RANK / RANK-L / OPG triad seems to be involved in the mechanisms of bone balance and destruction, as well as associated with tumor growth in odontogenic myxomas. Key words:Odontogenic myxoma, dental follicle, RANK, RANK-L, OPG, osteoclastogenesis. PMID:29680857

  5. Oxidized low density lipoprotein increases RANKL level in human vascular cells. Involvement of oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Mazière, Cécile, E-mail: maziere.cecile@chu-amiens.fr [Biochemistry Laboratory, South Hospital University, René Laennec Avenue, Amiens 80000 (France); Salle, Valéry [Internal Medicine, North Hospital University, Place Victor Pauchet, Amiens 80000 (France); INSERM U1088 (EA 4292), SFR CAP-Santé (FED 4231), University of Picardie – Jules Verne (France); Gomila, Cathy; Mazière, Jean-Claude [Biochemistry Laboratory, South Hospital University, René Laennec Avenue, Amiens 80000 (France)

    2013-10-18

    Highlights: •Oxidized LDL enhances RANKL level in human smooth muscle cells. •The effect of OxLDL is mediated by the transcription factor NFAT. •UVA, H{sub 2}O{sub 2} and buthionine sulfoximine also increase RANKL level. •All these effects are observed in human fibroblasts and endothelial cells. -- Abstract: Receptor Activator of NFκB Ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) have been shown to play a role not only in bone remodeling but also in inflammation, arterial calcification and atherosclerotic plaque rupture. In human smooth muscle cells, Cu{sup 2+}-oxidized LDL (CuLDL) 10–50 μg/ml increased reactive oxygen species (ROS) and RANKL level in a dose-dependent manner, whereas OPG level was not affected. The lipid extract of CuLDL reproduced the effects of the whole particle. Vivit, an inhibitor of the transcription factor NFAT, reduced the CuLDL-induced increase in RANKL, whereas PKA and NFκB inhibitors were ineffective. LDL oxidized by myeloperoxidase (MPO-LDL), or other pro-oxidant conditions such as ultraviolet A (UVA) irradiation, incubation with H{sub 2}O{sub 2} or with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis{sub ,} also induced an oxidative stress and enhanced RANKL level. The increase in RANKL in pro-oxidant conditions was also observed in fibroblasts and endothelial cells. Since RANKL is involved in myocardial inflammation, vascular calcification and plaque rupture, this study highlights a new mechanism whereby OxLDL might, by generation of an oxidative stress, exert a deleterious effect on different cell types of the arterial wall.

  6. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    Energy Technology Data Exchange (ETDEWEB)

    Okito, Asuka [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Akiyama, Masako [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Ono, Takashi [Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan)

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

  7. Exogenous IFN-beta regulates the RANKL-c-Fos-IFN-beta signaling pathway in the collagen antibody-induced arthritis model.

    Science.gov (United States)

    Zhao, Rong; Chen, Ni-Nan; Zhou, Xiao-Wei; Miao, Ping; Hu, Chao-Ying; Qian, Liu; Yu, Qi-Wen; Zhang, Ji-Ying; Nie, Hong; Chen, Xue-hua; Li, Pu; Xu, Rong; Xiao, Lian-Bo; Zhang, Xin; Liu, Jian-Ren; Zhang, Dong-Qing

    2014-12-10

    Although a variety of drugs have been used to treat the symptoms of rheumatoid arthritis (RA), none of them are able to cure the disease. Interferon β (IFN-β) has pleiotropic effects on RA, but whether it can be used to treat RA remains globally controversial. Thus, in this study we tested the effects of IFN-β on RA patients and on collagen antibody-induced arthritis (CAIA) model mice. The cytokine and auto-antibody expression profiles in the serum and synovial fluid (SF) from RA patients were assessed using enzyme-linked immunosorbent assay (ELISA) and compared with the results from osteoarthritis (OA) patients. Exogenous IFN-β was administered to RA patients and CAIA model mice, and the therapeutic effects were evaluated. Endogenous IFN-β expression in the joint bones of CAIA model mice was evaluated by quantitative real-time PCR (qRT-PCR). The effects of exogenous IFN-β on CAIA model mice were assessed using a clinical scoring system, hematoxylin eosin and safranin-O with fast green counterstain histology, molybdenum target X-ray, and tartrate-resistant acid phosphatase (TRAP) staining. The RANKL-RANK signaling pathway was analyzed using qRT-PCR. The RAW 264.7 cell line was differentiated into osteoclasts with RANKL stimulation and then treated with exogenous IFN-β. The expression of inflammatory cytokines (IFN-γ, IL-17, MMP-3, and RANKL) and auto-antibodies (CII antibodies, RF-IgM, and anti-CCP/GPI) were significantly higher in RA compared with OA patients. After IFN-β intervention, some clinical symptoms in RA patients were partially alleviated, and the expression of IFN-γ, IL-17, MMP-3, and OPG) returned to normal levels. In the CAIA model, the expression of endogenous IFN-β in the joint bones was decreased. After IFN-β administration, the arthritis scores were decreased; synovial inflammation, cartilage, and bone destruction were clearly attenuated; and the expression of c-Fos and NFATc1 were reduced, while RANKL and TRAF6 expression was

  8. Schisantherin A suppresses osteoclast formation and wear particle-induced osteolysis via modulating RANKL signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    He, Yi; Zhang, Qing; Shen, Yi; Chen, Xia; Zhou, Feng; Peng, Dan, E-mail: xyeypd@163.com

    2014-07-04

    Highlights: • Schisantherin A suppresses osteoclasts formation and function in vitro. • Schisantherin A impairs RANKL signaling pathway. • Schisantherin A suppresses osteolysis in vivo. • Schisantherin A may be used for treating osteoclast related diseases. - Abstract: Receptor activator of NF-κB ligand (RANKL) plays critical role in osteoclastogenesis. Targeting RANKL signaling pathways has been a promising strategy for treating osteoclast related bone diseases such as osteoporosis and aseptic prosthetic loosening. Schisantherin A (SA), a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, has been used as an antitussive, tonic, and sedative agent, but its effect on osteoclasts has been hitherto unknown. In the present study, SA was found to inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, SA inhibited OSCAR, cathepsin K and TRAP in a dose dependent manner. Further signal transduction studies revealed that SA down-regulate RANKL-induced nuclear factor-kappaB (NF-κB) signaling activation by suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the NF-κB transcriptional activity. Moreover, SA also decreased the RANKL-induced MAPKs signaling pathway, including JNK and ERK1/2 posphorylation while had no obvious effects on p38 activation. Finally, SA suppressed the NF-κB and MAPKs subsequent gene expression of NFATc1 and c-Fos. In vivo studies, SA inhibited osteoclast function and exhibited bone protection effect in wear-particle-induced bone erosion model. Taken together, SA could attenuate osteoclast formation and wear particle-induced osteolysis by mediating RANKL signaling pathways. These data indicated that SA is a promising therapeutic natural compound for the treatment of osteoclast-related prosthesis loosening.

  9. Expression profile of osteoprotegerin, RANK and RANKL genes in the femoral head of patients with avascular necrosis.

    Science.gov (United States)

    Samara, Stavroula; Dailiana, Zoe; Chassanidis, Christos; Koromila, Theodora; Papatheodorou, Loukia; Malizos, Konstantinos N; Kollia, Panagoula

    2014-02-01

    Femoral head avascular necrosis (AVN) is a recalcitrant disease of the hip that leads to joint destruction. Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B (RANK) and RANK ligand (RANKL) regulate the balance between osteoclasts-osteoblasts. The expression of these genes affects the maturation and function of osteoblasts-osteoclasts and bone remodeling. In this study, we investigated the molecular pathways leading to AVN by studying the expression profile of OPG, RANK and RANKL genes. Quantitative Real Time-PCR was performed for evaluation of OPG, RANK and RANKL expression. Analysis was based on parallel evaluation of mRNA and protein levels in normal/necrotic sites of 42 osteonecrotic femoral heads (FHs). OPG and RANKL protein levels were estimated by western blotting. The OPG mRNA levels were higher (insignificantly) in the necrotic than the normal site (p > 0.05). Although the expression of RANK and RANKL was significantly lower than OPG in both sites, RANK and RANKL mRNA levels were higher in the necrotic part than the normal (p < 0.05). Protein levels of OPG and RANKL showed no remarkable divergence. Our results indicate that differential expression mechanisms for OPG, RANK and RANKL that could play an important role in the progress of bone remodeling in the necrotic area, disturbing bone homeostasis. This finding may have an effect on the resulting bone destruction and the subsequent collapse of the hip joint. Copyright © 2013. Published by Elsevier Inc.

  10. Clinical prognostic significance and pro-metastatic activity of RANK/RANKL via the AKT pathway in endometrial cancer.

    Science.gov (United States)

    Wang, Jing; Liu, Yao; Wang, Lihua; Sun, Xiao; Wang, Yudong

    2016-02-02

    RANK/RANKL plays a key role in metastasis of certain malignant tumors, which makes it a promising target for developing novel therapeutic strategies for cancer. However, the prognostic value and pro-metastatic activity of RANK in endometrial cancer (EC) remain to be determined. Thus, the present study investigated the effect of RANK on the prognosis of EC patients, as well as the pro-metastatic activity of EC cells. The results indicated that those with high expression of RANK showed decreased overall survival and progression-free survival. Statistical analysis revealed the positive correlations between RANK/RANKL expression and metastasis-related factors. Additionally, RANK/RANKL significantly promoted cell migration/invasion via activating AKT/β-catenin/Snail pathway in vitro. However, RANK/RANKL-induced AKT activation could be suppressed after osteoprotegerin (OPG) treatment. Furthermore, the combination of medroxyprogesterone acetate (MPA) and RANKL could in turn attenuate the effect of RANKL alone. Similarly, MPA could partially inhibit the RANK-induced metastasis in an orthotopic mouse model via suppressing AKT/β-catenin/Snail pathway. Therefore, therapeutic inhibition of MPA in RANK/RANKL-induced metastasis was mediated by AKT/β-catenin/Snail pathway both in vitro and in vivo, suggesting a potential target of RANK for gene-based therapy for EC.

  11. Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

    Directory of Open Access Journals (Sweden)

    Jung-Yoon Choe

    2015-01-01

    Full Text Available The aim of this study was to clarify the role of monosodium urate (MSU crystals in receptor activator of nuclear factor kB ligand- (RANKL- RANK-induced osteoclast formation. RAW 264.7 murine macrophage cells were incubated with MSU crystals or RANKL and differentiated into osteoclast-like cells as confirmed by staining for tartrate-resistant acid phosphatase (TRAP and actin ring, pit formation assay, and TRAP activity assay. MSU crystals in the presence of RANKL augmented osteoclast differentiation, with enhanced mRNA expression of NFATc1, cathepsin K, carbonic anhydrase II, and matrix metalloproteinase-9 (MMP-9, in comparison to RAW 264.7 macrophages incubated in the presence of RANKL alone. Treatment with both MSU crystals and RANKL induced osteoclast differentiation by activating downstream molecules in the RANKL-RANK pathway including tumor necrosis factor receptor-associated factor 6 (TRAF-6, JNK, c-Jun, and NFATc1. IL-1b produced in response to treatment with both MSU and RANKL is involved in osteoclast differentiation in part through the induction of TRAF-6 downstream of the IL-1b pathway. This study revealed that MSU crystals contribute to enhanced osteoclast formation through activation of RANKL-mediated pathways and recruitment of IL-1b. These findings suggest that MSU crystals might be a pathologic causative agent of bone destruction in gout.

  12. Modification and translocation of Rac/Rop guanosine 5'-triphosphate-binding proteins of Scoparia dulcis in response to stimulation with methyl jasmonate.

    Science.gov (United States)

    Mitamura, Toshiaki; Yamamura, Yoshimi; Kurosaki, Fumiya

    2011-01-01

    Translocation of two Rac/Rop guanosine 5'-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.

  13. Expression of osteoprotegerin, RNAK and RANKL genes in femoral head avascular necrosis and related signaling pathway.

    Science.gov (United States)

    Miao, Qingtang; Hao, Sibin; Li, Hongmei; Sun, Fang; Wang, Xueling

    2015-01-01

    Femoral head avascular necrosis (AVN) causes the damage of hip joint and related dysfunctions, thus consisting of a clinical challenge. Osteoprotegerin (OPG), receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) all regulate the formation of bones via gene transcriptional regulation for the balance between osteoblasts and osteoclasts. This study thus investigated the expressional profiles of OPG, RANK and RANKL genes in AVN patients, and explored related molecular mediating pathways. Real-time qPCR was used to measure the gene expression of OPG, RANK and RANKL genes in AVN femoral head tissue samples from 42 patients, along with normal tissues. Western blotting analysis was performed to quantify protein levels of OPG and RANKL. There was a trend but not statistically significant elevation of mRNA levels of OPG in femoral head AVN tissues compared to normal tissues (P>0.05). The expression of RNAK and RNAKL, however, was significantly elevated in necrotic tissues (P<0.05). No significant difference in protein levels of OPG or RANKL between groups. The expression of OPG, RANK and RANKL genes exert a crucial role in the progression of AVN, suggesting their roles in mediating bone homeostasis and potential effects on bone destruction.

  14. RANKL-Targeted Therapies: The Next Frontier in the Treatment of Male Osteoporosis

    Directory of Open Access Journals (Sweden)

    Alicia K. Morgans

    2011-01-01

    Full Text Available Male osteoporosis is an increasingly recognized problem in aging men. A common cause of male osteoporosis is hypogonadism. Thousands of men with prostate cancer are treated with androgen deprivation therapy, a treatment that dramatically reduces serum testosterone and causes severe hypogonadism. Men treated with androgen deprivation therapy experience a decline in bone mineral density and have an increased rate of fracture. This paper describes prostate cancer survivors as a model of hypogonadal osteoporosis and discusses the use of RANKL-targeted therapies in osteoporosis. Denosumab, the only RANKL-targeted therapy currently available, increases bone mineral density and decreases fracture rate in men with prostate cancer. Denosumab is also associated with delayed time to first skeletal-related event and an increase in bone metastasis-free survival in these men. It is reasonable to investigate the use of RANKL-targeted therapy in male osteoporosis in the general population.

  15. 25-hydroxycholesterol promotes RANKL-induced osteoclastogenesis through coordinating NFATc1 and Sp1 complex in the transcription of miR-139-5p

    International Nuclear Information System (INIS)

    Zhang, Lishan; Lv, Yinping; Xian, Guozhe; Lin, Yanliang

    2017-01-01

    25-hydroxycholesterol (25-HC) is implicated in many processes, including lipid metabolism and the immune response. However, the role of 25-HC in RANKL-induced osteoclastogenesis remains largely unknown. Our results showed that 25-HC inhibited miR-139-5p expression in mouse bone marrow macrophages (BMMs) cultured in receptor activator of NF-κB ligand (RANKL) and monocyte macrophage colony-stimulating factor (M-CSF). Further investigation suggested that 25-HC promoted the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and Sp1, especially in the presence of RANKL and M-CSF. Meanwhile, 25-HC induced nuclear translocation of NFATc1, resulting in the interaction between NFATc1 and Sp1 that was confirmed by co-immunoprecipitation. Chromatin immunoprecipitation assay indicated that Sp1 could bind to miR-139-5p promoter, but NFATc1 had no binding capacity. Although forming NFATc1/Sp1 complex increased its binding to miR-139-5p promoter, the complex inhibited the transcriptional activity of Sp1. Inhibition of NFATc1 increase the expression of miR-139-5p, which might be due to the release of free Sp1 that could bind to the promoter of miR-139-5p. Enforced expression of miR-139-5p impaired osteoclastogenesis induced by co-treatment with 25-HC and RANKL. These results suggested that 25-HC induced the interaction between NFATc1 and Sp1, reducing the level of free Sp1 to inhibit miR-139-5p expression and promote osteoclastogenesis. - Highlights: • 25-hydroxycholesterol inhibited miR-139-5p expression in bone marrow macrophages. • 25-hydroxycholesterol promoted the expression of NFATc1 and Sp1. • 25-hydroxycholesterol induced the interaction between NFATc1 and Sp1. • NFATc1/Sp1 complex inhibited the transcription of miR-139-5p. • MiR-139-5p impaired osteoclastogenesis induced by 25-hydroxycholesterol and RANKL.

  16. Changes in RANKL during the first two years after cART initiation in HIV-infected cART naïve adults

    DEFF Research Database (Denmark)

    Mathiesen, Inger Hee Mabuza; Salem, Mohammad; Gerstoft, Jan

    2017-01-01

    accelerated bone loss could be mediated by increased soluble RANKL (sRANKL) levels associated with CD4+ T cell recovery. METHODS: We used multiplex immunoassays to determine sRANKL and OPG concentrations in plasma from 48 HIV patients at baseline and 12, 24, 48 and 96 weeks after cART initiation. RESULTS......: Soluble RANKL changed significantly over time (overall p = 0.02) with 25% decrease (95% CI: -42 to -5) at week 24 compared to baseline and stabilized at a lower level thereafter. We found no correlation between CD4+ T cell count increment and changes in sRANKL or between percentage change in BMD...... and changes in sRANKL. CONCLUSION: In this study there was no indication that the accelerated bone loss after cART initiation was mediated by early changes in sRANKL due to CD4+ T cell recovery. Future studies should focus on the initial weeks after initiation of cART. TRIAL REGISTRATION: Clinical...

  17. Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway

    International Nuclear Information System (INIS)

    Yu, Mingxiang; Chen, Xianying; Lv, Chaoyang; Yi, Xilu; Zhang, Yao; Xue, Mengjuan; He, Shunmei; Zhu, Guoying; Wang, Hongfu

    2014-01-01

    Highlights: • Curcumol suppresses osteoclasts differentiation in vitro. • Curcumol impairs JNK/AP-1 signaling pathway. • Curcumol may be used for treating osteoclast related diseases. - Abstract: Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with both bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases

  18. Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mingxiang, E-mail: yu.mingxiang@zs-hospital.sh.cn [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Chen, Xianying [Department of Endocrinology and Metabolism, Hainan Provincial Nong Ken Hospital, Hainan (China); Lv, Chaoyang [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Yi, Xilu [Department of Endocrinology and Metabolism, Shanghai Songjiang District Central Hospital, Shanghai (China); Zhang, Yao; Xue, Mengjuan; He, Shunmei [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Zhu, Guoying [Institute of Radiation Medicine, Fudan University, Shanghai (China); Wang, Hongfu, E-mail: hfwang@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, Shanghai (China)

    2014-05-02

    Highlights: • Curcumol suppresses osteoclasts differentiation in vitro. • Curcumol impairs JNK/AP-1 signaling pathway. • Curcumol may be used for treating osteoclast related diseases. - Abstract: Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with both bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases.

  19. RANKL/Osteoprotegerin System and Bone Turnover in Hashimoto Thyroiditis.

    Science.gov (United States)

    Konca Degertekin, Ceyla; Turhan Iyidir, Ozlem; Aktas Yılmaz, Banu; Elbeg, Sehri; Pasaoglu, Ozge Tugce; Pasaoglu, Hatice; Cakır, Nuri; Arslan, Metin

    2016-10-01

    Hypothyroidism is associated with changes in bone metabolism. The impact of hypothyroidism and the associated autoimmunity on the mediators of bone turnover in Hashimoto's thyroiditis (HT) is not known. In this study, we assessed the levels of OPG, RANKL, and IL-6 along with markers of bone formation as osteocalcin (OC) and markers of bone resorption as type 1 collagen C telopeptide (CTX) and tartrate-resistant acid phosphatase isoform 5b (TRAcP 5b) in 30 hypothyroid and 30 euthyroid premenopausal HT patients and 20 healthy premenopausal controls. We found that TRAcP 5b (p = 0.006), CTX (p = 0.01), OC (p = 0.017), and IL-6 (p Thyroid autoimmunity might have a unique impact on OPG/RANKL levels apart from the resultant hypothyroidism.

  20. Echinocystic acid inhibits RANKL-induced osteoclastogenesis by regulating NF-κB and ERK signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jian-hui, E-mail: jianhui_yangxa@163.com [Rehabilitation Center, First Affiliated Hospital of Health Science Center, Xi’an Jiaotong University, Xi’an, 710061, Shaanxi Province (China); Li, Bing [Department of Dermatology, the 451st Hospital of People’s Liberation Army, Xi’an 710054, Shaanxi Province (China); Wu, Qiong; Lv, Jian-guo; Nie, Hui-Yong [Rehabilitation Center, First Affiliated Hospital of Health Science Center, Xi’an Jiaotong University, Xi’an, 710061, Shaanxi Province (China)

    2016-09-02

    Receptor activator of nuclear factor-κB ligand (RANKL) is a key factor in the differentiation and activation of osteoclasts. Echinocystic acid (EA), a pentacyclic triterpene isolated from the fruits of Gleditsia sinensis Lam, was reported to prevent reduction of bone mass and strength and improve the cancellous bone structure and biochemical properties in ovariectomy rats. However, the molecular mechanism of EA on the osteoclast formation has not been reported. The purpose of this study was to investigate the effects and mechanism of EA on RANKL-induced osteoclastogenesis. Our results showed that EA inhibited the formation of osteoclast, as well as the expression of osteoclastogenesis-related marker proteins in bone marrow macrophages (BMMs). At molecular levels, EA inhibited RANKL-induced NF-κB activation and ERK phosphorylation in BMMs. In conclusion, the present study demonstrated that EA can suppress osteoclastogenesis in vitro. Moreover, we clarified that these inhibitory effects of EA occur through suppression of NF-κB and ERK activation. Therefore, EA may be a potential agent in the treatment of osteoclast-related diseases such as osteoporosis. - Highlights: • EA inhibited the formation of osteoclast in BMMs. • EA inhibits the expression of osteoclastogenesis-related marker proteins in BMMs. • EA inhibits RANKL-induced NF-κB activation in BMMs. • EA inhibits RANKL-induced ERK phosphorylation in BMMs.

  1. The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

    Science.gov (United States)

    Wu, Wei-Jie; Kim, Min Seuk; Ahn, Byung-Yong

    2015-10-01

    To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

  2. cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin.

    Directory of Open Access Journals (Sweden)

    Der-Chih Yang

    Full Text Available BACKGROUND: Mesenchymal stem cells (MSCs are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown. METHODS AND FINDINGS: We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX and forskolin enhances adipogenesis, the gene expression of PPARgamma2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-kappaB Ligand to Osteoprotegerin (RANKL/OPG gene expression - the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish. CONCLUSIONS: Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression.

  3. Periapical fluid RANKL and IL-8 are differentially regulated in pulpitis and apical periodontitis.

    Science.gov (United States)

    Rechenberg, Dan-K; Bostanci, Nagihan; Zehnder, Matthias; Belibasakis, Georgios N

    2014-09-01

    The dental pulp space can become infected due to a breach in the surrounding hard tissues. This leads to inflammation of the pulp (pulpitis), soft tissue breakdown, and finally to bone loss around the root apex (apical periodontitis). The succession of the molecular events leading to apical periodontitis is currently not known. The main inflammatory mediator associated with neutrophil chemotaxis is interleukin-8 (IL-8), and with bone resorption the dyad of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). The levels of RANKL, OPG and IL-8 were studied in periapical tissue fluid of human teeth (n = 48) diagnosed with symptomatic irreversible pulpitis (SIP) and asymptomatic apical periodontitis (AAP). SIP represents the starting point, and AAP an established steady state of the disease. Periapical tissue fluid samples were collected using paper points and then evaluated using enzyme-linked immunosorbent assays (ELISAs). Target protein levels per case were calibrated against the corresponding total protein content, as determined fluorometrically. RANKL was expressed at significantly higher levels in SIP compared to AAP (P apical periodontitis, periapical bone resorption signaling, as determined by RANKL, occurs prior to inflammatory cell recruitment signaling, as determined by IL-8. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Parallel Assessment of Bone Mineral Density and RANKL/OPG Ratio in Saudi Females

    Directory of Open Access Journals (Sweden)

    AI Hassan

    2016-02-01

    Full Text Available Background: Osteoporosis is a significant risk factor for morbidity, and its high prevalence among Saudi women should be considered to be a public health problem. Quantitative ultrasound was recommended for bone mineral density (BMD screening. Receptor activator of nuclear factor kappa-B ligand (RANKL and osteoprotegerin (OPG and their ratio are critical for physiological bone remodelling, and related abnormalities may lead to several osteopathies. Methods: The BMD of 499 Saudi females aged 20 to 65 years was measured using quantitative ultrasound from the beginning of October 2013 to the end of March 2014 at the female medical unit of Taibah University, Madinah, KSA. Possible associated risk factors for low BMD were studied. Blood RANKL and OPG were measured by enzyme-linked immunosorbent assay (ELISA. Results: No significant differences were found between participants with normal and low BMD regarding the studied risk factors. However, there was a significant association (p < 0.05 between BMD and regular physical activity among participants aged 20 years to less than 35 years, and women aged 35–50 years with higher body mass index (BMI had higher BMD. The RANKL/OPG ratio was inversely associated (p = 0.04 with BMD. Conclusions: Regular physical activity is crucial for maximizing BMD in young females and decreasing the possibility of developing osteoporosis with ageing. The RANKL/OPG ratio might be considered a useful and easy-to-use tool for the prediction of low BMD.

  5. Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells

    International Nuclear Information System (INIS)

    Matayoshi, Takaaki; Goto, Tetsuya; Fukuhara, Eiji; Takano, Hiroshi; Kobayashi, Shigeru; Takahashi, Tetsu

    2005-01-01

    The present study was designed to evaluate the effects of neuropeptide substance P (Sp) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the Sp receptor, neurokinin-1 receptor (NK 1 -R). The addition of Sp stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by Sp or NK 1 -R antagonists. Increased expression of the receptor activator of nuclear factor κB ligand (Rankle) in synovial fibroblastic cells after the addition of Sp was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium

  6. [Correlation of RANKL/OPG, dickkopf-1 and bone marrow edema in rheumatoid arthritis with the complaint of knee pain].

    Science.gov (United States)

    Li, Min; Wu, Xiao-hui; Yin, Geng; Xie, Qi-bing

    2015-03-01

    To investigate the correlation of the receptor activator of nuclear factor kappa-B ligand (RANKL)/serum osteoprotegerin (OPG) system, Dickkopf-1 (DKK-1) and bone marrow edema (BME) in rheumatoid arthritis (RA) with the complaint of knee pain. The clinical data of 50 cases of RA with the complaint of knee pain were collected. According to MRI finding, half of them (25 cases) had bone marrow edema (BME). Each patient received the measurement of serum OPG, RANKL, DKK-1, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), anti cyclic citrullinated peptide antibody (CCP), rheumatoid factort(RF). The clinical symptoms, disease activity score 28 (DAS28), were compared between BME and non-BME griups, and the correlation between RANKL/OPG system, DKK-1 and BME of RA was analyzed. Compared with non BME group, BME group had shorter course (P=0.000), higher DAS28 score (P=0.009), CRP (P=0:000), RF (P=0.033) and CCP (P=0.012). lower level of serum OPG (P=0.000), higher level of RANKL (P=0.000), RANKL/OPG (P=0.000), and DKK-1 (P=0.001). The severity of bone marrow edema was correlated with the serum RANKL (volume r(s)=0.31, P=0.027; degree r(s)=0.33, P=0.022), RANKL/OPG (volume r(s)=0.29, P=0.039; degree r(s)=0.28, P=0.043), DKK-1 (volume r(s)=0.33, P=0.021; degree r(s)=0.34, P=0.019). BME is one of the early signs of bone erosion in RA, there were more active inflammation, autoantibodies, and osteocasts in RA patients with BME.

  7. Biotechnological approach for systemic delivery of membrane Receptor Activator of NF-κB Ligand (RANKL) active domain into the circulation

    Science.gov (United States)

    Cappariello, Alfredo; Paone, Riccardo; Maurizi, Antonio; Capulli, Mattia; Rucci, Nadia; Muraca, Maurizio; Teti, Anna

    2015-01-01

    Deficiency of Receptor Activator of NF-κB Ligand (RANKL) prevents osteoclast formation causing osteopetrosis. RANKL is a membrane-bound protein cleaved into active soluble (s)RANKL by metalloproteinase 14 (MMP14). We created a bio-device that harbors primary osteoblasts, cultured on 3D hydroxyapatite scaffolds carrying immobilized MMP14 catalytic domain. Scaffolds were sealed in diffusion chambers and implanted in RANKL-deficient mice. Mice received 1 or 2 diffusion chambers, once or twice and were sacrificed after 1 or 2 months from implants. A progressive increase of body weight was observed in the implanted groups. Histological sections of tibias of non-implanted mice were negative for the osteoclast marker Tartrate-Resistant Acid Phosphatase (TRAcP), consistent with the lack of osteoclasts. In contrast, tibias excised from implanted mice showed TRAcP-positive cells in the bone marrow and on the bone surface, these latter morphologically similar to mature osteoclasts. In mice implanted with 4 diffusion chambers total, we noted the highest number and size of TRAcP-positive cells, with quantifiable eroded bone surface and significant reduction of trabecular bone volume. These data demonstrate that our bio-device delivers effective sRANKL, inducing osteoclastogenesis in RANKL-deficient mice, supporting the feasibility of an innovative experimental strategy to treat systemic cytokine deficiencies. PMID:25678116

  8. Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency.

    Science.gov (United States)

    Saada, Ann; Ben-Shalom, Efrat; Zyslin, Rivka; Miller, Chaya; Mandel, Hanna; Elpeleg, Orly

    2003-10-24

    Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.

  9. Protective effect of zinc supplementation against cadmium-induced oxidative stress and the RANK/RANKL/OPG system imbalance in the bone tissue of rats

    International Nuclear Information System (INIS)

    Brzóska, Malgorzata M.; Rogalska, Joanna

    2013-01-01

    It was investigated whether protective influence of zinc (Zn) against cadmium (Cd)-induced disorders in bone metabolism may be related to its antioxidative properties and impact on the receptor activator of nuclear factor (NF)-κΒ (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system. Numerous indices of oxidative/antioxidative status, and Cd and Zn were determined in the distal femur of the rats administered Zn (30 and 60 mg/l) or/and Cd (5 and 50 mg/l) for 6 months. Soluble RANKL (sRANKL) and OPG were measured in the bone and serum. Zn supplementation importantly protected from Cd-induced oxidative stress preventing protein, DNA, and lipid oxidation in the bone. Moreover, Zn protected from the Cd-induced increase in sRANKL concentration and the sRANKL/OPG ratio, and decrease in OPG concentration in the bone and serum. Numerous correlations were noted between indices of the oxidative/antioxidative bone status, concentrations of sRANKL and OPG in the bone and serum, as well as the bone concentrations of Zn and Cd, and previously reported by us in these animals (Brzóska et al., 2007) indices of bone turnover and bone mineral density. The results allow us to conclude that the ability of Zn to prevent from oxidative stress and the RANK/RANKL/OPG system imbalance may be implicated in the mechanisms of its protective impact against Cd-induced bone damage. This paper is the first report from an in vivo study providing evidence that beneficial Zn impact on the skeleton under exposure to Cd is related to the improvement of the bone tissue oxidative/antioxidative status and mediating the RANK/RANKL/OPG system. - Highlights: • Cd induces oxidative stress in the bone tissue. • Cd disturbs bone metabolism via disorder of the RANK/RANKL/OPG system balance. • Zn supplementation protects from Cd-induced oxidative stress in the bone tissue. • Zn protects from the RANK/RANKL/OPG system imbalance caused by Cd in the bone tissue. • Enhanced Zn intake protects from Cd

  10. Protective effect of zinc supplementation against cadmium-induced oxidative stress and the RANK/RANKL/OPG system imbalance in the bone tissue of rats

    Energy Technology Data Exchange (ETDEWEB)

    Brzóska, Malgorzata M., E-mail: Malgorzata.Brzoska@umb.edu.pl; Rogalska, Joanna

    2013-10-01

    It was investigated whether protective influence of zinc (Zn) against cadmium (Cd)-induced disorders in bone metabolism may be related to its antioxidative properties and impact on the receptor activator of nuclear factor (NF)-κΒ (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system. Numerous indices of oxidative/antioxidative status, and Cd and Zn were determined in the distal femur of the rats administered Zn (30 and 60 mg/l) or/and Cd (5 and 50 mg/l) for 6 months. Soluble RANKL (sRANKL) and OPG were measured in the bone and serum. Zn supplementation importantly protected from Cd-induced oxidative stress preventing protein, DNA, and lipid oxidation in the bone. Moreover, Zn protected from the Cd-induced increase in sRANKL concentration and the sRANKL/OPG ratio, and decrease in OPG concentration in the bone and serum. Numerous correlations were noted between indices of the oxidative/antioxidative bone status, concentrations of sRANKL and OPG in the bone and serum, as well as the bone concentrations of Zn and Cd, and previously reported by us in these animals (Brzóska et al., 2007) indices of bone turnover and bone mineral density. The results allow us to conclude that the ability of Zn to prevent from oxidative stress and the RANK/RANKL/OPG system imbalance may be implicated in the mechanisms of its protective impact against Cd-induced bone damage. This paper is the first report from an in vivo study providing evidence that beneficial Zn impact on the skeleton under exposure to Cd is related to the improvement of the bone tissue oxidative/antioxidative status and mediating the RANK/RANKL/OPG system. - Highlights: • Cd induces oxidative stress in the bone tissue. • Cd disturbs bone metabolism via disorder of the RANK/RANKL/OPG system balance. • Zn supplementation protects from Cd-induced oxidative stress in the bone tissue. • Zn protects from the RANK/RANKL/OPG system imbalance caused by Cd in the bone tissue. • Enhanced Zn intake protects from Cd

  11. Immunolocalization of RANK and RANKL along the root surface and in the periodontal membrane of human primary and permanent teeth

    DEFF Research Database (Denmark)

    Bille, Marie-Louise Bastholm; Thomsen, Bjarke; Andersen, Thomas Levin

    2012-01-01

    Abstract Objective. Root resorption, impaired tooth eruption and early tooth loss have been described in relation to diseases that involve defects in the RANK-RANKL-OPG-expression. The aim of the present immunhistochemical study was to localize and compare the reactions for RANK and membrane...... in odontoblasts and in cells along denticles in one primary tooth. RANK was located in mononuclear cells in the pulp and in multinucleated odontoclasts along resorbed root surfaces and along resorbed dentin surfaces in the pulp in primary teeth and one permanent tooth. Conclusions. This study demonstrated RANK...... positivity in resorption areas in primary and permanent teeth. RANKL was positive in the pulp of one primary tooth. RANK expression in odontoclasts and RANKL expression in the pulp may indicate that RANK/RANKL play a role during resorption....

  12. Tumor Necrosis Factor α Stimulates Osteoclast Differentiation by a Mechanism Independent of the Odf/Rankl–Rank Interaction

    Science.gov (United States)

    Kobayashi, Kanichiro; Takahashi, Naoyuki; Jimi, Eijiro; Udagawa, Nobuyuki; Takami, Masamichi; Kotake, Shigeru; Nakagawa, Nobuaki; Kinosaki, Masahiko; Yamaguchi, Kyoji; Shima, Nobuyuki; Yasuda, Hisataka; Morinaga, Tomonori; Higashio, Kanji; Martin, T. John; Suda, Tatsuo

    2000-01-01

    Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases. PMID:10637272

  13. Low-magnitude high-frequency vibration inhibits RANKL-induced osteoclast differentiation of RAW264.7 cells.

    Science.gov (United States)

    Wu, Song-Hui; Zhong, Zhao-Ming; Chen, Jian-Ting

    2012-01-01

    Osteoclasts are the key participants in regulation of bone mass. Low-magnitude high-frequency vibration (LMHFV) has been found to be anabolic to bone in vivo. This study aimed to investigate the effect of LMHFV on osteoclast differentiation in vitro. Murine monocyte cell line RAW264.7 cells in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) were treated with or without LMHFV at 45 Hz (0.3 g) for 15 min day(-1). Tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and actin ring formation were evaluated. Expression of the osteoclast-specific genes, such as cathepsin K, matrix metallopeptidase-9 (MMP-9) and TRAP, were analyzed using real time-PCR. c-Fos, an osteoclast-specific transcription factor, was determined using Western blot. We found that LMHFV significantly decreased the number of RANKL-induced TRAP-positive MNCs (P<0.01), and inhibited the actin ring formation. The mRNA expression of the cathepsin K, MMP-9 and TRAP were down-regulated by LMHFV intervention (all P<0.001). Furthermore, LMHFV also inhibited the expression of c-Fos protein in the RANKL-treated RAW264.7 cells (P<0.05). Our results suggest that LMHFV can inhibit the RANKL-induced osteoclast differentiation of RAW264.7 cells, which give some new insight into the anabolic effects of LMHFV on bone.

  14. Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

    Directory of Open Access Journals (Sweden)

    Jerome Mauris

    2009-09-01

    Full Text Available Human PMS2 (hPMS2 homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X(2E(X(4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.We examined the effect ATP had on the Mn(++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5 s(-1 and 4.2+/-0.3x10(-5 s(-1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X(2E(X(4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.ATP stimulated the Mn(++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X(2E(X(4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++ induced nicking activity.

  15. Fucoidan, a Sulfated Polysaccharide, Inhibits Osteoclast Differentiation and Function by Modulating RANKL Signaling

    Directory of Open Access Journals (Sweden)

    Young Woo Kim

    2014-10-01

    Full Text Available Multinucleated osteoclasts differentiate from hematopoietic progenitors of the monocyte/macrophage lineage. Because of its pivotal role in bone resorption, regulation of osteoclast differentiation is a potential therapeutic approach to the treatment of erosive bone disease. In this study, we have found that fucoidan, a sulfated polysaccharide extracted from brown seaweed, inhibited osteoclast differentiation. In particular, addition of fucoidan into the early stage osteoclast cultures significantly inhibited receptor activator of nuclear factor kappa B (NF-κB ligand (RANKL-induced osteoclast formation, thus suggesting that fucoidan affects osteoclast progenitors. Furthermore, fucoidan significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases (MAPKs such as JNK, ERK, and p38, and also c-Fos and NFATc1, which are crucial transcription factors for osteoclastogenesis. In addition, the activation of NF-κB, which is an upstream transcription factor modulating NFATc1 expression, was alleviated in the fucoidan-treated cells. These results collectively suggest that fucoidan inhibits osteoclastogenesis from bone marrow macrophages by inhibiting RANKL-induced p38, JNK, ERK and NF-κB activation, and by downregulating the expression of genes that partake in both osteoclast differentiation and resorption.

  16. Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

    International Nuclear Information System (INIS)

    Nishida, Shozo; Tsubaki, Masanobu; Hoshino, Mayumi; Namimatsu, Ayumi; Uji, Hiromi; Yoshioka, Shohei; Tanimori, Yoshihiro; Yanae, Masashi; Iwaki, Masahiro; Irimajiri, Kiyohiro

    2005-01-01

    Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in

  17. 1,25-Dihydroxyvitamin D3 Inhibits the RANKL Pathway and Impacts on the Production of Pathway-Associated Cytokines in Early Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Jing Luo

    2013-01-01

    Full Text Available Objectives. To study effects of 1,25-dihydroxyvitamin D3 (1,25(OH2D3 on RANKL signaling pathway and pathway-associated cytokines in patients with rheumatoid arthritis (RA. Methods. Receptor activator of nuclear factor-kappa B ligand (RANKL, osteoprotegerin (OPG, IFN-γ, IL-6, TNF-α, IL-17, and IL-4 were examined in 54 patients with incipient RA using a cytometric bead array (CBA or an enzyme-linked immunosorbent assay (ELISA. Results. After 72 hours of incubation of peripheral blood mononuclear cells (PBMCs with 1,25(OH2D3 in RA patients, the levels of RANKL, TNF-α, IL-17 and IL-6 significantly decreased compared to those of the control. 1,25(OH2D3 had no significantly impact on the levels of OPG, RANKL/OPG, and IL-4. Conclusions. The present study demonstrated that 1,25(OH2D3 reduced the production of RANKL and the secretion of TNF-α, IL-17, and IL-6 in PBMCs of RA patients, which indicated that 1,25(OH2D3 might be able to decrease damage of cartilage and bone in RA patients by regulating the expression of RANKL signaling pathway and pathway-associated cytokines.

  18. Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy.

    Science.gov (United States)

    Dufresne, Sébastien S; Boulanger-Piette, Antoine; Bossé, Sabrina; Argaw, Anteneh; Hamoudi, Dounia; Marcadet, Laetitia; Gamu, Daniel; Fajardo, Val A; Yagita, Hideo; Penninger, Josef M; Russell Tupling, A; Frenette, Jérôme

    2018-04-24

    Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANK mko ) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL

  19. Effect of Bisphosphonates on the Levels of Rankl and Opg in Gingival Crevicular Fluid of Patients With Periodontal Disease and Post-menopausal Osteoporosis.

    Science.gov (United States)

    Verde, María E; Bermejo, Daniela; Gruppi, Adriana; Grenón, Miriam

    2015-12-01

    The Receptor activator of nuclear factor-kappa B ligand (RANKL)/RANK/Osteoprotegerine (OPG) system has been proposed as essential for osteoclast biology and identified as key part in regulating the physiology and pathology of the skeletal system. The study of the RANKL/RANK/OPG system has increased the understanding of the mechanisms involved in the bone remodeling process, especially in postmenopausal osteoporosis and periodontal disease. Bisphosphonates have become the mainstay of the treatment and prevention of post-menopausal osteoporosis. They inhibit the formation and dissolution of calcium phosphate crystals in bone and also osteoclasts, thus reducing bone turnover.Current investigations relate osteoporosis with the appearance and progression of periodontal disease. Although the etiology of both is different, the bone loss present in both shares several characteristics. Thus, therapy used for osteoporosis can be considered of value in the treatment of periodontal disease. The aim of this study was to evaluate the levels of RANKL, OPG and their relationship in gingival crevicular fluid (GCF) in patients with periodontal disease and postmenopausal osteoporosis/ osteopenia in relation to consumption of bisphosphonates. We studied 66 periodontal active sites obtained from 17 post- menopausal women patients aged between 45-70 years old with osteoporosis/osteopenia and periodontal disease. GCF samples were collected using sterile filter paper strips. To determine the concentration of RANKL and OPG, a commercial ELISA assay was used. The values of RANKL, OPG and their ratio (RANKL/ OPG) were compared with Mann-Whitney U Test. The values of RANKL, OPG and their ratio obtained in patients with osteoporosis/osteopenia and periodontal disease with or without bisphosphonates treatment showed no differences. Bisphosphonates do not alter the concentration of RANKL and OPG and their ratio in the GCF of patients with osteoporosis/ osteopenia and periodontal disease

  20. Osteoprotegerin inhibits bone resorption and prevents tumor development in a xenogenic model of Ewing's sarcoma by inhibiting RANKL

    Science.gov (United States)

    Picarda, Gaëlle; Matous, Etienne; Amiaud, Jérôme; Charrier, Céline; Lamoureux, François; Heymann, Marie-Françoise; Tirode, Franck; Pitard, Bruno; Trichet, Valérie; Heymann, Dominique; Redini, Françoise

    2013-01-01

    Ewing's sarcoma (ES) associated with high osyeolytic lesions typically arises in the bones of children and adolescents. The development of multi-disciplinary therapy has increased current long-term survival rates to greater than 50% but only 20% for high risk group patients (relapse, metastases, etc.). Among new therapeutic approaches, osteoprotegerin (OPG), an anti-bone resorption molecule may represent a promising candidate to inhibit RANKL-mediated osteolytic component of ES and consequently to limit the tumor development. Xenogenic orthotopic models of Ewing's sarcoma were induced by intra-osseous injection of human TC-71 ES cells. OPG was administered in vivo by non-viral gene transfer using an amphiphilic non ionic block copolymer. ES bearing mice were assigned to controls (no treatment, synthetic vector alone or F68/empty pcDNA3.1 plasmid) and hOPG treated groups. A substantial but not significant inhibition of tumor development was observed in the hOPG group as compared to control groups. Marked bone lesions were revealed by micro-computed tomography analyses in control groups whereas a normal bone micro-architecture was preserved in the hOPG treated group. RANKL over-expressed in ES animal model was expressed by tumor cells rather than by host cells. However, TRAIL present in the tumor microenvironment may interfere with OPG effect on tumor development and bone remodeling via RANKL inhibition. In conclusion, the use of a xenogenic model of Ewing's sarcoma allowed discriminating between the tumor and host cells responsible for the elevation of RANKL production observed in this tumor and demonstrated the relevance of blocking RANKL by OPG as a promising therapy in ES. PMID:26909278

  1. Carrier-free 8-azidoadenosine 5'-[γ-32P]triphosphate

    International Nuclear Information System (INIS)

    Sabbatini, G.P.; Holt, C. von

    1987-01-01

    The authors found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed the synthesis in a single-step procedure carrier-free 8-azidoadenosine 5'-[γ- 32 P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[γ- 32 P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue. 14 refs.; 4 figs.; 1 table

  2. Assessment of the relationship between melatonin, hormones of the pituitary-ovarian, -thyroid and -adrenocortical axes, and osteoprotegerin and its ligand sRANKL in girls with anorexia nervosa

    Directory of Open Access Journals (Sweden)

    Zofia Ostrowska

    2013-05-01

    Full Text Available Background: It has been suggested that disturbances in melatonin (MEL secretion might play a role in osteoporosis development in females with anorexia nervosa (AN. It might be hypothesized that changes in the levels of hormones of the pituitary-ovarian, -thyroid and -adrenocortical axes might mediate the potential relationship between MEL and bone tissue.Aim: We investigated whether a relationship existed between MEL and LH, FSH-E2, TSH-FT3, FT4 and ACTH-cortisol axes in girls with AN. We also aimed to establish whether such a relationship might adversely affect the balance of the OPG/sRANKL system.Material/Methods: Eighty-six girls with AN and 21 healthy subjects aged 12.6 to 18.2 years participated in the study. The serum levels of hormones as well as OPG and sRANKL were determined by radioimmunoassay (RIA, immunoradiometric assay (IRMA or enzyme-linked immunosorbent assay (ELISA methods.Discussion: Our study participants with AN showed a significant reduction in body mass and body mass index (BMI, a decrease in LH, E2 and FT3 concentrations, increased MEL concentration at 02.00 hours and increased amplitude between its nocturnal and morning levels (Δ MEL2.00/9.00 as well as an increase in cortisol concentration. These changes were associated with a significant increase of OPG and sRANKL levels and a decrease in the OPG/sRANKL ratio. BMI values correlated positively with LH, FSH, E2, FT3 and the OPG/sRANKL ratio while the correlation between BMI and cortisol was negative. Δ MEL2.00/9.00 correlated positively with cortisol and negatively with LH, FSH, E2, FT3 concentrations and the OPG/sRANKL ratio. A positive correlation was observed between LH, E2 and the OPG/sRANKL ratio as well as between cortisol and sRANKL while the correlation between LH and OPG as well as between cortisol and the OPG/sRANKL ratio was negative. E2 and LH were shown to be significant and independent predictors of Δ MEL2.00/9.00. LH turned out to be a

  3. Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

    International Nuclear Information System (INIS)

    Dhar, A.; Paul, A.K.; Shukla, S.D.

    1990-01-01

    Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

  4. Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Se Jeong [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Gu, Dong Ryun [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Jin, Su Hyun [Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Park, Keun Ha [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Lee, Seoung Hoon, E-mail: leesh2@wku.ac.kr [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Wonkwang Institute of Biomaterials and Implant, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of)

    2016-06-17

    Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

  5. Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

    International Nuclear Information System (INIS)

    Oh, Se Jeong; Gu, Dong Ryun; Jin, Su Hyun; Park, Keun Ha; Lee, Seoung Hoon

    2016-01-01

    Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

  6. Relation of soluble RANKL and osteoprotegerin levels in blood and gingival crevicular fluid to the degree of root resorption after orthodontic tooth movement

    International Nuclear Information System (INIS)

    Tyrovola, J.B.; Halazonetis, D.J.; Makou, M.; Perrea, D.; Dontas, I.; Vlachos, I.S.

    2010-01-01

    The aim of the present study was the determination of the levels of osteoprotegerin and soluble receptor activator of nuclear factor-(KB) ligand (RANKL) in blood serum and in gingival crevicular fluid relative to the degree of orthodontic root resorption in a rat model. Blood samples and gingival crevicular fluid were collected from fourteen 6-month-old male Wistar rats weighing 350-500 g. A 25-g closed orthodontic coil spring was inserted between each upper right first molar and the upper incisors. After 21 days of loading, both upper first molars (treated and control) were extracted and studied under microcomputed tomography scanning. Statistical analysis demonstrated a positive linear correlation between the initial concentration of RANKL in blood serum and the degree of root resorption. The ratio of the initial concentrations of osteoprotegerin to RANKL in blood serum proved to be an independent prognostic factor of the degree of root resorption. The initial concentration of RANKL in gingival crevicular fluid showed a negative correlation to the initial concentration of RANKL in blood serum and for a finite range of initial concentrations of osteoprotegerin in gingival crevicular fluid, the dental root seemed protected against extreme external root resorption. Finally, the concentration of osteoprotegerin in blood serum decreased significantly in cases of severe root resorption. (author)

  7. Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

    International Nuclear Information System (INIS)

    Dowling, J.N.; Saha, A.K.; Glew, R.H.

    1987-01-01

    Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP 3 ) was explored. When neutrophil phosphoinositides were labeled with 32 P, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP 2 ) over 2 h. Treatment of [ 3 H]inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP 2 . Following fMLP stimulation, the fractional reduction in PIP 2 and the fractional increase in IP 3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP 3 was reduced by ACP pre-treatment. The reduction in IP 3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP 2 available for hydrolysis. However, some loss of IP 3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP 2 , the prognitor of IP 3 , and by hydrolyzing IP 3 itself

  8. Reaction of ammonium triphosphate with gadolinium nitrate in aqueous solution at 273K

    International Nuclear Information System (INIS)

    Rodicheva, G.V.; Tananaev, I.V.; Romanova, N.M.

    1982-01-01

    The solubility in the system (NW 4 ) 5 P 3 O 10 -Gd(NO 3 ) 3 - H 2 O (273 K) is studied. Depending on the reagent ratio formation of the compounds Gd 5 (P 3 O 10 ) 3 x22H 2 O, NH 4 Gd 3 (P 3 O 10 ) 2 x12H 2 O and (NH 4 ) 3 Gd 4 (P 3 O 10 ) 3 x14H 2 O is established. Gadolinium triphosphates, separated from solution, are studied using the methods of paper chromatography, X-ray diffractometry, thermography. Simultaneously with thermal dehydration of gadolinium triphosphates the processes of triphosphate decomposition and phosphate anion condensation take place. A mixture of crystalline ortho-phosphate and long- chain polyphosphate of gadolinium is the final product of thermal decomposition (1063 K) of normal and doubl e ammonium- containing gadolinium triphosphates [ru

  9. A terbium(III)-organic framework for highly selective sensing of cytidine triphosphate.

    Science.gov (United States)

    Zhao, Xi Juan; He, Rong Xing; Li, Yuan Fang

    2012-11-21

    Highly selective sensing of cytidine triphosphate (CTP) against other triphosphate nucleosides including ATP, GTP and UTP is successfully achieved with a luminescent terbium(III)-organic framework (TbOF) of [Tb(2)(2,3-pzdc)(2)(ox)(H(2)O)(2)](n) (2,3-pzdc(2-) = 2,3-pyrazinedicarboxylate, ox(2-) = oxalate).

  10. EFFECT OF INFLIXIMAB ON PARAMETERS OF REMODELING OF ARTERIAL BLOODSTREAM, RANKL AND OSTEOPROTEGERIN LEVELS IN PATIENTS WITH RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    Larisa Aleksandrovna Knyazeva

    2013-01-01

    Full Text Available Objective. To study the effect of infliximab (INF on serum levels of RANKL and osteoprotegerin (OPG, as well as on structural and functional properties of the vascular wall in patients with rheumatoid arthritis (RA.Material and Methods. A total of 79 RA patients who corresponded to the classification criteria ACR (1987 or ACR/EULAR (2010 and were seronegative for IgM rheumatoid factor (RF were examined. The mean age of patients was 43.6±8.5 years. The serum levels of OPG and RANKL were determined by ELISA (Biomedica, Austria; the common carotid arteries (CCAs were visualized using an Acuson X/10 ultrasonic complex equipped with a 7 MHz linear sensor in the β-mode prior to therapy and after 12-month therapy with INF.Results and Discussion. An increased OPG level was observed mostly in patients with RA duration up to 1 year; an increase in RANKL level was pronounced stronger in patients with PA duration over 2 years. The disturbance of structural and functional properties of the arterial bloodstream was revealed, manifesting itself as an increase in the intimamedia complex thickness, diameter and rigidity index of CCA that were stronger pronounced in patients with late onset RA. A correlation analysis showed the presence of reliable relationship between the RANKL and OPG levels and CCA remodeling parameters. INF therapy showed high clinical effectiveness and correction effect on the RANKL/OPG system. In addition, it was accompanied by a reduction of signs of CCA remodeling, which was stronger pronounced in patients with early RA.Conclusion. The results prove the reasonability of using INF at early stages of RA in order to optimize the therapy and achieve more efficient control of cardiovascular complications.

  11. Bone metabolism and RANKL/RANK/OPG trail in periodontal disease

    Directory of Open Access Journals (Sweden)

    Czupkallo Lukasz

    2016-12-01

    Full Text Available Periodontal disease is an inflammatory disease of multifactorial etiology. In order for it to appear there must come to an imbalance between the effects of pathogens and host defense mechanisms. As a result of its course the destruction of structures supporting the teeth appears (periodontium, cement, bone, and consequently leads to teeth loosening and loss. In recent years, the participation of RANKL/RANK/OPG in bone remodeling process was highligted.

  12. Mechanical loading prevents the stimulating effect of IL-1{beta} on osteocyte-modulated osteoclastogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kulkarni, Rishikesh N.; Bakker, Astrid D.; Everts, Vincent [Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam (Netherlands); Klein-Nulend, Jenneke, E-mail: j.kleinnulend@acta.nl [Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam (Netherlands)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Osteocyte incubation with IL-1{beta} stimulated osteocyte-modulated osteoclastogenesis. Black-Right-Pointing-Pointer Conditioned medium from IL-1{beta}-treated osteocytes increased osteoclastogenesis. Black-Right-Pointing-Pointer IL-1{beta} upregulated RANKL and downregulated OPG gene expression by osteocytes. Black-Right-Pointing-Pointer CYR61 is upregulated in mechanically stimulated osteocytes. Black-Right-Pointing-Pointer Mechanical loading of osteocytes may abolish IL-1{beta}-induced osteoclastogenesis. -- Abstract: Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1{beta} (IL-1{beta}), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1{beta} affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. MLO-Y4 osteocytes were pre-incubated with IL-1{beta} (0.1-1 ng/ml) for 24 h. Cells were either or not subjected to mechanical loading by 1 h pulsating fluid flow (PFF; 0.7 {+-} 0.3 Pa, 5 Hz) in the presence of IL-1{beta} (0.1-1 ng/ml). Conditioned medium was collected after 1 h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1{beta}-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1{beta}-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF. Incubation of osteocytes with IL-1{beta}, as well as conditioned medium from static IL-1{beta}-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL

  13. Mechanical loading prevents the stimulating effect of IL-1β on osteocyte-modulated osteoclastogenesis

    International Nuclear Information System (INIS)

    Kulkarni, Rishikesh N.; Bakker, Astrid D.; Everts, Vincent; Klein-Nulend, Jenneke

    2012-01-01

    Highlights: ► Osteocyte incubation with IL-1β stimulated osteocyte-modulated osteoclastogenesis. ► Conditioned medium from IL-1β-treated osteocytes increased osteoclastogenesis. ► IL-1β upregulated RANKL and downregulated OPG gene expression by osteocytes. ► CYR61 is upregulated in mechanically stimulated osteocytes. ► Mechanical loading of osteocytes may abolish IL-1β-induced osteoclastogenesis. -- Abstract: Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1β (IL-1β), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1β affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. MLO-Y4 osteocytes were pre-incubated with IL-1β (0.1–1 ng/ml) for 24 h. Cells were either or not subjected to mechanical loading by 1 h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) in the presence of IL-1β (0.1–1 ng/ml). Conditioned medium was collected after 1 h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1β-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1β-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF. Incubation of osteocytes with IL-1β, as well as conditioned medium from static IL-1β-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL-1β-treated osteocytes prevented osteoclast formation. Incubation with IL-1β upregulated RANKL and downregulated OPG gene expression by static osteocytes. PFF upregulated

  14. RANKL regulates differentiation of microfold cells in mouse nasopharynx-associated lymphoid tissue (NALT).

    Science.gov (United States)

    Mutoh, Mami; Kimura, Shunsuke; Takahashi-Iwanaga, Hiromi; Hisamoto, Meri; Iwanaga, Toshihiko; Iida, Junichiro

    2016-04-01

    Murine nasopharynx-associated lymphoid tissue (NALT), located at the base of the nasal cavity, serves as a major site for the induction of mucosal immune responses against airway antigens. The follicle-associated epithelium (FAE) covering the luminal surface of NALT is characterized by the presence of microfold cells (M cells), which take up and transport luminal antigens to lymphocytes. Glycoprotein 2 (GP2) has recently been identified as a reliable marker for M cells in Peyer's patches of the intestine. However, the expression of GP2 and other functional molecules in the M cells of NALT has not yet been examined. We have immunohistochemically detected GP2-expressing cells in the FAE of NALT and the simultaneous expression of other intestinal M-cell markers, namely Tnfaip2, CCL9, and Spi-B. These cells have been further identified as M cells because of their higher uptake capacity of luminal microbeads. Electron microscopic observations have shown that GP2-expressing cells on the FAE display morphological features typical of M cells: they possess short microvilli and microfolds on the luminal surface and are closely associated with intraepithelial lymphocytes. We have also found that the receptor activator of nuclear factor kappa-B ligand (RANKL) is expressed by stromal cells underneath the FAE, which provides its receptor RANK. The administration of RANKL markedly increases the number of GP2(+)Tnfaip2(+) cells on the NALT FAE and that of intestinal M cells. These results suggest that GP2(+)Tnfaip2(+) cells in NALT are equivalent to intestinal M cells, and that RANKL-RANK signaling induces their differentiation.

  15. The effect of leptin replacement on parathyroid hormone, RANKL-osteoprotegerin axis, and Wnt inhibitors in young women with hypothalamic amenorrhea.

    Science.gov (United States)

    Foo, Joo-Pin; Polyzos, Stergios A; Anastasilakis, Athanasios D; Chou, Sharon; Mantzoros, Christos S

    2014-11-01

    Recombinant leptin (metreleptin) treatment restores bone mineral density in women with hypothalamic amenorrhea (HA), a condition characterized by hypoleptinemia, which has adverse impact on bone health. The objective of the study was to investigate how metreleptin exerts its positive effect on bone metabolism in humans. This was a randomized, double-blinded, placebo-controlled study. The study was conducted at Beth Israel Deaconess Medical Center (Boston, Massachusetts). Women (n = 18) with HA and hypoleptinemia for at least 6 months were randomized to receive either metreleptin or placebo for 36 weeks. Serum samples were obtained at baseline and 12, 24, and 36 weeks of treatment. Circulating levels of leptin, intact PTH (iPTH), receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), sclerostin, dickkopf-1, and fibroblast growth factor-23. Metreleptin administration significantly increased leptin levels throughout the treatment period (P = .001). iPTH decreased over the 36 weeks of treatment (P = .01). There was a trend toward a decrease in serum RANKL and increase in serum OPG in the metreleptin-treated group. The RANKL to OPG ratio was significantly decreased within the metreleptin (P = .04) but not the placebo group. Metreleptin had no effect on serum sclerostin, dickkopf-1, and fibroblast growth factor-23. Metreleptin treatment over 36 weeks decreases iPTH and RANKL to OPG ratio levels in hypoleptinemic women with HA.

  16. The Effect of RANKL/OPG Balance on Reducing Implant Complications

    Directory of Open Access Journals (Sweden)

    Elizabeth R. Kapasa

    2017-09-01

    Full Text Available Despite the phenomenal success of implants particularly in the realms of dentistry and orthopaedics, there are still challenges to overcome. The failure of implants resulting from infection, prosthetic loosening, and non-union continue to be the most notorious examples. The cascade of fracture healing and bone repair, especially with the presence of an implant, is complex because it involves a multifaceted immune response alongside the intricate process of bone formation and remodelling. Bone loss is a serious clinical problem that is frequently accompanied by chronic inflammation, illustrating that there is a convoluted relationship between inflammation and bone erosion. The effects of pro-inflammatory factors play a significant role in initiating and maintaining osteoclastogenesis that results in bone resorption by osteoclasts. This is because there is a disruption of the relative ratio between Receptor Activator of Nuclear Factor κB-Ligand (RANKL and osteoprotegerin (OPG, which is central to modulating bone repair and remodelling. This review aims to provide a background to the bone remodelling process, the bone repair cascade post-implantation, and the associated complications. Furthermore, current clinical solutions that can influence bone formation via either internal or extrinsic mechanisms will be described. These efficacious treatments for osteolysis via targeting the RANKL/OPG ratio may be crucial to reducing the incidence of related implant failures in the future.

  17. Individual and combining effects of anti-RANKL monoclonal antibody and teriparatide in ovariectomized mice

    Directory of Open Access Journals (Sweden)

    Naoto Tokuyama

    2015-06-01

    Full Text Available We examined the individual and combined effects of teriparatide and anti-RANKL (receptor activator of nuclear factor κB ligand monoclonal antibody in ovariectomized mice. Three-month-old female C57BL/6 mice were ovariectomized (OVX or sham operated. Four weeks after OVX, they were assigned to 3 different groups to receive anti-RANKL monoclonal antibody (Ab alone (5 mg/kg single injection at 4 weeks after OVX, Ab group, teriparatide alone (80 μg/kg daily injection for 4 weeks from 4 weeks after OVX, PTH group, or mAb plus teriparatide (Ab + PTH group. Mice were sacrificed 8 weeks after OVX. Bone mineral density (BMD was measured at the femur and lumbar spine. Hind limbs were subjected to histological and histomorphometric analysis. Serum osteocalcin and CTX-I levels were measured to investigate the bone turnover. Compared with Ab group, Ab + PTH group showed a significant increase in BMD at distal femur and femoral shaft. Cortical bone volume was significantly increased in PTH and Ab + PTH groups compared with Ab group. Bone turnover in Ab + PTH group was suppressed to the same degree as in Ab group. The number of TRAP-positive multinucleated cells was markedly reduced in Ab and Ab + PTH groups. These results suggest that combined treatment of teriparatide with anti-RANKL antibody has additive effects on BMD in OVX mice compared with individual treatment.

  18. Safflower bud inhibits RANKL-induced osteoclast differentiation and prevents bone loss in ovariectomized mice.

    Science.gov (United States)

    Choi, Joo-Hee; Lim, Seul-Ki; Kim, Dong-Il; Park, Min-Jung; Kim, Young-Kuk; Lee, An-Chul; Kim, Young-Min; Yang, Soo-Jin; Park, Jong-Hwan

    2017-10-15

    The powder and extract of safflower seeds are known to be effective in the prevention of bone loss in ovariectomized animals. However, the inhibitory effect and molecular mechanisms of safflower bud (SB), the germinated safflower, on bone destruction is unclear. The present study was designed to investigate the inhibitory effect and molecular mechanism of SB on osteoclastic differentiation and on bone loss in ovarietomized (OVX) mice. Osteoclastogenesis was determined by TRAP staining, F-actin ring formation, and bone resorption assay. NF-κB and MAPKs activation was analyzed by transfection assay and Western blot, respectively. Real-time PCR was performed to examine the expression of osteoclastogenesis-related genes. Histological changes, increases in TRAP-positive cells, and cathepsin K expression were examined in the metaphysis of OVX mice. Density of bone marrow was evaluated by µCT. SB inhibited the RANKL-induced differentiation of BMDMs into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by SB in RANKL-treated BMDMs. In addition, SB decreased the activation of NF-κB and MAPKs and the expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. Feeding of SB-included diet prevented bone loss in OVX mice. The number of TRAP-positive cells and level of protein expression of cathepsin K was reduced and bone mineral density was increased in the metaphysis of mice fed SB compared with OVX mice. These findings suggest that SB can be a preventive and therapeutic candidate for destructive bone diseases. Copyright © 2017. Published by Elsevier GmbH.

  19. The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

    Science.gov (United States)

    Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C

    2009-04-01

    To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

  20. Oral sucrose for heel lance enhances adenosine triphosphate use in preterm neonates with respiratory distress.

    Science.gov (United States)

    Angeles, Danilyn M; Asmerom, Yayesh; Boskovic, Danilo S; Slater, Laurel; Bacot-Carter, Sharon; Bahjri, Khaled; Mukasa, Joseph; Holden, Megan; Fayard, Elba

    2015-01-01

    To examine the effects of oral sucrose on procedural pain, and on biochemical markers of adenosine triphosphate utilization and oxidative stress in preterm neonates with mild to moderate respiratory distress. Preterm neonates with a clinically required heel lance that met study criteria (n = 49) were randomized into three groups: (1) control (n = 24), (2) heel lance treated with placebo and non-nutritive sucking (n = 15) and (3) heel lance treated with sucrose and non-nutritive sucking (n = 10). Plasma markers of adenosine triphosphate degradation (hypoxanthine, xanthine and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the Premature Infant Pain Profile. Data were analyzed using repeated measures analysis of variance, chi-square and one-way analysis of variance. We found that in preterm neonates who were intubated and/or were receiving ⩾30% FiO2, a single dose of oral sucrose given before a heel lance significantly increased markers of adenosine triphosphate use. We found that oral sucrose enhanced adenosine triphosphate use in neonates who were intubated and/or were receiving ⩾30% FiO2. Although oral sucrose decreased pain scores, our data suggest that it also increased energy use as evidenced by increased plasma markers of adenosine triphosphate utilization. These effects of sucrose, specifically the fructose component, on adenosine triphosphate metabolism warrant further investigation.

  1. STAT6 silencing induces hepatocellular carcinoma-derived cell apoptosis and growth inhibition by decreasing the RANKL expression.

    Science.gov (United States)

    Qing, Tian; Yamin, Zhang; Guijie, Wang; Yan, Jin; Zhongyang, Shen

    2017-08-01

    Signal transducer and activator of transcription-6 (STAT6) is highly expressed in various human cancers and considered a regulator of multiple biological processes in cancers, including cell apoptosis. Evidence has indicated that STAT6 predicts a worse prognosis in hepatocellular carcinoma (HCC) patients. The objective of this study was to investigate the effects and mechanism of STAT6 in human HCC cells. We found that STAT6 silencing significantly inhibited HepG2 and Hep3B cell survival and proliferation. We observed that depletion of STAT6 increased HepG2 and Hep3B cell apoptosis by using a histone DNA ELISA detection kit. STAT6 silencing induced expression of apoptosis-associated genes Bax and caspase-3/7 and inhibited anti-apoptosis gene Bcl-2 levels. We also observed that STAT6 silencing downregulated the expression of receptor activator of NF-κB ligand (RANKL). Our results demonstrated that treatment with pcDNA3.1-RANKL abolished STAT6 depletion-induced HepG2 and Hep3B cell apoptosis and growth inhibition. Based on these findings, we believe that RANKL plays a major role in STAT6-induced HCC cell apoptosis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Ionizing Radiation Stimulates Expression of Pro-Osteoclastogenic Genes in Marrow and Skeletal Tissue

    Science.gov (United States)

    Alwood, J. S.; Shahnazari, M.; Chicana, B.; Schreurs, A. S.; Kumar, A.; Bartolini, A.; Shirazi-Fard, Y.; Globus, R. K.

    2015-01-01

    Exposure to ionizing radiation can cause rapid mineral loss and increase bone-resorbing osteoclasts within metabolically-active, cancellous-bone tissue leading to structural deficits. To better understand mechanisms involved in rapid, radiation-induced bone loss, we determined the influence of total-body irradiation on expression of select cytokines known both to stimulate osteoclastogenesis and contribute to inflammatory bone disease. Adult (16wk), male C57BL/6J mice were exposed to either 2Gy gamma rays (137Cs, 0.8Gy/min) or heavy ions (56Fe, 600MeV, 0.50-1.1Gy/min); this dose corresponds to either a single fraction of radiotherapy (typical total dose is =10Gy) or accumulates over long-duration, interplanetary missions. Serum, marrow, and mineralized tissue were harvested 4hrs-7d later. Gamma irradiation caused a prompt (2.6-fold within 4hrs) and persistent (peaking at 4.1-fold within 1d) rise in the expression of the obligate osteoclastogenic cytokine, receptor activator of nuclear factor kappaB-ligand (Rankl) within marrow cells over controls. Similarly, Rankl expression peaked in marrow cells within 3d of iron exposure (9.2-fold). Changes in Rankl expression induced by gamma irradiation preceded and overlapped with a rise in expression of other pro-osteoclastic cytokines in marrow (e.g., monocyte chemotactic protein-1 increased 11.9-fold, tumor necrosis factor-alpha increased 1.7- fold over controls). Marrow expression of the RANKL decoy receptor, osteoprotegerin (Opg), also rose after irradiation (11.3-fold). The ratio Rankl/Opg in marrow was increased 1.8-fold, a net pro-resorption balance. As expected, radiation increased a serum marker of resorption (tartrate resistant acid phosphatase) and led to cancellous bone loss (16% decrease in bone volume/total volume) through reduced trabecular struts. We conclude that total-body irradiation (gamma or heavy-ion) caused temporal, concerted regulation of gene expression within marrow and mineralized tissue for

  3. Polymorphisms in the RANK/RANKL Genes and Their Effect on Bone Specific Prognosis in Breast Cancer Patients

    Directory of Open Access Journals (Sweden)

    Alexander Hein

    2014-01-01

    Full Text Available The receptor activator of NF-κB (RANK pathway is involved in bone health as well as breast cancer (BC pathogenesis and progression. Whereas the therapeutic implication of this pathway is established for the treatment of osteoporosis and bone metastases, the application in adjuvant BC is currently investigated. As genetic variants in this pathway have been described to influence bone health, aim of this study was the prognostic relevance of genetic variants in RANK and RANKL. Single nucleotide polymorphisms in RANK(L (rs1054016/rs1805034/rs35211496 were genotyped and analyzed with regard to bone metastasis-free survival (BMFS, disease-free survival, and overall survival for a retrospective cohort of 1251 patients. Cox proportional hazard models were built to examine the prognostic influence in addition to commonly established prognostic factors. The SNP rs1054016 seems to influence BMFS. Patients with two minor alleles had a more favorable prognosis than patients with at least one common allele (HR 0.37 (95% CI: 0.17, 0.84, whereas other outcome parameters remained unaffected. rs1805034 and rs35211496 had no prognostic relevance. The effect of rs1054016(RANKL adds to the evidence that the RANK pathway plays a role in BC pathogenesis and progression with respect to BMFS, emphasizing the connection between BC and bone health.

  4. Inhibitory effects of methyl-3,5-di-O-caffeoyl-epi-quinate on RANKL-induced osteoclast differentiation.

    Science.gov (United States)

    Kim, Tae Hoon; Ihn, Hye Jung; Kim, Kiryeong; Cho, Hye-Sung; Shin, Hong-In; Bae, Yong Chul; Park, Eui Kyun

    2018-04-09

    In this study, we have shown that methyl-3,5-di-O-caffeoyl-epi-quinate, a naturally occurring compound isolated from Ainsliaea acerifolia, inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and the expression of osteoclast marker genes. Methyl-3,5-di-O-caffeoyl-epi-quinate also inhibited RANKL-induced activation of p38, Akt and extracellular signal-regulated kinase (ERK) as well as the expression of nuclear factor of activated T-cell (NFATc1), the key regulator of osteoclast differentiation. Negative regulators for osteoclast differentiation was upregulated by methyl-3,5-di-O-caffeoyl-epi-quinate. Collectively, our results suggested that methyl-3,5-di-O-caffeoyl-epi-quinate suppresses osteoclast differentiation via downregulation of RANK signaling pathways and NFATc1. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang-Hyun; Jang, Hae-Dong, E-mail: haedong@hnu.kr

    2015-02-15

    Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and

  6. Stability of [5-3H]uridine-5'-triphosphate

    International Nuclear Information System (INIS)

    Brabec, D.

    1980-01-01

    The effect of temperature, the solvent systems, evaporation, volume reduction, etc. on the decomposition rate of [5- 3 H]uridine-5'-triphosphate was investigated. The decomposition rates and optimum storage conditions were established. The possibility of reducing the duration of the purification and separation process was examined. (author)

  7. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7040...

  8. Synthesis of high specific activity tritium labelled [2-3H]-adenosine-5'-triphosphate

    International Nuclear Information System (INIS)

    Jaiswal, D.K.; Morimoto, H.; Trump, E.L.; Williams, P.G.; Wemmer, D.E.

    1996-01-01

    A procedure for high level tritium labelling at the C2-H position of adenosine 5'-triphosphate ([2- 3 H]-ATP, 1), based on the tritiodehalogenation reaction of 2-bromoadenosine 5'-triphosphate (2) has been elaborated. This precursor was prepared in a six-step synthesis from guanosine. The tritiodehalogenation of (2) for three hours over palladium oxide in phosphate buffer yielded tritium labelled ATP with high specific activity, in good chemical yield. (author)

  9. Combined Inhibition of the BMP pathway and the RANK-RANKL axis in a Mixed Lytic/blastic Prostate Cancer Lesion

    Science.gov (United States)

    Virk, Mandeep S.; Alaee, Farhang; Petrigliano, Frank A.; Sugiyama, Osamu; Chatziioannou, Arion F.; Stout, David; Dougall, William C.; Lieberman, Jay R.

    2010-01-01

    The purpose of this study was to investigate the influence of combined inhibition of RANKL (receptor activator of nuclear factor kappa-B ligand) and bone morphogenetic protein (BMP) activity in a mixed lytic/blastic prostate cancer lesion in bone. Human prostate cancer cells (C4 2b) were injected into immunocompromised mice using an intratibial injection model to create mixed lytic/blastic lesions. RANK-Fc, a recombinant RANKL antagonist, was injected subcutaneously three times a week (10mg/kg) to inhibit RANKL and subsequent formation, function and survival of osteoclasts. Inhibition of BMP activity was achieved by transducing prostate cancer cells ex vivo with a retroviral vector expressing noggin (retronoggin; RN). There were three treatment groups (RANK-Fc treatment, RN treatment and combined RN and RANK-Fc treatment) and two control groups (untreated control and empty vector control for the RN treatment group). The progression of bone lesion and tumor growth was evaluated using plain radiographs, hind limb tumor size, 18F-Fluorodeoxyglucose and 18F-fluoride micro PET-CT, histology and histomorphometry. Treatment with RANK-Fc alone inhibited osteolysis and transformed a mixed lytic/blastic lesion into an osteoblastic phenotype. Treatment with RN alone inhibited the osteoblastic component in a mixed lytic/blastic lesion and resulted in formation of smaller osteolytic bone lesion with smaller soft tissue size. The animals treated with both RN and RANK-Fc demonstrated delayed development of bone lesions, inhibition of osteolysis, small soft tissue tumors and preservation of bone architecture with less tumor induced new bone formation. This study suggests that combined inhibition of the RANKL and the BMP pathway may be an effective biologic therapy to inhibit the progression of established mixed lytic/blastic prostate cancer lesions in bone. PMID:21073986

  10. Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease

    OpenAIRE

    Mikihito Kajiya; Gabriela Giro; Martin A. Taubman; Xiaozhe Han; Marcia P.A. Mayer; Toshihisa Kawai

    2010-01-01

    Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, ...

  11. The Effects of Aronia melanocarpa 'Viking' Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling.

    Science.gov (United States)

    Ghosh, Mithun; Kim, In Sook; Lee, Young Min; Hong, Seong Min; Lee, Taek Hwan; Lim, Ji Hong; Debnath, Trishna; Lim, Beong Ou

    2018-03-08

    This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa 'Viking' (AM) and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS) are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL) were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK) and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun- N -terminal kinase (JNK) and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1) protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β₃. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB)-mediated c-Fos and NFATc1 signaling pathway.

  12. ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency.

    Science.gov (United States)

    Sauer, Aisha V; Mrak, Emanuela; Hernandez, Raisa Jofra; Zacchi, Elena; Cavani, Francesco; Casiraghi, Miriam; Grunebaum, Eyal; Roifman, Chaim M; Cervi, Maria C; Ambrosi, Alessandro; Carlucci, Filippo; Roncarolo, Maria Grazia; Villa, Anna; Rubinacci, Alessandro; Aiuti, Alessandro

    2009-10-08

    Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.

  13. Geraniin suppresses RANKL-induced osteoclastogenesis in vitro and ameliorates wear particle-induced osteolysis in mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Fei; Zhai, Zanjing; Jiang, Chuan; Liu, Xuqiang; Li, Haowei; Qu, Xinhua [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Ouyang, Zhengxiao [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Department of Orthopaedics, Hunan Provincial Tumor Hospital and Tumor Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410013 (China); Fan, Qiming; Tang, Tingting [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Qin, An, E-mail: dr.qinan@gmail.com [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Gu, Dongyun, E-mail: dongyungu@gmail.com [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Engineering Research Center of Digital Medicine and Clinical Translation, Ministry of Education of PR China (China); School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030 (China)

    2015-01-01

    Wear particle-induced osteolysis and subsequent aseptic loosening remains the most common complication that limits the longevity of prostheses. Wear particle-induced osteoclastogenesis is known to be responsible for extensive bone erosion that leads to prosthesis failure. Thus, inhibition of osteoclastic bone resorption may serve as a therapeutic strategy for the treatment of wear particle induced osteolysis. In this study, we demonstrated for the first time that geraniin, an active natural compound derived from Geranium thunbergii, ameliorated particle-induced osteolysis in a Ti particle-induced mouse calvaria model in vivo. We also investigated the mechanism by which geraniin exerts inhibitory effects on osteoclasts. Geraniin inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, evidenced by reduced osteoclast formation and suppressed osteoclast specific gene expression. Specially, geraniin inhibited actin ring formation and bone resorption in vitro. Further molecular investigation demonstrated geraniin impaired osteoclast differentiation via the inhibition of the RANKL-induced NF-κB and ERK signaling pathways, as well as suppressed the expression of key osteoclast transcriptional factors NFATc1 and c-Fos. Collectively, our data suggested that geraniin exerts inhibitory effects on osteoclast differentiation in vitro and suppresses Ti particle-induced osteolysis in vivo. Geraniin is therefore a potential natural compound for the treatment of wear particle induced osteolysis in prostheses failure. - Highlights: • Geraniin suppresses osteoclasts formation and function in vitro. • Geraniin impairs RANKL-induced nuclear factor-κB and ERK signaling pathway. • Geraniin suppresses osteolysis in vivo. • Geraniin may be used for treating osteoclast related diseases.

  14. Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

    Science.gov (United States)

    Grossmann, K; Friedrich, H; Seitz, U

    1980-01-01

    The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I. PMID:7470092

  15. Etude de l'influence du TNFSF11 (RANKL) sur le développment et homéostasie des organes lymphoïdes secondaires

    OpenAIRE

    Cordeiro , Olga

    2015-01-01

    RANKL and RANK are members of the TNF-superfamily and TNF-receptor superfamily, respectively. They are known to play an important role in the regulation of bone mass and in the development and the function of the immune system. However questions still remain. We have used genetically modified mice to address some of these questions, in particular by using a mouse whose lymph node marginal reticular stromal cells lack RANKL. The results obtained during this PhD provide important new insights i...

  16. Tenuigenin inhibits RANKL-induced osteoclastogenesis by down-regulating NF-κB activation and suppresses bone loss in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Shuo [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China); Department of Orthopedics, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410012 (China); Li, Xianan [Department of Orthopedics, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410012 (China); Cheng, Liang [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China); Wu, Hongwei [Department of Orthopedics, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410012 (China); Zhang, Can [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China); Li, Kanghua, E-mail: lkh8738@sina.com [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China)

    2015-10-30

    Tenuigenin, a major active component of polygala tenuifolia root, has been used to treat patients with insomnia, dementia, and neurosis. In this study, we aimed to investigate the effects of tenuigenin on osteoclastogenesis and clarify the possible mechanism. We showed that tenuigenin inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption without cytotoxicity, which was further demonstrated by reduced osteoclast specific gene expression such as TRAP, c-Src, ATP6v0d2, etc. Moreover, the inhibitory effect of tenuigenin was associated with impaired NF-κB activity owing to delayed degradation/regeneration of IkBa and inhibition of p65 nuclear translocation. Consistent with the in vitro results, micro-ct scanning and analysis data showed that tenuigenin suppressed RANKL-induced bone loss in an animal model. Taken together, our data demonstrate that tenuigenin inhibit osteoclast formation and bone resorption both in vitro and in vivo, and comprise a potential therapeutic alternative for osteoclast-related disorders such as osteoporosis and cancer-induced bone destruction. - Highlights: • Tenuigenin suppresses osteoclasts formation, survival and function in vitro. • Tenuigenin impairs NF-κB activation. • Tenuigenin suppresses RANKL-induced bone lose in vivo. • Tenuigenin may be used for treating osteoclast related diseases.

  17. Tenuigenin inhibits RANKL-induced osteoclastogenesis by down-regulating NF-κB activation and suppresses bone loss in vivo

    International Nuclear Information System (INIS)

    Yang, Shuo; Li, Xianan; Cheng, Liang; Wu, Hongwei; Zhang, Can; Li, Kanghua

    2015-01-01

    Tenuigenin, a major active component of polygala tenuifolia root, has been used to treat patients with insomnia, dementia, and neurosis. In this study, we aimed to investigate the effects of tenuigenin on osteoclastogenesis and clarify the possible mechanism. We showed that tenuigenin inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption without cytotoxicity, which was further demonstrated by reduced osteoclast specific gene expression such as TRAP, c-Src, ATP6v0d2, etc. Moreover, the inhibitory effect of tenuigenin was associated with impaired NF-κB activity owing to delayed degradation/regeneration of IkBa and inhibition of p65 nuclear translocation. Consistent with the in vitro results, micro-ct scanning and analysis data showed that tenuigenin suppressed RANKL-induced bone loss in an animal model. Taken together, our data demonstrate that tenuigenin inhibit osteoclast formation and bone resorption both in vitro and in vivo, and comprise a potential therapeutic alternative for osteoclast-related disorders such as osteoporosis and cancer-induced bone destruction. - Highlights: • Tenuigenin suppresses osteoclasts formation, survival and function in vitro. • Tenuigenin impairs NF-κB activation. • Tenuigenin suppresses RANKL-induced bone lose in vivo. • Tenuigenin may be used for treating osteoclast related diseases.

  18. Effects of high glucose and advanced glycation end products on the expressions of sclerostin and RANKL as well as apoptosis in osteocyte-like MLO-Y4-A2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Ken-ichiro, E-mail: ken1nai@med.shimane-u.ac.jp; Yamaguchi, Toru, E-mail: yamaguch@med.shimane-u.ac.jp; Kanazawa, Ippei, E-mail: ippei.k@med.shimane-u.ac.jp; Sugimoto, Toshitsugu, E-mail: sugimoto@med.shimane-u.ac.jp

    2015-05-29

    In diabetes mellitus (DM), high glucose (HG) and advanced glycation end products (AGEs) are involved in bone quality deterioration. Osteocytes produce sclerostin and receptor activator of nuclear factor-kB ligand (RANKL) and regulate osteoblast and osteoclast function. However, whether HG or AGEs directly affect osteocytes and regulate sclerostin and RANKL production is unknown. Here, we examined the effects of HG, AGE2, and AGE3 on the expression of sclerostin and RANKL and on apoptosis in osteocyte-like MLO-Y4-A2 cells. Treatment of the cells with 22 mM glucose, 100 μg/mL either AGE2 or AGE3 significantly increased the expression of sclerostin protein and mRNA; however, both AGEs, but not glucose, significantly decreased the expression of RANKL protein and mRNA. Moreover, treatment of the cells with HG, AGE2, or AGE3 for 72 h induced significant apoptosis. These detrimental effects of HG, AGE2, and AGE3 on sclerostin and RANKL expressions and on apoptosis were antagonized by pretreatment of the cells with 10{sup −8} M human parathyroid hormone (PTH)-(1–34). Thus, HG and AGEs likely suppress bone formation by increasing sclerostin expression in osteocytes, whereas AGEs suppress bone resorption by decreasing RANKL expression. Together, these processes may cause low bone turnover in DM. In addition, HG and AGEs may cause cortical bone deterioration by inducing osteocyte apoptosis. PTH may effectively treat these pathological processes and improve osteocyte function. - Highlights: • AGEs are involved in bone quality deterioration in diabetes mellitus (DM). • AGEs increased sclerostin as well as apoptosis, and decreased RANKL in osteocytes. • The effects of AGEs on osteocyte function were antagonized by human PTH-(1–34). • AGEs may cause low bone turnover and cortical porosity in DM. • PTH may be effective in bone quality deterioration by improving osteocyte function.

  19. Impaired bone remodeling in children with osteogenesis imperfecta treated and untreated with bisphosphonates: the role of DKK1, RANKL, and TNF-α.

    Science.gov (United States)

    Brunetti, G; Papadia, F; Tummolo, A; Fischetto, R; Nicastro, F; Piacente, L; Ventura, A; Mori, G; Oranger, A; Gigante, I; Colucci, S; Ciccarelli, M; Grano, M; Cavallo, L; Delvecchio, M; Faienza, M F

    2016-07-01

    In this study, we investigated the bone cell activity in patients with osteogenesis imperfecta (OI) treated and untreated with neridronate. We demonstrated the key role of Dickkopf-1 (DKK1), receptor activator of nuclear factor-κB ligand (RANKL), and tumor necrosis factor alpha (TNF-α) in regulating bone cell of untreated and treated OI subjects. These cytokines could represent new pharmacological targets for OI. Bisphosphonates are widely used in the treatment of children with osteogenesis imperfecta (OI) with the objective of reducing the risk of fractures. Although bisphosphonates increase bone mineral density in OI subjects, the effects on fracture incidence are conflicting. The aim of this study was to investigate the mechanisms underlying bone cell activity in subjects with mild untreated forms of OI and in a group of subjects with severe OI treated with cycles of intravenous neridronate. Sclerostin, DKK1, TNF-α, RANKL, osteoprotegerin (OPG), and bone turnover markers were quantified in serum of 18 OI patients (12 females, mean age 8.86 ± 3.90), 8 of which were receiving cyclic intravenous neridronate, and 21 sex- and age-matched controls. The effects on osteoblastogenesis and OPG expression of media conditioned by the serum of OI patients and anti-DKK1 neutralizing antibody were evaluated. Osteoclastogenesis was assessed in cultures from patients and controls. DKK1 and RANKL levels were significantly increased both in untreated and in treated OI subjects with respect to controls. The serum from patients with high DKK1 levels inhibited both osteoblast differentiation and OPG expression in vitro. High RANKL and low OPG messenger RNA (mRNA) levels were found in lymphomonocytes from patients. High amounts of TNF-α were expressed by monocytes, and an elevated percentage of circulating CD11b-CD51/CD61+ osteoclast precursors was observed in patients. Our study demonstrated the key role of DKK1, RANKL, and TNF-α in regulating bone cell activity of subjects

  20. Effects of Wenyangbushen formula on the expression of VEGF, OPG, RANK and RANKL in rabbits with steroid-induced femoral head avascular necrosis.

    Science.gov (United States)

    Song, Hong-Mei; Wei, Ying-Chen; Li, Nan; Wu, Bin; Xie, Na; Zhang, Kun-Mu; Wang, Shi-Zhong; Wang, He-Ming

    2015-12-01

    The present study aimed to investigate the effects of Wenyangbushen formula on the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), osteoprotegerin (OPG), receptor activator of nuclear factor (NF)‑κβ ligand (RANK), and RANK ligand (RANKL) in a rabbit model of steroid‑induced avascular necrosis of the femoral head (SANFH). The present study also aimed to examine the potential mechanism underlying the effect of this formula on the treatment of SANFH. A total of 136 New Zealand rabbits were randomly divided into five groups: Normal group, model group, and three groups treated with the traditional Chinese medicine (TCM), Wenyangbushen decoction, at a low, moderate and high dose, respectively. The normal group and positive control group were intragastrically administered with saline. The TCM groups were treated with Wenyangbushen decoction at the indicated dosage. Following treatment for 8 weeks, the mRNA and protein expression levels of VEGF, OPG, RANK and RANKL in the femoral head tissues were determined using reverse transcription‑quantitative polymerase chain reaction and western blot analyses, respectively. The data revealed that Wenyangbushen decoction effectively promoted the growth of bone cells, osteoblasts and chondrocytes, and prevented cell apoptosis in the SANFH. The mRNA and protein expression levels of OPG and VEGF were increased, while the levels of RANK and RANKL were reduced in the necrotic tissue of the model group, compared with those in the normal rabbits. Wenyangbushen treatment prevented these changes, manifested by an upregulation in the expression levels of VEGF and OPG, and downregulation in the expression levels of RANK and RANKL in a dose‑dependent manner. It was concluded that treatment with Wenyangbushen formula alleviated necrosis of the femoral head induced by steroids. It was observed to promote bone cell, osteoblast and chondrocyte growth, as well as prevent cell apoptosis. In addition, it

  1. Parallel Assessment of Bone Mineral Density and RANKL/OPG Ratio in Saudi Females

    OpenAIRE

    AI Hassan; SA Eltarhouny; HE Hashem; SA Algaidi; AR Abdallah; AM Sandokji

    2016-01-01

    Background: Osteoporosis is a significant risk factor for morbidity, and its high prevalence among Saudi women should be considered to be a public health problem. Quantitative ultrasound was recommended for bone mineral density (BMD) screening. Receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) and their ratio are critical for physiological bone remodelling, and related abnormalities may lead to several osteopathies. Methods: The BMD of 499 Saudi female...

  2. Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    Smith, C.A.; Hanawalt, P.C.

    1978-01-01

    The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [ 3 H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3 H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg 2+ -dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

  3. The Effects of Aronia melanocarpa ‘Viking’ Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling

    Directory of Open Access Journals (Sweden)

    Mithun Ghosh

    2018-03-01

    Full Text Available This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa ‘Viking’ (AM and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK, c-Jun-N-terminal kinase (JNK and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1 protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β3. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB-mediated c-Fos and NFATc1 signaling pathway.

  4. The key role of proinflammatory cytokines, matrix proteins, RANKL/OPG and Wnt/β-catenin in bone healing of hip arthroplasty patients.

    Science.gov (United States)

    Cassuto, Jean; Folestad, Agnetha; Göthlin, Jan; Malchau, Henrik; Kärrholm, Johan

    2018-02-01

    We still lack understanding of why some implants fail while most remain stable after decades of use. Proinflammatory cytokines, matrix proteins and bone regulating cytokines of the RANKL/OPG (receptor activator of nuclear factor kappa B ligand/osteoprotegerin) and Wnt/β-catenin pathways are mandatory for normal bone repair but their spatial and temporal role in the healing of primary total hip arthroplasties (THA) has not been previously shown. Twenty-four osteoarthritis patients with one-sided well-fixed primary THA were prospectively monitored during 18years (18Y) with repeated blood samples, clinical variables and radiographs. Eighty-one healthy donors divided in three age- and gender-matched groups and twenty osteoarthritis patients awaiting THA and serving as control of the validity of stored plasma in THA patients, were included. Plasma was analyzed for C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-1β, tumor necrosis factor (TNF)-α, osteopontin (OPN), secreted protein acidic and rich in cysteine (SPARC/osteonectin), osteocalcin (OC), bone specific alkaline phosphatase (BALP), N-terminal propeptide of collagen type I (P1NP), RANKL, OPG, the Wnt agonistic ligands (Wnt)-1 and Wnt-3a, and the Wnt antagonists sclerostin, Dickkopf (Dkk)-1, Dkk-3, Dkk-4, secreted frizzled related protein (sFRP)-1, sFRP-3 and Wnt inhibitory factor-1 (Wif-1). Inflammatory mediators in arthroplasty patients (CRP, IL-6, OPN) increased significantly on day one after surgery vs preoperative value (PR) and healthy subjects and returned to baseline at 6W. TNF-α did not change relative preoperative level or healthy subjects. SPARC and OC increased in a biphasic fashion with the primary phase beginning shortly after surgery and lasting 3M (SPARC) and 2Y (OC) while the secondary phase peaked at 1Y (SPARC) and 13Y (OC), with both returning to basal level at 15Y. BALP peaked at 3M after surgery with a return to basal level at 2Y followed by a continuous increase from 5Y until 18Y. P

  5. Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.

    Directory of Open Access Journals (Sweden)

    Jinhu Xiong

    Full Text Available The cytokine receptor activator of nuclear factor kappa B ligand (RANKL, encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

  6. RANK/RANKL/OPG Signalization Implication in Periodontitis: New Evidence from a RANK Transgenic Mouse Model

    Directory of Open Access Journals (Sweden)

    Bouchra Sojod

    2017-05-01

    Full Text Available Periodontitis is based on a complex inflammatory over-response combined with possible genetic predisposition factors. The RANKL/RANK/OPG signaling pathway is implicated in bone resorption through its key function in osteoclast differentiation and activation, as well as in the inflammatory response. This central element of osteo-immunology has been suggested to be perturbed in several diseases, including periodontitis, as it is a predisposing factor for this disease. The aim of the present study was to validate this hypothesis using a transgenic mouse line, which over-expresses RANK (RTg and develops a periodontitis-like phenotype at 5 months of age. RTg mice exhibited severe alveolar bone loss, an increased number of TRAP positive cells, and disorganization of periodontal ligaments. This phenotype was more pronounced in females. We also observed dental root resorption lacunas. Hyperplasia of the gingival epithelium, including Malassez epithelial rests, was visible as early as 25 days, preceding any other symptoms. These results demonstrate that perturbations of the RANKL/RANK/OPG system constitute a core element of periodontitis, and more globally, osteo-immune diseases.

  7. RANK/RANKL/OPG Signalization Implication in Periodontitis: New Evidence from a RANK Transgenic Mouse Model

    Science.gov (United States)

    Sojod, Bouchra; Chateau, Danielle; Mueller, Christopher G.; Babajko, Sylvie; Berdal, Ariane; Lézot, Frédéric; Castaneda, Beatriz

    2017-01-01

    Periodontitis is based on a complex inflammatory over-response combined with possible genetic predisposition factors. The RANKL/RANK/OPG signaling pathway is implicated in bone resorption through its key function in osteoclast differentiation and activation, as well as in the inflammatory response. This central element of osteo-immunology has been suggested to be perturbed in several diseases, including periodontitis, as it is a predisposing factor for this disease. The aim of the present study was to validate this hypothesis using a transgenic mouse line, which over-expresses RANK (RTg) and develops a periodontitis-like phenotype at 5 months of age. RTg mice exhibited severe alveolar bone loss, an increased number of TRAP positive cells, and disorganization of periodontal ligaments. This phenotype was more pronounced in females. We also observed dental root resorption lacunas. Hyperplasia of the gingival epithelium, including Malassez epithelial rests, was visible as early as 25 days, preceding any other symptoms. These results demonstrate that perturbations of the RANKL/RANK/OPG system constitute a core element of periodontitis, and more globally, osteo-immune diseases. PMID:28596739

  8. Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPα-interaction

    International Nuclear Information System (INIS)

    Lundberg, Pernilla; Koskinen, Cecilia; Baldock, Paul A.; Loethgren, Hanna; Stenberg, Asa; Lerner, Ulf H.; Oldenborg, Per-Arne

    2007-01-01

    Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPα) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPα-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPα strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP) + osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47 -/- mice were strongly reduced, and bones of CD47 -/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPα interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47 -/- mice

  9. Paracrine-mediated osteoclastogenesis by the osteosarcoma MG63 cell line: is RANKL/RANK signalling really important?

    Science.gov (United States)

    Costa-Rodrigues, J; Teixeira, C A; Fernandes, M H

    2011-08-01

    Although in the past little attention has been paid to the influence of osteosarcoma cells in osteoclast function, recent studies suggest a close relationship between osteosarcoma aggressiveness and osteoclastic activity. The present study addresses the paracrine effects of MG63 cells, a human osteosarcoma-derived cell line, on the differentiation of peripheral blood osteoclast precursor cells (PBMC). PBMC were cultured for 21 days in the presence of conditioned media from MG63 cell cultures (CM) collected at 48 h (CM_MG1), 7 days (CM_MG2) and 14 days (CM_MG3). MG63 cell cultures displayed the expression of ALP and BMP-2 and, also, the osteoclastogenic genes M-CSF and RANKL, although with a low expression of RANKL. PBMC cultures supplemented with CM presented an evident osteoclastogenic behavior, which was dependent on the culture period of the MG63 cells. The inductive effect appeared to be more relevant for the differentiation and activation genes, c-myc and c-src, and lower for genes associated with osteoclast function. In addition, PBMC cultures displayed increased functional parameters, including calcium phosphate resorbing activity. Assessment of the PBMC cultures in the presence of U0126, PDTC, and indomethacin suggested that in addition to MEK and NFkB pathways, other signaling mechanisms, probably not involving RANKL/RANK interaction, might be activated in the presence of conditioned medium from MG63. In conclusion, MG63 cell line appears to induce a significant paracrine-mediated osteoclastogenic response. Understanding the mechanisms underlying the interaction of osteosarcoma cells and osteoclasts may contribute to the development of new potential approaches in the treatment of such bone metabolic diseases.

  10. Anti-RANKL treatment inhibits erosive joint destruction and lowers inflammation but has no effect on bone formation in the delayed-type hypersensitivity arthritis (DTHA) model

    DEFF Research Database (Denmark)

    Atkinson, Sara Marie; Bleil, Janine; Maier, Rene

    2016-01-01

    . Periarticular bone formation was observed from day 10. Induction of new bone formation indicated by enhanced Runx2, collagen X, osteocalcin, MMP2, MMP9, and MMP13 mRNA expression was observed only between days 8 and 11. Anti-RANKL treatment resulted in a modest reduction in paw and ankle swelling...... and bone formation were analyzed by mRNA deep sequencing. Serum concentrations of tartrate-resistant acid phosphatase 5b, carboxy-terminal telopeptide I (CTX-I), matrix metalloproteinase 3 (MMP3), and serum amyloid P component (SAP) were determined by enzyme-linked immunosorbent assay. Anti......-RANKL monoclonal antibody treatment was initiated at the time of immunization. Results: Bone destruction (MMP3 serum levels, cathepsin B activity, and RANKL mRNA) peaked at day 3 after arthritis induction, followed by a peak in cartilage destruction and bone erosion on day 5 after arthritis induction...

  11. Model Linking Plasma and Intracellular Tenofovir/Emtricitabine with Deoxynucleoside Triphosphates.

    Directory of Open Access Journals (Sweden)

    Xinhui Chen

    Full Text Available The coformulation of the nucleos(tide analogs (NA tenofovir (TFV disoproxil fumarate (TDF and emtricitabine (FTC is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP and FTC-triphosphate (FTC-TP. Such complexities manifest in nonlinear intracellular pharmacokinetics (PK. In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD study among forty subjects receiving daily TDF/FTC (300 mg/200 mg from the first-dose to pharmacological intracellular steady-state (30 days. TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM. Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP and deoxycytidine triphosphate (dCTP production were 1020 fmol/106 cells (130% and 44.4 pmol/106 cells (82.5%, resulting in (90% prediction interval 11% (0.45%, 53% and 14% (2.6%, 35% reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP. Simulation

  12. Follicle-Stimulating Hormone Increases the Risk of Postmenopausal Osteoporosis by Stimulating Osteoclast Differentiation.

    Directory of Open Access Journals (Sweden)

    Jie Wang

    Full Text Available The objectives of this study were to observe the changes in follicle-stimulating hormone (FSH and bone mineral density (BMD in postmenopausal women, to research the relationship between FSH and postmenopausal osteoporosis, and to observe the effects of FSH on osteoclast differentiation in RAW264.7 cells.We analyzed 248 postmenopausal women with normal bone metabolism. A radioimmunoassay (RIA was used to detect serum FSH, luteinizing hormone (LH, and estradiol (E2. Dual-energy X-ray absorptiometry was used to measure forearm BMD. Then, we analyzed the age-related changes in serum FSH, LH and E2. Additionally, FSH serum concentrations were compared between a group of postmenopausal women with osteoporosis and a control group. Osteoclasts were induced from RAW264.7 cells in vitro by receptor activator of nuclear factor kappa B ligand (RANKL, and these cells were treated with 0, 5, 10, and 20 ng/ml FSH. After the osteoclasts matured, tartrate-resistant acid phosphatase (TRAP staining was used to identify osteoclasts, and the mRNA expression levels of genes involved in osteoclastic phenotypes and function, such as receptor activator of NF-κB (Rank, Trap, matrix metalloproteinase-9 (Mmp-9 and Cathepsin K, were detected in different groups using real-time PCR (polymerase chain reaction.1. FSH serum concentrations in postmenopausal women with osteoporosis increased notably compared with the control group. 2. RANKL induced RAW264.7 cell differentiation into mature osteoclasts in vitro. 3. FSH increased mRNA expression of genes involved in osteoclastic phenotypes and function, such as Rank, Trap, Mmp-9 and Cathepsin K, in a dose-dependent manner.The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro.

  13. Picrasidine I from Picrasma Quassioides Suppresses Osteoclastogenesis via Inhibition of RANKL Induced Signaling Pathways and Attenuation of ROS Production

    Directory of Open Access Journals (Sweden)

    Lingbo Kong

    2017-10-01

    Full Text Available Background/Aims: Osteoporosis is a metabolic bone disorder that tortures about millions of people worldwide. Recent study demonstrated agents derived from picrasma quassioides is a promising drug for targets multiple signaling pathways. However its potential in treatment of bone loss has not been fully understood. Methods: The bone marrow macrophages (BMMs were cultured and induced with M-CSF and RANKL followed by picrasidine I (PI treatment. Then the effects of PI on osteoclast formation were evaluated by counting tartrate-resistant acid phosphatase (TRAP-positive multinucleated cells. Moreover, effects of PI on bone resorption activity of mature osteoclast were studied through bone resorption pit counting and actin ring structure analysis. Further, the involved potential signaling pathways cross-talking were investigated by performed Western blotting and quantitative real-time PCR examination. Results: Results demonstrated PI strongly inhibited RANKL induced osteoclast formation from its precursors. Mechanistically, the inhibitory effect of PI on osteoclast differentiation was due to the suppression of osteoclastogenic transcription factors, c-Fos and NFATc1. Moreover, PI markedly blocked the RANKL-induced osteoclastogenesis by attenuating MAPKs and NF-κB signaling pathways. In addition, PI decreased the ROS generation in osteoclast and osteoblast. Conclusion: Taken together our data demonstrate that PI has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-κB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

  14. RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

    International Nuclear Information System (INIS)

    Sundaram, Kumaran; Nishimura, Riko; Senn, Joseph; Youssef, Rimon F.; London, Steven D.; Reddy, Sakamuri V.

    2007-01-01

    Osteoclast differentiation is tightly regulated by receptor activator of NF-κB ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+ 1 to - 1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to + 1 bp to - 446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from - 446 bp to - 1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (- 1123 bp to - 1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in

  15. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    NARCIS (Netherlands)

    Makarchikov, A.F.; Wins, P.; Janssen, E.E.W.; Wieringa, B.; Grisar, T.; Bettendorff, L.

    2002-01-01

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate

  16. Th17-cells in atopic dermatitis stimulate orthodontic root resorption.

    Science.gov (United States)

    Yamada, K; Yamaguchi, M; Asano, M; Fujita, S; Kobayashi, R; Kasai, K

    2013-10-01

    The aim of this study was to investigate how atopic dermatitis (AD) contributes to root resorption during orthodontic tooth movement. Atopic dermatitis model mice and wild-type mice were subjected to an excessive orthodontic force (OF) to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and RANKL proteins were determined in the periodontal ligament (PDL) by an immunohistochemical analysis. Furthermore, the effects of the compression force on co-cultures of CD4(+) cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin. The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6, and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. Th17 cells may therefore be associated with the deterioration of root resorption of AD mice, and may explain why AD patients are more susceptible to root resorption than healthy individuals when an excessive OF is applied. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Andrographolide Inhibits Ovariectomy-Induced Bone Loss via the Suppression of RANKL Signaling Pathways

    Science.gov (United States)

    Wang, Tao; Liu, Qian; Zhou, Lin; Yuan, Jin Bo; Lin, Xixi; Zeng, Rong; Liang, Xiaonan; Zhao, Jinmin; Xu, Jiake

    2015-01-01

    Osteoporosis is a debilitating skeletal disorder with an increased risk of low-energy fracture, which commonly occurs among postmenopausal women. Andrographolide (AP), a natural product isolated from Andrographis paniculata, has been found to have anti-inflammatory, anti-cancer, anti-asthmatic, and neuro-protective properties. However, its therapeutic effect on osteoporosis is unknown. In this study, an ovariectomy (OVX) mouse model was used to evaluate the therapeutic effects of AP on post-menopausal osteoporosis by using micro-computed tomography (micro-CT). Bone marrow-derived osteoclast culture was used to examine the inhibitory effect of AP on osteoclastogenesis. Real time PCR was employed to examine the effect of AP on the expression of osteoclast marker genes. The activities of transcriptional factors NF-κB and NFATc1 were evaluated using a luciferase reporter assay, and the IκBα protein level was analyzed by Western blot. We found that OVX mice treated with AP have greater bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) compared to vehicle-treated OVX mice. AP inhibited RANKL-induced osteoclastogenesis, the expression of osteoclast marker genes including cathepsin K (Ctsk), TRACP (Acp5), and NFATc1, as well as the transcriptional activities of NF-κB and NFATc1. In conclusion, our results suggest that AP inhibits estrogen deficiency-induced bone loss in mice via the suppression of RANKL-induced osteoclastogensis and NF-κB and NFATc1 activities and, thus, might have therapeutic potential for osteoporosis. PMID:26593901

  18. Andrographolide Inhibits Ovariectomy-Induced Bone Loss via the Suppression of RANKL Signaling Pathways.

    Science.gov (United States)

    Wang, Tao; Liu, Qian; Zhou, Lin; Yuan, Jin Bo; Lin, Xixi; Zeng, Rong; Liang, Xiaonan; Zhao, Jinmin; Xu, Jiake

    2015-11-17

    Osteoporosis is a debilitating skeletal disorder with an increased risk of low-energy fracture, which commonly occurs among postmenopausal women. Andrographolide (AP), a natural product isolated from Andrographis paniculata, has been found to have anti-inflammatory, anti-cancer, anti-asthmatic, and neuro-protective properties. However, its therapeutic effect on osteoporosis is unknown. In this study, an ovariectomy (OVX) mouse model was used to evaluate the therapeutic effects of AP on post-menopausal osteoporosis by using micro-computed tomography (micro-CT). Bone marrow-derived osteoclast culture was used to examine the inhibitory effect of AP on osteoclastogenesis. Real time PCR was employed to examine the effect of AP on the expression of osteoclast marker genes. The activities of transcriptional factors NF-κB and NFATc1 were evaluated using a luciferase reporter assay, and the IκBα protein level was analyzed by Western blot. We found that OVX mice treated with AP have greater bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) compared to vehicle-treated OVX mice. AP inhibited RANKL-induced osteoclastogenesis, the expression of osteoclast marker genes including cathepsin K (Ctsk), TRACP (Acp5), and NFATc1, as well as the transcriptional activities of NF-κB and NFATc1. In conclusion, our results suggest that AP inhibits estrogen deficiency-induced bone loss in mice via the suppression of RANKL-induced osteoclastogensis and NF-κB and NFATc1 activities and, thus, might have therapeutic potential for osteoporosis.

  19. Andrographolide Inhibits Ovariectomy-Induced Bone Loss via the Suppression of RANKL Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Tao Wang

    2015-11-01

    Full Text Available Osteoporosis is a debilitating skeletal disorder with an increased risk of low-energy fracture, which commonly occurs among postmenopausal women. Andrographolide (AP, a natural product isolated from Andrographis paniculata, has been found to have anti-inflammatory, anti-cancer, anti-asthmatic, and neuro-protective properties. However, its therapeutic effect on osteoporosis is unknown. In this study, an ovariectomy (OVX mouse model was used to evaluate the therapeutic effects of AP on post-menopausal osteoporosis by using micro-computed tomography (micro-CT. Bone marrow-derived osteoclast culture was used to examine the inhibitory effect of AP on osteoclastogenesis. Real time PCR was employed to examine the effect of AP on the expression of osteoclast marker genes. The activities of transcriptional factors NF-κB and NFATc1 were evaluated using a luciferase reporter assay, and the IκBα protein level was analyzed by Western blot. We found that OVX mice treated with AP have greater bone volume (BV/TV, trabecular thickness (Tb.Th, and trabecular number (Tb.N compared to vehicle-treated OVX mice. AP inhibited RANKL-induced osteoclastogenesis, the expression of osteoclast marker genes including cathepsin K (Ctsk, TRACP (Acp5, and NFATc1, as well as the transcriptional activities of NF-κB and NFATc1. In conclusion, our results suggest that AP inhibits estrogen deficiency-induced bone loss in mice via the suppression of RANKL-induced osteoclastogensis and NF-κB and NFATc1 activities and, thus, might have therapeutic potential for osteoporosis.

  20. Influence of Simultaneous Targeting of the Bone Morphogenetic Protein Pathway and RANK-RANKL Axis in Osteolytic Prostate Cancer Lesion in Bone

    Science.gov (United States)

    Virk, Mandeep S.; Petrigliano, Frank A.; Liu, Nancy Q.; Chatziioannou, Arion F.; Stout, David; Kang, Christine O.; Dougall, William C.; Lieberman, Jay R.

    2009-01-01

    Metastasis to bone is the leading cause of morbidity and mortality in advanced prostate cancer patients. Considering the complex reciprocal interactions between the tumor cells and the bone microenvironment, there is increasing interest in developing combination therapies targeting both the tumor growth and the bone microenvironment. In this study, we investigated the effect of simultaneous blockade of BMP pathway and RANK-RANKL axis in an osteolytic prostate cancer lesion in bone. We used a retroviral vector encoding noggin (Retronoggin) to antagonize the effect of BMPs and RANK: Fc, which is a recombinant RANKL antagonist was used to inhibit RANK-RANKL axis. The tumor growth and bone loss were evaluated using plain radiographs, hind limb tumor measurements, micro PET-CT (18F- fluorodeoxyglucose [FDG] and 18F-fluoride tracer), and histology. Tibias implanted with PC-3 cells developed pure osteolytic lesions at 2 weeks with progressive increase in cortical bone destruction at successive time points. Tibias implanted with PC-3 cells over expressing noggin (Retronoggin) resulted in reduced tumor size and decreased bone loss compared to the implanted tibias in untreated control animals. RANK: Fc administration inhibited the formation of osteoclasts, delayed the development of osteolytic lesions, decreased bone loss and reduced tumor size in tibias implanted with PC-3 cells. The combination therapy with RANK: Fc and noggin over expression effectively delayed the radiographic development of osteolytic lesions, and decreased the bone loss and tumor burden compared to implanted tibias treated with noggin over expression alone. Furthermore, the animals treated with the combination strategy exhibited decreased bone loss (micro CT) and lower tumor burden (FDG micro PET) compared to animals treated with RANK: Fc alone. Combined blockade of RANK-RANKL axis and BMP pathway resulted in reduced tumor burden and decreased bone loss compared to inhibition of either individual

  1. Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice.

    Directory of Open Access Journals (Sweden)

    J Preston Campbell

    2012-07-01

    Full Text Available Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.

  2. Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice.

    Science.gov (United States)

    Campbell, J Preston; Karolak, Matthew R; Ma, Yun; Perrien, Daniel S; Masood-Campbell, S Kathryn; Penner, Niki L; Munoz, Steve A; Zijlstra, Andries; Yang, Xiangli; Sterling, Julie A; Elefteriou, Florent

    2012-07-01

    Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s) remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.

  3. Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate

    International Nuclear Information System (INIS)

    Koka, P.

    1984-01-01

    A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme, to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h -1 mg -1 ) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, up to 26 nucleotides in length in relation to DNA size markers, but these are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [γ- 32 P]ATP, and UV-irradiated DNA-cellulose contained exogenous [γ- 32 P], which eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions. Higher (X5) concentrations of ADP and adenosine 5'-(β, γ-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity. In addition, there is an apparent hydrolysis of ATP during photoreactivation as measured by the release of 32 P from [γ- 32 P]ATP

  4. Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation.

    Directory of Open Access Journals (Sweden)

    Qian Feng

    Full Text Available Upon viral infections, pattern recognition receptors (PRRs recognize pathogen-associated molecular patterns (PAMPs and stimulate an antiviral state associated with the production of type I interferons (IFNs and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5' triphosphate (5'ppp-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3, a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5'ppp. We show here that a 5'ppp-containing cloverleaf (CL RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.

  5. Periodontal tissue activation by vibration: intermittent stimulation by resonance vibration accelerates experimental tooth movement in rats.

    Science.gov (United States)

    Nishimura, Makoto; Chiba, Mirei; Ohashi, Toshiro; Sato, Masaaki; Shimizu, Yoshiyuki; Igarashi, Kaoru; Mitani, Hideo

    2008-04-01

    Accelerating the speed of orthodontic tooth movement should contribute to the shortening of the treatment period. This would be beneficial because long treatment times are a negative aspect of orthodontic treatment. In this study, we evaluated the effects of mechanical stimulation by resonance vibration on tooth movement, and we showed the cellular and molecular mechanisms of periodontal ligament responses. The maxillary first molars of 6-week-old male Wistar rats were moved to the buccal side by using an expansive spring for 21 days (n = 6, control group), and the amount of tooth movement was measured. Additional vibrational stimulation (60 Hz, 1.0 m/s(2)) was applied to the first molars by using a loading vibration system for 8 minutes on days 0, 7, and 14 during orthodontic tooth movement (n = 6, experimental group). The animals were killed under anesthesia, and each maxilla was dissected. The specimens were fixed, decalcified, and embedded in paraffin. Sections were used for immunohistochemical analysis of receptor activator of NF kappa B ligand (RANKL) expression. The number of osteoclasts in the alveolar bone was counted by using TRAP staining, and the amount of root resorption was measured in sections stained with hematoxylin and eosin. The average resonance frequency of the maxillary first molar was 61.02 +/- 8.38 Hz. Tooth movement in the experimental group was significantly greater than in the control group (P vibration might accelerate orthodontic tooth movement via enhanced RANKL expression in the periodontal ligament without additional damage to periodontal tissues such as root resorption.

  6. Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Bajaj, Mamta; Moriyama, Hideaki

    2007-01-01

    The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V M of 1.8 Å 3 Da −1

  7. Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase

    International Nuclear Information System (INIS)

    Porta, Jason; Kolar, Carol; Kozmin, Stanislav G.; Pavlov, Youri I.; Borgstahl, Gloria E. O.

    2006-01-01

    X-ray crystallographic analysis of human inosine triphosphate pyrophosphohydrolase provided the secondary structure and active-site structure at 1.6 Å resolution in an orthorhombic crystal form. The structure gives a framework for future structure–function studies employing site-directed mutagenesis and for the identification of substrate/product-binding sites. The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 Å resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix α1, the loop before α6 and helix α7 to cap off the active site when substrate is bound

  8. Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo: Impact on oxidative stress and RANKL/OPG signals

    International Nuclear Information System (INIS)

    Tolba, Mai F.; El-Serafi, Ahmed T.; Omar, Hany A.

    2017-01-01

    Glucocorticoid-induced osteoporosis (GIO) is one of the most common causes of secondary osteoporosis. Given that glucocorticoids are considered as a main component of the treatment protocols for a variety of inflammation and immune-mediated diseases besides its use as adjuvant to several chemotherapeutic agents, it is crucial to find ways to overcome this critical adverse effect. Caffeic acid phenethyl ester (CAPE), which is a natural compound derived from honeybee propolis displayed promising antiosteoporotic effects against mechanical bone injury in various studies. The current work aimed at investigating the potential protective effect of CAPE against GIO in vivo with emphasis on the modulation of oxidative status and receptor activator of NF-kB ligand (RANKL)/osteoprotegrin (OPG) signaling. The results showed that CAPE opposed dexamethasone (DEX)-mediated alterations in bone histology and tartarate-resistant acid phosphatase (TRAP) activity. In addition, CAPE restored oxidative balance, Runt-related transcription factor 2 (RunX2) expression and reduced caspase-3 activity in femur tissues. Co-administration of CAPE with DEX normalized RANKL/OPG ratio and Akt activation indicating a reduction in DEX-osteoclastogenesis. In conclusion, concurrent treatment of CAPE with DEX exhibited promising effects in the protection against DEX-induced osteoporosis through opposing osteoclastogenesis and protecting osteoblasts. The potent antioxidant activity of CAPE is, at least in part, involved in its anti-apoptotic effects and modulation of RunX2 and RANKL/OPG signals. The use of CAPE-enriched propolis formulas is strongly recommended for patients on chronic glucocorticoid therapy to help in the attenuation of GIO. - Highlights: • Caffeic acid phenethyl ester (CAPE) counteracts DEX-induced osteoporosis. • CAPE hinders DEX-induced alterations in oxidation parameters as GSH, SOD and MDA. • CAPE opposes osteoclastogenesis via suppressing RANL/OPG ratio and Akt signals.

  9. Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo: Impact on oxidative stress and RANKL/OPG signals

    Energy Technology Data Exchange (ETDEWEB)

    Tolba, Mai F. [Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo 11566 (Egypt); Chapman University, Irvine 92618, CA (United States); El-Serafi, Ahmed T. [Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272 (United Arab Emirates); Department of Medical Biochemistry, Faculty of Medicine, Suez Canal University, Ismailia (Egypt); Omar, Hany A., E-mail: hanyomar@sharjah.ac.ae [Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272 (United Arab Emirates); Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514 (Egypt)

    2017-06-01

    Glucocorticoid-induced osteoporosis (GIO) is one of the most common causes of secondary osteoporosis. Given that glucocorticoids are considered as a main component of the treatment protocols for a variety of inflammation and immune-mediated diseases besides its use as adjuvant to several chemotherapeutic agents, it is crucial to find ways to overcome this critical adverse effect. Caffeic acid phenethyl ester (CAPE), which is a natural compound derived from honeybee propolis displayed promising antiosteoporotic effects against mechanical bone injury in various studies. The current work aimed at investigating the potential protective effect of CAPE against GIO in vivo with emphasis on the modulation of oxidative status and receptor activator of NF-kB ligand (RANKL)/osteoprotegrin (OPG) signaling. The results showed that CAPE opposed dexamethasone (DEX)-mediated alterations in bone histology and tartarate-resistant acid phosphatase (TRAP) activity. In addition, CAPE restored oxidative balance, Runt-related transcription factor 2 (RunX2) expression and reduced caspase-3 activity in femur tissues. Co-administration of CAPE with DEX normalized RANKL/OPG ratio and Akt activation indicating a reduction in DEX-osteoclastogenesis. In conclusion, concurrent treatment of CAPE with DEX exhibited promising effects in the protection against DEX-induced osteoporosis through opposing osteoclastogenesis and protecting osteoblasts. The potent antioxidant activity of CAPE is, at least in part, involved in its anti-apoptotic effects and modulation of RunX2 and RANKL/OPG signals. The use of CAPE-enriched propolis formulas is strongly recommended for patients on chronic glucocorticoid therapy to help in the attenuation of GIO. - Highlights: • Caffeic acid phenethyl ester (CAPE) counteracts DEX-induced osteoporosis. • CAPE hinders DEX-induced alterations in oxidation parameters as GSH, SOD and MDA. • CAPE opposes osteoclastogenesis via suppressing RANL/OPG ratio and Akt signals.

  10. RANKL/RANK/OPG cytokine receptor system: mRNA expression pattern in BPH, primary and metastatic prostate cancer disease.

    Science.gov (United States)

    Christoph, Frank; König, Frank; Lebentrau, Steffen; Jandrig, Burkhard; Krause, Hans; Strenziok, Romy; Schostak, Martin

    2018-02-01

    The cytokine system RANKL (receptor activator of NF-κB ligand), its receptor RANK and the antagonist OPG (osteoprotegerin) play a critical role in bone turnover. Our investigation was conducted to describe the gene expression at primary tumour site in prostate cancer patients and correlate the results with Gleason Score and PSA level. Seventy-one samples were obtained from prostate cancer patients at the time of radical prostatectomy and palliative prostate resection (n = 71). Patients with benign prostate hyperplasia served as controls (n = 60). We performed real-time RT-PCR after microdissection of the samples. The mRNA expression of RANK was highest in tumour tissue from patients with bone metastases (p BPH or locally confined tumours, also shown in clinical subgroups distinguished by Gleason Score (BPH tissue but did not exceed as much as in the tumour tissue. We demonstrated that RANK, RANKL and OPG are directly expressed by prostate cancer cells at the primary tumour site and showed a clear correlation with Gleason Score, serum PSA level and advanced disease. In BPH, mRNA expression is also detectable, but RANK expression does not exceed as much as compared to tumour tissue.

  11. Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Bajaj, Mamta [School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States); Moriyama, Hideaki, E-mail: hmoriyama2@unl.edu [Department of Chemistry, e-Toxicology and Biotechnology, University of Nebraska-Lincoln, Hamilton Hall, Lincoln, Nebraska 68588-0304 (United States); School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States)

    2007-05-01

    The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V{sub M} of 1.8 Å{sup 3} Da{sup −1}.

  12. Studies on Transcriptional Incorporation of 5'-N-Triphosphates of 5'-Amino-5'-Deoxyribonucleosides.

    Directory of Open Access Journals (Sweden)

    Weronika Kotkowiak

    Full Text Available In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleosides (5'NH NTPs. The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5'NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5'NH NTPs incorporation. Based on experimental data it is postulated that 5'NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleoside and the exact mechanism of transcriptional inhibition.

  13. Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways

    Directory of Open Access Journals (Sweden)

    Le Xuan

    2009-01-01

    Full Text Available Background: Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption. This bacterium exerts its pathogenic effect indirectly through multiple virulence factors, such as lipopolysaccharides, fimbriae, and proteases. Another possible pathogenic path may be through a direct interaction with the host′s soft and hard tissues (e.g., alveolar bone, which could lead to periodontitis. Aims and Objectives: The aim of the present study was to investigate the direct effect of live and heat-inactivated P gingivalis on bone resorption, using an in vitro osteoblast culture model. Results: Optical microscopy and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide MTT assay revealed that live P gingivalis induced osteoblast detachment and reduced their proliferation. This effect was specific to live bacteria and was dependent on their concentration. Live P gingivalis increased IL-6 mRNA expression and protein production and downregulated RANKL and OPG mRNA expression. The effect of live P gingivalis on bone resorption was strengthened by an increase in MMP-9 expression and its activity. This increase was accompanied by an increase in TIMP-1 and TIMP-2 mRNA expression and protein production by osteoblasts infected with live P gingivalis. Conclusion: Overall, the results suggest that direct contact of P gingivalis with osteoblasts induces bone resorption through an inflammatory pathway that involves IL-6, RANKL/OPG, and MMP-9/TIMPs.

  14. Effect of different superovulation stimulation protocols on adenosine triphosphate concentration in rabbit oocytes.

    Science.gov (United States)

    Cortell, Carmela; Salvetti, Pascal; Joly, Thierry; Viudes-de-Castro, Maria Pilar

    2015-08-01

    Ovarian stimulation protocols are used usually to increase the number of oocytes collected. The determination of how oocyte quality may be affected by these superovulation procedures, therefore, would be very useful. There is a high correlation between oocyte ATP concentration and developmental competence of the resulting embryo. The aim of this study was to evaluate the effect of follicle stimulating hormone (FSH) origin and administration protocols on oocyte ATP content. Rabbit does were distributed randomly into four groups: (i) a control group; (ii) the rhFSH3 group: females were injected, every 24 h over 3 days, with 0.6 μl of rhFSH diluted in polyvinylpyrrolidone (PVP); (iii) the pFSH3 group: females were injected every 24 h over 3 days with 11.4 μg of pFSH diluted in PVP; and (iv) the pFSH5 group: females were injected twice a day for 5 days with 11.4 μg of pFSH diluted in saline serum. Secondly, the effect of pFSH5 protocol on developmental potential was evaluated. Developmental competence of oocytes from the control and pFSH5 groups was examined. Differences in superovulation treatments were found for ATP levels. In the pFSH5 group, the ATP level was significantly lower than that of the other groups (5.63 ± 0.14 for pFSH group versus 6.42 ± 0.13 and 6.19 ± 0.15 for rhFSH3 and pFSH3, respectively; P ATP production, it is possible that, after this superovulation treatment, oocyte metabolism would be affected.

  15. Azilsartan increases levels of IL-10, down-regulates MMP-2, MMP-9, RANKL/RANK, Cathepsin K and up-regulates OPG in an experimental periodontitis model.

    Directory of Open Access Journals (Sweden)

    Aurigena Antunes de Araújo

    Full Text Available AIMS: The aim of this study was to evaluate the effects of azilsartan (AZT on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs, receptor activator of nuclear factor κB ligand (RANKL, receptor activator of nuclear factor κB (RANK, osteoprotegerin (OPG, cyclooxygenase-2 (COX-2, and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis. MATERIALS AND METHODS: Male Wistar albino rats were randomly divided into 5 groups of 10 rats each: (1 nonligated, water; (2 ligated, water; (3 ligated, 1 mg/kg AZT; (4 ligated, 5 mg/kg AZT; and (5 ligated, 10 mg/kg AZT. All groups were treated with saline or AZT for 10 days. Periodontal tissues were analyzed by histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1β, IL-10, TNF-α, myeloperoxidase (MPO, and glutathione (GSH were determined by ELISA. RESULTS: Treatment with 5 mg/kg AZT resulted in reduced MPO (p<0.05 and IL-1β (p<0.05, increased levels of IL-10 (p<0.05, and reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and increased expression of OPG. CONCLUSIONS: These findings reveal that AZT increases anti-inflammatory cytokines and GSH and decreases bone loss in ligature-induced periodontitis in rats.

  16. Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

    NARCIS (Netherlands)

    Verschuur, A. C.; van Gennip, A. H.; Muller, E. J.; Voûte, P. A.; Vreken, P.; van Kuilenburg, A. B.

    1999-01-01

    Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood

  17. Microcontroller-assisted compensation of adenosine triphosphate levels: instrument and method development.

    Science.gov (United States)

    Hu, Jie-Bi; Chen, Ting-Ru; Chen, Yu-Chie; Urban, Pawel L

    2015-01-30

    In order to ascertain optimum conditions for biocatalytic processes carried out in vitro, we have designed a bio-opto-electronic system which ensures real-time compensation for depletion of adenosine triphosphate (ATP) in reactions involving transfer of phosphate groups. The system covers ATP concentration range of 2-48 μM. The report demonstrates feasibility of the device operation using apyrase as the ATP-depleting enzyme.

  18. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    Science.gov (United States)

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  19. Intercalation of gaseous thiols and sulfides into Ag+ ion-exchanged aluminum dihydrogen triphosphate.

    Science.gov (United States)

    Hayashi, Aki; Saimen, Hiroki; Watanabe, Nobuaki; Kimura, Hitomi; Kobayashi, Ayumi; Nakayama, Hirokazu; Tsuhako, Mitsutomo

    2005-08-02

    Ag(+) ion-exchanged layered aluminum dihydrogen triphosphate (AlP) with the interlayer distance of 0.85 nm was synthesized by the ion-exchange of proton in triphosphate with Ag(+) ion. The amount of exchanged Ag(+) ion depended on the concentration of AgNO(3) aqueous solution. Ag(+) ion-exchanged AlP adsorbed gaseous thiols and sulfides into the interlayer region. The adsorption amounts of thiols were more than those of sulfides, thiols with one mercapto group > thiol with two mercapto groups > sulfides, and depended on the amount of exchanged Ag(+) ion in the interlayer region. The thiols with one mercapto group were intercalated to expand the interlayer distance of Ag(+) ion-exchanged AlP, whereas there was no expansion in the adsorption of sulfide. In the case of thiol with two mercapto groups, there was observed contraction of the interlayer distance through the bridging with Ag(+) ions of the upper and lower sides of the interlayer region.

  20. Synthesis of adenosine triphosphate tritiated in position 2 and 8

    International Nuclear Information System (INIS)

    Cossery, Jean-Michel

    1986-01-01

    Adenosine triphosphate or ATP is an important molecule present at the cellular level in many fundamental biochemical mechanism, and the study of its metabolism is therefore of particular interest. In this thesis for pharmacy graduation, the author first describes the different steps of synthesis and purification leading to chloride-2-ATP, a precursor of the final tritiated molecule. Then, the author explains the tritiation of this molecule to obtain an ATP tritiated in position 2 and in position 8 [fr

  1. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors.

    Science.gov (United States)

    Bettendorff, Lucien; Wins, Pierre

    2009-06-01

    Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

  2. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    Science.gov (United States)

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  3. Synthesis and study of the triphosphate salt LiSr2P3O10·8H2O

    International Nuclear Information System (INIS)

    Sotnikova-Yuzhik, V.A.; Peslyak, G.V.

    1995-01-01

    Lithium triphosphate interaction with strontium nitrate in aqueous solution at 0.3 mole% concentration and 20 deg C is studied. Formation of crystal hydrate LiSr 2 P 3 O 10 ·8H 2 O and amorphous phase of variable composition Li 2,5-0,5x P 3 O 10 ·6H 2 O (0.20≤x≤0.55) is determined. Data on the stability of binary lithium-strontium triphosphate at storage, sequence of chemical and phase transitions under heating are obtained. 5 refs., 4 figs., 3 tabs

  4. Metabolic Recruitment and Directed Evolution of Nucleoside Triphosphate Uptake in Escherichia coli.

    Science.gov (United States)

    Pezo, Valérie; Hassan, Camille; Louis, Dominique; Sargueil, Bruno; Herdewijn, Piet; Marlière, Philippe

    2018-05-18

    We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

  5. Histomorphometric analysis and immunolocalization of RANKL and OPG during the alveolar healing process in female ovariectomized rats treated with oestrogen or raloxifene.

    Science.gov (United States)

    Luvizuto, Eloá Rodrigues; Queiroz, Thallita Pereira; Dias, Sheila Mônica Damásio; Okamoto, Tetuo; Dornelles, Rita Cássia Menegati; Garcia, Idelmo Rangel; Okamoto, Roberta

    2010-01-01

    To investigate the effects of bone-resorption inhibitors (oestrogen and raloxifene) on the RANKL/OPG balance during the chronology of the alveolar healing process in ovariectomized (OVX) rats by means of immunocolocalization and histomorphometric analysis. One hundred sixty female Wistar rats at 70 days of age were either OVX or sham-operated and divided into four groups: sham, OVX/Oil, OVX with E(2) replacement (17beta-estradiol, 400 microg/month), OVX with RLX treatment (1mg/kg bw/day). The 60-day treatment started 8 days after ovariectomy. The incisors were extracted to allow analysis of 7, 14, 21, 28 and 42 days of wound healing. After obtaining the histological samples, slides were stained with hematoxylin and eosin or subjected to immunocolocalization reaction for RANKL and OPG. Results were quantitatively evaluated. Histomorphometric analysis showed that the sham group presented the highest and OVX/Oil group the lowest mean bone formation value in the post-extraction period. The immunocolocalization analysis showed a larger increase in bone turnover at 7 postoperative days in OVX/Oil and sham groups and decreasing bone turnover in the other periods. The OVX/Oil group showed a large decrease in bone turnover at 14 postoperative days, a period demonstrated by mild cellular activity. OVX/E(2) and sham groups showed a decreased bone turnover at 28 postoperative days while OVX/RLX group showed a decreased bone turnover at 21 postoperative days. On the 42nd postoperative day, sham and OVX/RLX groups showed an established alveolar bone healing process. Ovariectomy delays the alveolar healing process and interferes with bone turnover through the balance between RANKL and OPG. Oestrogen replacement or raloxifene treatment did not totally recover the oestrogen-deficient state. However raloxifene treatment showed more satisfactory results than oestrogen replacement. Copyright 2009 Elsevier Ltd. All rights reserved.

  6. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  7. Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

    International Nuclear Information System (INIS)

    Park, Eui Kyun; Kim, Myung Sunny; Lee, Seung Ho; Kim, Kyung Hee; Park, Ju-Young; Kim, Tae-Ho; Lee, In-Seon; Woo, Je-Tae; Jung, Jae-Chang; Shin, Hong-In; Choi, Je-Yong; Kim, Shin-Yoon

    2004-01-01

    Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation

  8. Osteoprotegerin in bone metastases: mathematical solution to the puzzle.

    Directory of Open Access Journals (Sweden)

    Marc D Ryser

    Full Text Available Bone is a common site for cancer metastasis. To create space for their growth, cancer cells stimulate bone resorbing osteoclasts. Cytokine RANKL is a key osteoclast activator, while osteoprotegerin (OPG is a RANKL decoy receptor and an inhibitor of osteoclastogenesis. Consistently, systemic application of OPG decreases metastatic tumor burden in bone. However, OPG produced locally by cancer cells was shown to enhance osteolysis and tumor growth. We propose that OPG produced by cancer cells causes a local reduction in RANKL levels, inducing a steeper RANKL gradient away from the tumor and towards the bone tissue, resulting in faster resorption and tumor expansion. We tested this hypothesis using a mathematical model of nonlinear partial differential equations describing the spatial dynamics of OPG, RANKL, PTHrP, osteoclasts, tumor and bone mass. We demonstrate that at lower expression rates, tumor-derived OPG enhances the chemotactic RANKL gradient and osteolysis, whereas at higher expression rates OPG broadly inhibits RANKL and decreases osteolysis and tumor burden. Moreover, tumor expression of a soluble mediator inducing RANKL in the host tissue, such as PTHrP, is important for correct orientation of the RANKL gradient. A meta-analysis of OPG, RANKL and PTHrP expression in normal prostate, carcinoma and metastatic tissues demonstrated an increase in expression of OPG, but not RANKL, in metastatic prostate cancer, and positive correlation between OPG and PTHrP in metastatic prostate cancer. The proposed mechanism highlights the importance of the spatial distribution of receptors, decoys and ligands, and can be applied to other systems involving regulation of spatially anisotropic processes.

  9. Determination of adenosine disodium triphosphate using prulifloxacin-terbium(III) as a fluorescence probe by spectrofluorimetry

    International Nuclear Information System (INIS)

    Yu Fengshan; Li Lin; Chen Fang

    2008-01-01

    A new spectrofluorimetric method is developed for determination of adenosine disodium triphosphate (ATP). The interactions between prulifloxacin (PUFX)-Tb 3+ complex and adenosine disodium triphosphate has been studied by using UV-vis absorption and fluorescence spectra. Using prulifloxacin-Tb 3+ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the prulifloxacin-Tb 3+ complex at λ = 545 nm and the enhanced fluorescence intensity is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The dynamic range for the determination of ATP is 4.0 x 10 -7 to 2.0 x 10 -5 mol L -1 , and the detection limit (3 σ/k) is 1.7 x 10 -8 mol L -1 . This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in real pharmaceutical samples. The mechanism of fluorescence enhancement of prulifloxacin-Tb 3+ complex by ATP was also discussed

  10. Slight changes in the mechanical stimulation affects osteoblast- and osteoclast-like cells in co-culture.

    Science.gov (United States)

    Kadow-Romacker, Anke; Duda, Georg N; Bormann, Nicole; Schmidmaier, Gerhard; Wildemann, Britt

    2013-12-01

    Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1-5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells.

  11. Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

    Science.gov (United States)

    Gong, Wei; Dou, Huan; Liu, Xianqin; Sun, Lingyun; Hou, Yayi

    2012-10-01

    1. In the present study, we investigated the effects of technetium-99 conjugated with methylene diphosphonate ((99)Tc-MDP), an agent used in radionuclide therapy, on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and explored the underlying mechanisms. 2. The murine macrophage cell line RAW264.7 and bone marrow-derived-macrophages from C57BL/6 mice (BMM) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/mL)-induced osteoclastogenesis in RAW264.7 cells was investigated by flow cytometry and real-time reverse transcription-polymerase chain reaction (RT-PCR). To detect multinucleated osteoclast formation, RAW264.7 cells were induced with RANKL for 4 days, whereas BMM were induced by 50 ng/mL RANKL and 20 ng/mL macrophage colony-stimulating factor for 7 days, before being stained with tartrate-resistant acid phosphatase. 3. Osteoclastogenesis was evaluated using the osteoclast markers CD51, matrix metalloproteinase (MMP)-9 and cathepsin K. At 0.01 μg/mL, (99)Tc-MDP significantly inhibited RANKL-induced osteoclastogenesis without any cytotoxicity. In addition, (99)Tc-MDP abolished the appearance of multinucleated osteoclasts. 4. Real-time RT-PCR analysis of transcription factor expression revealed that (99)Tc-MDP inhibited the expression of c-Fos and nuclear factor of activated T cells. In addition, (99)Tc-MDP inhibited the expression of the inflammatory factors interleukin (IL)-6, tumour necrosis factor-α and IL-1β. Finally, (99)Tc-MDP inhibited the activation of mitogen-activated protein kinases in RAW264.7 cells following RANKL stimulation. 5. In conclusion, (99)Tc-MDP possesses anti-osteoclastogenic activity against RANKL-induced osteoclast formation. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.

  12. Determination of Zidovudine Triphosphate Intracellular Concentrations in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Individuals by Tandem Mass Spectrometry

    Science.gov (United States)

    Font, Eva; Rosario, Osvaldo; Santana, Jorge; García, Hermes; Sommadossi, Jean-Pierre; Rodriguez, Jose F.

    1999-01-01

    Nucleoside reverse transcriptase inhibitors (NRTIs) used against the human immunodeficiency virus (HIV) need to be activated intracellularly to their triphosphate moiety to inhibit HIV replication. Intracellular concentrations of these NRTI triphosphates, especially zidovudine triphosphate (ZDV-TP), are relatively low (low numbers of femtomoles per 106 cells) in HIV-infected patient peripheral blood mononuclear cells. Recently, several methods have used either high-performance liquid chromatography (HPLC) or solid-phase extraction (SPE) coupled with radioimmunoassay to obtain in vivo measurements of ZDV-TP. The limit of detection (LOD) by these methods ranged from 20 to 200 fmol/106 cells. In this report, we describe the development of a method to determine intracellular ZDV-TP concentrations in HIV-infected patients using SPE and HPLC with tandem mass spectrometry for analysis. The LOD by this method is 4.0 fmol/106 cells with a linear concentration range of at least 4 orders of magnitude from 4.0 to 10,000 fmol/106 cells. In hispanic HIV-infected patients, ZDV-TP was detectable even when the sampling time after drug administration was 15 h. Intracellular ZDV-TP concentrations in these patients ranged from 41 to 193 fmol/106 cells. The low LOD obtained with this method will provide the opportunity for further in vivo pharmacokinetic studies of intracellular ZDV-TP in different HIV-infected populations. Furthermore, this methodology could be used to perform simultaneous detection of two or more NRTIs, such as ZDV-TP and lamivudine triphosphate. PMID:10582890

  13. Substance P Promotes the Proliferation, but Inhibits Differentiation and Mineralization of Osteoblasts from Rats with Spinal Cord Injury via RANKL/OPG System.

    Directory of Open Access Journals (Sweden)

    Hai-Juan Liu

    Full Text Available Spinal cord injury (SCI causes a significant amount of bone loss, which results in osteoporosis (OP. The neuropeptide substance P (SP and SP receptors may play important roles in the pathogenesis of OP after SCI. To identify the roles of SP in the bone marrow mesenchymal stem cell derived osteoblasts (BMSC-OB in SCI rats, we investigated the expression of neurokinin-1 receptors (NK1R in BMSC-OB and the effects of SP on bone formation by development of BMSC-OB cultures. Sixty young male Sprague-Dawley rats were randomized into two groups: SHAM and SCI. The expression of NK1R protein in BMSC-OB was observed using immunohistochemistry and Western blot analysis. The dose- and time-dependent effects of SP on the proliferation, differentiation and mineralization of BMSC-OB and the expression of osteoblastic markers by in vitro experiments. The expression of NK1R in BMSC-OB was observed on plasma membranes and in cytoplasm. One week after osteogenic differentiation, the expression of NK1R was significantly increased after SCI at mRNA and protein levels. However, this difference was gradually attenuated at 2 or 3 weeks later. SP have the function to enhance cell proliferation, inhibite cell differentiation and mineralization at a proper concentration and incubation time, and this effect would be inhibited by adding SP or NK1R antagonist. The expression of RANKL/OPG was significantly increased in tibiae after SCI. Similarly, the RANKL/OPG expression in SCI rats was significantly increased when treating with 10-8 M SP. SP plays a very important role in the pathogenesis of OP after SCI. The direct effect of SP may lead to increased bone resorption through the RANKL/OPG axis after SCI. In addition, high expression of SP also results in the suppression of osteogenesis in SCI rats. Then, the balance between bone resorption and bone formation was broken and finally osteoporosis occurred.

  14. Macrolactin F inhibits RANKL-mediated osteoclastogenesis by suppressing Akt, MAPK and NFATc1 pathways and promotes osteoblastogenesis through a BMP-2/smad/Akt/Runx2 signaling pathway.

    Science.gov (United States)

    Li, Liang; Sapkota, Mahesh; Gao, Ming; Choi, Hyukjae; Soh, Yunjo

    2017-11-15

    The balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts. In the current study, macrolactin F (MF) was investigated for novel biological activity on the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages (BMMs). We found that RANKL-induced osteoclast formation and differentiation from BMMs was significantly inhibited by MF in a dose-dependent manner without cytotoxicity. RANKL-induced F-actin ring formation and bone resorption activity in BMMs which was attenuated by MF. In addition, MF suppressed the expression of osteoclast-related genes, including c-myc, RANK, tartrate-resistant acid phosphatase (TRAP), nuclear factor of activated T cells c1 (NFATc1), cathepsin K and matrix metalloproteinase 9 (MMP9). Furthermore, the protein expression NFATc1, c-Fos, MMP9, cathepsin K and phosphorylation of Jun N-terminal kinase (JNK), p38 and Akt were also down-regulated by MF treatment. Interestingly, MF promoted pre-osteoblast cell differentiation on Alizarin Red-mineralization activity, alkaline phosphatase (ALP) activity, and the expression of osteoblastogenic markers including Runx2, Osterix, Smad4, ALP, type I collagen alpha 1 (Col1α), osteopontin (OPN), and osteocalcin (OCN) via activation of the BMP-2/smad/Akt/Runx2 pathway on MC3T3-E1. Taken together, these results indicate that MF may be useful as a therapeutic agent to enhance bone health and treat osteoporosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Allium cepa L. and Quercetin Inhibit RANKL/Porphyromonas gingivalis LPS-Induced Osteoclastogenesis by Downregulating NF-κB Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Tatiane Oliveira

    2015-01-01

    Full Text Available Objectives. We evaluated the in vitro modulatory effects of Allium cepa L. extract (AcE and quercetin (Qt on osteoclastogenesis under inflammatory conditions (LPS-induced. Methods. RAW 264.7 cells were differentiated with 30 ng/mL of RANKL, costimulated with PgLPS (1 µg/mL, and treated with AcE (50–1000 µg/mL or Qt (1.25, 2.5, or 5 µM. Cell viability was determined by alamarBlue and protein assays. Nuclei morphology was analysed by DAPI staining. TRAP assays were performed as follows: p-nitrophenyl phosphate was used to determine the acid phosphatase activity of the osteoclasts and TRAP staining was used to evaluate the number and size of TRAP-positive multinucleated osteoclast cells. Von Kossa staining was used to measure osteoclast resorptive activity. Cytokine levels were measured on osteoclast precursor cell culture supernatants. Using western blot analysis, p-IκBα and IκBα degradation, inhibitor of NF-kappaB, were evaluated. Results. Both AcE and Qt did not affect cell viability and significantly reduced osteoclastogenesis compared to control. We observed lower production of IL-6 and IL-1α and an increased production of IL-3 and IL-4. AcE and Qt downregulated NF-κB pathway. Conclusion. AcE and Qt may be inhibitors of osteoclastogenesis under inflammatory conditions (LPS-induced via attenuation of RANKL/PgLPS-induced NF-κB activation.

  16. Feeding blueberry diets to young rats dose-dependently inhibits bone resorption through suppression of RANKL in stromal cells.

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    Full Text Available Previous studies have demonstrated that weanling rats fed AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB powder for two weeks beginning on postnatal day 21 (PND21 significantly increased bone formation at PND35. However, the minimal level of dietary BB needed to produce these effects is, as yet, unknown. The current study examined the effects of three different levels of BB diet supplementation (1, 3, and 5% for 35 days beginning on PND25 on bone quality, and osteoclastic bone resorption in female rats. Peripheral quantitative CT scan (pQCT of tibia, demonstrated that bone mineral density (BMD and content (BMC were dose-dependently increased in BB-fed rats compared to controls (P<0.05. Significantly increased bone mass after feeding 5% BB extracts was also observed in a TEN (total enteral nutrition rat model in which daily caloric and food intake was precisely controlled. Expression of RANKL (receptor activator of nuclear factor-κB ligand a protein essential for osteoclast formation was dose-dependently decreased in the femur of BB animals. In addition, expression of PPARγ (peroxisome proliferator-activated receptor γ which regulates bone marrow adipogenesis was suppressed in BB diet rats compared to non-BB diet controls. Finally, a set of in vitro cell cultures revealed that the inhibitory effect of BB diet rat serum on RANKL expression was more profound in mesenchymal stromal cells compared to its effect on mature osteoblasts, pre-adipocytes and osteocytes. These results suggest that inhibition of bone resorption may contribute to increased bone mass during early development after BB consumption.

  17. The immediate nucleotide precursor, guanosine triphosphate, in the riboflavin biosynthetic pathway

    International Nuclear Information System (INIS)

    Mitsuda, Hisateru; Nakajima, Kenji; Nadamoto, Tomonori

    1977-01-01

    In the present paper, the nucleotide precursor of riboflavin was investigated by experiments with labeled purines using non-growing cells of Eremothecium ashbyii. The added purines, at 10 -4 M, were effectively incorporated into riboflavin at an early stage of riboflavin biosynthesis under the experimental conditions. In particular, both labeled xanthine and labeled guanine were specifically transported to guanosine nucleotides, GMP, GDP, GDP-Mannose and GTP, in the course of the riboflavin biosynthesis. A comparison of specific activities of labeled guanosine nucleotides and labeled riboflavin indicated that the nucleotide precursor of riboflavin is guanosine triphosphate. From the results obtained, a biosynthetic pathway of riboflavin is proposed. (auth.)

  18. Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

    Science.gov (United States)

    Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

    2017-11-01

    5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Extracellular adenosine 5'-triphosphate concentrations changes in rat spinal cord associated with the activation of urinary bladder afferents. A microdialysis study.

    Science.gov (United States)

    Rocha, Jeová Nina

    2016-01-01

    To determine adenosine 5'-triphosphate levels in the interstice of spinal cord L6-S1 segment, under basal conditions or during mechanical and chemical activation of urinary bladder afferents. A microdialysis probe was transversally implanted in the dorsal half of spinal cord L6-S1 segment in female rats. Microdialysate was collected at 15 minutes intervals during 135 minutes, in anesthetized animals. Adenosine 5'-triphosphate concentrations were determined with a bioluminescent assay. In one group of animals (n=7) microdialysate samples were obtained with an empty bladder during a 10-minutes bladder distension to 20 or 40cmH2O with either saline, saline with acetic acid or saline with capsaicin. In another group of animals (n=6) bladder distention was performed and the microdialysis solution contained the ectonucleotidase inhibitor ARL 67156. Basal extracellular adenosine triphosphate levels were 110.9±35.34fmol/15 minutes, (mean±SEM, n=13), and bladder distention was associated with a significant increase in adenosine 5'-triphosphate levels which was not observed after bladder distention with saline solution containing capsaicin (10µM). Microdialysis with solution containing ARL 67156 (1mM) was associated with significantly higher extracellular adenosine 5'-triphosphate levels and no further increase in adenosine 5'-triphosphate was observed during bladder distension. Adenosine 5'-triphosphate was present in the interstice of L6-S1 spinal cord segments, was degraded by ectonucleotidase, and its concentration increased following the activation of bladder mechanosensitive but not of the chemosensitive afferents fibers. Adenosine 5'-triphosphate may originate either from the central endings of bladder mechanosensitive primary afferent neurons, or most likely from intrinsic spinal neurons, or glial cells and its release appears to be modulated by capsaicin activated bladder primary afferent or by adenosine 5'-triphosphate itself. Determinar as concentra

  20. Effects of adenosine triphosphate concentration on motor force regulation during skeletal muscle contraction

    Science.gov (United States)

    Wei, J.; Dong, C.; Chen, B.

    2017-04-01

    We employ a mechanical model of sarcomere to quantitatively investigate how adenosine triphosphate (ATP) concentration affects motor force regulation during skeletal muscle contraction. Our simulation indicates that there can be negative cross-bridges resisting contraction within the sarcomere and higher ATP concentration would decrease the resistance force from negative cross-bridges by promoting their timely detachment. It is revealed that the motor force is well regulated only when ATP concentration is above a certain level. These predictions may provide insights into the role of ATP in regulating coordination among multiple motors.

  1. Molecular structure of tetraaqua adenosine 5'-triphosphate aluminium(III) complex: A study involving Raman spectroscopy, theoretical DFT and potentiometry

    Science.gov (United States)

    Tenório, Thaís; Silva, Andréa M.; Ramos, Joanna Maria; Buarque, Camilla D.; Felcman, Judith

    2013-03-01

    The Alzheimer's disease is one of the most common neurodegenerative diseases that affect elderly population, due to the formation of β-amyloid protein aggregate and several symptoms, especially progressive cognitive decline. The result is a decrease in capture of glucose by cells leading to obliteration, meddling in the Krebs cycle, the principal biochemical route to the energy production leading to a decline in the levels of adenosine 5'-triphosphate. Aluminium(III) is connected to Alzheimer's and its ion provides raise fluidity of the plasma membrane, decrease cell viability and aggregation of amyloid plaques. Studies reveal that AlATP complex promotes the formation of reactive fibrils of β-amyloid protein and independent amyloidogenic peptides, suggesting the action of the complex as a chaperone in the role pathogenic process. In this research, one of complexes formed by Al(III) and adenosine 5'-triphosphate in aqueous solution is analyzed by potentiometry, Raman spectroscopy and ab initio calculations. The value of the log KAlATP found was 9.21 ± 0.01 and adenosine 5'-triphosphate should act as a bidentate ligand in the complex. Raman spectroscopy and potentiometry indicate that donor atoms are the oxygen of the phosphate β and the oxygen of the phosphate γ, the terminal phosphates. Computational calculations using Density Functional Theory, with hybrid functions B3LYP and 6-311++G(d,p) basis set regarding water solvent effects, have confirmed the results. Frontier molecular orbitals, electrostatic potential contour surface, electrostatic potential mapped and Mulliken charges of the title molecule were also investigated.

  2. Metabolic costs of force generation for constant-frequency and catchlike-inducing electrical stimulation in human tibialis anterior muscle

    DEFF Research Database (Denmark)

    Ratkevicius, Aivaras; Quistorff, Bjørn

    2002-01-01

    -frequency trains, catchlike-inducing trains produced a faster force generation and were more effective in maintaining the force--time integral as well as peak force. However, ATP costs of force generation were similar for the catchlike-inducing and constant-frequency stimulation (6.7 plus/minus 1.1 and 6.6 plus......Metabolic costs of force generation were compared for constant-frequency and catchlike-inducing electrical stimulation. Repetitive catchlike-inducing trains consisted of 2 interpulse intervals (IPIs) at 12.5 ms, 1 IPI at 25 ms, and 5 IPIs at 50 ms. Constant-frequency trains consisted of 8 IPIs...... at 37.5 ms. One train was delivered to the peroneal nerve every 2.5 s for 36 times under ischemic conditions. Anaerobic adenosine triphosphate (ATP) turnover was determined using 31-phosphorus magnetic resonance spectroscopy (P-MRS) of the human tibialis anterior muscle. Compared with constant...

  3. Enhancement of osteogenic differentiation and proliferation in human mesenchymal stem cells by a modified low intensity ultrasound stimulation under simulated microgravity.

    Directory of Open Access Journals (Sweden)

    Sardar M Z Uddin

    Full Text Available Adult stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. Lack of mechanical signals due to disuse can inhibit osteogenesis and induce adipogenesis of mesenchymal stem cells (MSCs. Long-term bed rest due to both brain/spinal cord injury and space travel can lead to disuse osteoporosis that is in part caused by a reduced number of osteoblasts. Thus, it is essential to provide proper mechanical stimulation for cellular viability and osteogenesis, particularly under disuse conditions. The objective of this study was to examine the effects of low intensity pulsed ultrasound (LIPUS on the osteogenic differentiation of adipose-derived human stem cells (Ad-hMSC in simulated microgravity conditions. Cells were cultured in a 1D clinostat to simulate microgravity (SMG and treated with LIPUS at 30mW/cm(2 for 20 min/day. It was hypothesized that the application of LIPUS to SMG cultures would restore osteogenesis in Ad-hMSCs. The results showed significant increases in ALP, OSX, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures of Ad-MSC compared to non-treated cultures. LIPUS also restored OSX, RUNX2 and RANKL expression in osteoblast cells. SMG significantly reduced ALP positive cells by 70% (p<0.01 and ALP activity by 22% (p<0.01, while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix collagen and mineralization was assessed by Sirius red and Alizarin red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50% (p<0.001 and mineralization by 45% (p<0.001 in LIPUS treated-SMG cultures relative to SMG-only cultures. The data suggest that LIPUS treatment can restore normal osteogenic differentiation of MSCs from disuse by daily short duration stimulation.

  4. Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely re...

  5. Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver.

    Science.gov (United States)

    Nagashima, Kazuki; Sawa, Shinichiro; Nitta, Takeshi; Prados, Alejandro; Koliaraki, Vasiliki; Kollias, George; Nakashima, Tomoki; Takayanagi, Hiroshi

    2017-11-04

    The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin + CD31 - cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin + CD31 - cells. Tnfsf11 fl/Δ Col6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

    International Nuclear Information System (INIS)

    Miyamoto, Kana; Ninomiya, Ken; Sonoda, Koh-Hei; Miyauchi, Yoshiteru; Hoshi, Hiroko; Iwasaki, Ryotaro; Miyamoto, Hiroya

    2009-01-01

    Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.

  7. Increased deoxythymidine triphosphate levels is a feature of relative cognitive decline

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Frederiksen, Jane H; Olsen, Maria Nathalie Angleys

    2015-01-01

    Mitochondrial bioenergetics, mitochondrial reactive oxygen species (ROS) and cellular levels of nucleotides have been hypothesized as early indicators of Alzheimer's disease (AD). Utilizing relative decline of cognitive ability as a predictor of AD risk, we evaluated the correlation between change...... of cognitive ability and mitochondrial bioenergetics, ROS and cellular levels of deoxyribonucleotides. Change of cognitive abilities, scored at ages of approximately 20 and 57 was determined for a cohort of 1985 male participants. Mitochondrial bioenergetics, mitochondrial ROS and whole-cell levels...... of deoxyribonucleotide triphosphates were measured in peripheral blood mononuclear cells (PBMCs) from a total of 103 selected participants displaying the most pronounced relative cognitive decline and relative cognitive improvement. We show that relative cognitive decline is associated with higher PBMC content...

  8. Mitochondrially-Encoded Adenosine Triphosphate Synthase 6 Gene Haplotype Variation among World Population during 2003-2013

    OpenAIRE

    Steven Steven; Yoni F Syukriani; Julius B Dewanto

    2016-01-01

    Background: Adaptation and natural selection serve as an important part of evolution. Adaptation in molecular level can lead to genetic drift which causes mutation of genetic material; one of which is polymorphism of mitochondrial DNA (mtDNA). The aim of this study is to verify the polymorphism of mitochondrially-encoded Adenosine Triphosphate synthase6gene (MT-ATP6) as one of mtDNA building blocks among tropic, sub-tropic, and polar areas. Methods: This descriptive quantitative research used...

  9. Betulinic acid, a bioactive pentacyclic triterpenoid, inhibits skeletal-related events induced by breast cancer bone metastases and treatment

    International Nuclear Information System (INIS)

    Park, Se Young; Kim, Hyun-Jeong; Kim, Ki Rim; Lee, Sun Kyoung; Lee, Chang Ki; Park, Kwang-Kyun; Chung, Won-Yoon

    2014-01-01

    Many breast cancer patients experience bone metastases and suffer skeletal complications. The present study provides evidence on the protective and therapeutic potential of betulinic acid on cancer-associated bone diseases. Betulinic acid is a naturally occurring triterpenoid with the beneficial activity to limit the progression and severity of cancer, diabetes, cardiovascular diseases, atherosclerosis, and obesity. We first investigated its effect on breast cancer cells, osteoblastic cells, and osteoclasts in the vicious cycle of osteolytic bone metastasis. Betulinic acid reduced cell viability and the production of parathyroid hormone-related protein (PTHrP), a major osteolytic factor, in MDA-MB-231 human metastatic breast cancer cells stimulated with or without tumor growth factor-β. Betulinic acid blocked an increase in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin ratio by downregulating RANKL protein expression in PTHrP-treated human osteoblastic cells. In addition, betulinic acid inhibited RANKL-induced osteoclastogenesis in murine bone marrow macrophages and decreased the production of resorbed area in plates with a bone biomimetic synthetic surface by suppressing the secretion of matrix metalloproteinase (MMP)-2, MMP-9, and cathepsin K in RANKL-induced osteoclasts. Furthermore, oral administration of betulinic acid inhibited bone loss in mice intra-tibially inoculated with breast cancer cells and in ovariectomized mice causing estrogen deprivation, as supported by the restored bone morphometric parameters and serum bone turnover markers. Taken together, these findings suggest that betulinic acid may have the potential to prevent bone loss in patients with bone metastases and cancer treatment-induced estrogen deficiency. - Highlights: • Betulinic acid reduced PTHrP production in human metastatic breast cancer cells. • Betulinic acid blocked RANKL/OPG ratio in PTHrP-stimulated human osteoblastic cells. • Betulinic

  10. Betulinic acid, a bioactive pentacyclic triterpenoid, inhibits skeletal-related events induced by breast cancer bone metastases and treatment

    Energy Technology Data Exchange (ETDEWEB)

    Park, Se Young; Kim, Hyun-Jeong; Kim, Ki Rim; Lee, Sun Kyoung; Lee, Chang Ki; Park, Kwang-Kyun, E-mail: biochelab@yuhs.ac; Chung, Won-Yoon, E-mail: wychung@yuhs.ac

    2014-03-01

    Many breast cancer patients experience bone metastases and suffer skeletal complications. The present study provides evidence on the protective and therapeutic potential of betulinic acid on cancer-associated bone diseases. Betulinic acid is a naturally occurring triterpenoid with the beneficial activity to limit the progression and severity of cancer, diabetes, cardiovascular diseases, atherosclerosis, and obesity. We first investigated its effect on breast cancer cells, osteoblastic cells, and osteoclasts in the vicious cycle of osteolytic bone metastasis. Betulinic acid reduced cell viability and the production of parathyroid hormone-related protein (PTHrP), a major osteolytic factor, in MDA-MB-231 human metastatic breast cancer cells stimulated with or without tumor growth factor-β. Betulinic acid blocked an increase in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin ratio by downregulating RANKL protein expression in PTHrP-treated human osteoblastic cells. In addition, betulinic acid inhibited RANKL-induced osteoclastogenesis in murine bone marrow macrophages and decreased the production of resorbed area in plates with a bone biomimetic synthetic surface by suppressing the secretion of matrix metalloproteinase (MMP)-2, MMP-9, and cathepsin K in RANKL-induced osteoclasts. Furthermore, oral administration of betulinic acid inhibited bone loss in mice intra-tibially inoculated with breast cancer cells and in ovariectomized mice causing estrogen deprivation, as supported by the restored bone morphometric parameters and serum bone turnover markers. Taken together, these findings suggest that betulinic acid may have the potential to prevent bone loss in patients with bone metastases and cancer treatment-induced estrogen deficiency. - Highlights: • Betulinic acid reduced PTHrP production in human metastatic breast cancer cells. • Betulinic acid blocked RANKL/OPG ratio in PTHrP-stimulated human osteoblastic cells. • Betulinic

  11. Nutritional State-Dependent Ghrelin Activation of Vasopressin Neurons via Retrograde Trans-Neuronal–Glial Stimulation of Excitatory GABA Circuits

    Science.gov (United States)

    Haam, Juhee; Halmos, Katalin C.; Di, Shi

    2014-01-01

    Behavioral and physiological coupling between energy balance and fluid homeostasis is critical for survival. The orexigenic hormone ghrelin has been shown to stimulate the secretion of the osmoregulatory hormone vasopressin (VP), linking nutritional status to the control of blood osmolality, although the mechanism of this systemic crosstalk is unknown. Here, we show using electrophysiological recordings and calcium imaging in rat brain slices that ghrelin stimulates VP neurons in the hypothalamic paraventricular nucleus (PVN) in a nutritional state-dependent manner by activating an excitatory GABAergic synaptic input via a retrograde neuronal–glial circuit. In slices from fasted rats, ghrelin activation of a postsynaptic ghrelin receptor, the growth hormone secretagogue receptor type 1a (GHS-R1a), in VP neurons caused the dendritic release of VP, which stimulated astrocytes to release the gliotransmitter adenosine triphosphate (ATP). ATP activation of P2X receptors excited presynaptic GABA neurons to increase GABA release, which was excitatory to the VP neurons. This trans-neuronal–glial retrograde circuit activated by ghrelin provides an alternative means of stimulation of VP release and represents a novel mechanism of neuronal control by local neuronal–glial circuits. It also provides a potential cellular mechanism for the physiological integration of energy and fluid homeostasis. PMID:24790191

  12. Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

    International Nuclear Information System (INIS)

    Crew, Jennifer R.; Falzari, Kanakeshwari; DiMario, Joseph X.

    2010-01-01

    Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-γ co-activator-1 (PGC-1α) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

  13. Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

    Directory of Open Access Journals (Sweden)

    Andressa Vilas Boas Nogueira

    2014-01-01

    Full Text Available The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS of low (CTSL and high (CTSH magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2 and prostaglandin E2 (PGE2 was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG and receptor activator of nuclear factor kappa-B ligand (RANKL were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P<0.05 increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

  14. Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Simonova, Anna; Bialek-Pietras, M.; Olejniczak, A.; Lesnikowski, Z. J.; Hocek, Michal

    2017-01-01

    Roč. 27, č. 21 (2017), s. 4786-4788 ISSN 0960-894X R&D Projects: GA ČR GBP206/12/G151 Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 Keywords : nucleotides * nucleoside triphosphates * carboranes * DNA polymerase * oligonucleotides Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.454, year: 2016

  15. Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: adenosylcobalamin destruction and formation of a nucleotide-based radical.

    Science.gov (United States)

    Lohman, Gregory J S; Gerfen, Gary J; Stubbe, Joanne

    2010-02-23

    Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP) in cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [1'-(2)H]F(2)CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

  16. Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence.

    NARCIS (Netherlands)

    Pellegrini, P.; Sauerwein, R.W.; Finlayson, T.; McLeod, J.; Covell, D.A.; Maier, T.; Machida, C.A.

    2009-01-01

    INTRODUCTION: Enamel decalcification is a common problem in orthodontics. The objectives of this randomized clinical study were to enumerate and compare plaque bacteria surrounding 2 bracket types, self-ligating (SL) vs elastomeric ligating (E), and to determine whether adenosine triphosphate

  17. Mechanism of action of minoxidil in the treatment of androgenetic alopecia is likely mediated by mitochondrial adenosine triphosphate synthase-induced stem cell differentiation.

    Science.gov (United States)

    Goren, A; Naccarato, T; Situm, M; Kovacevic, M; Lotti, T; McCoy, J

    2017-01-01

    Topical minoxidil is the only topical drug approved by the US Food and Drug Administration (FDA) for the treatment of androgenetic alopecia. However, the exact mechanism by which minoxidil stimulates anagen phase and promotes hair growth is not fully understood. In the late telegen phase of the hair follicle growth cycle, stem cells located in the bulge region differentiate and re-enter anagen phase, a period of growth lasting 2-6 years. In androgenetic alopecia, the anagen phase is shortened and a progressive miniaturization of hair follicles occurs, eventually leading to hair loss. Several studies have demonstrated that minoxidil increases the amount of intracellular Ca2+, which has been shown to up-regulate the enzyme adenosine triphosphate (ATP) synthase. A recent study demonstrated that ATP synthase, independent of its role in ATP synthesis, promotes stem cell differentiation. As such, we propose that minoxidil induced Ca2+ influx can increase stem cell differentiation and may be a key factor in the mechanism by which minoxidil facilitates hair growth. Based on our theory, we provide a roadmap for the development of a new class of drugs for the treatment of androgenetic alopecia.

  18. Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2014-01-01

    Full Text Available The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

  19. Paradoxical action of fulvestrant in estradiol-induced regression of tamoxifen-stimulated breast cancer.

    Science.gov (United States)

    Osipo, Clodia; Gajdos, Csaba; Liu, Hong; Chen, Bin; Jordan, V Craig

    2003-11-05

    Long-term tamoxifen treatment of breast cancer can result in tamoxifen-stimulated breast cancer, in which estrogen inhibits tumor growth after tamoxifen withdrawal. We investigated the molecular mechanism(s) of estradiol-induced tumor regression by using an in vivo model of tamoxifen-stimulated human breast cancer. Growth of parental estradiol-stimulated MCF-7E2 and long-term tamoxifen-stimulated MCF-7TAMLT xenografts in athymic mice was measured during treatment with vehicle, estradiol, estradiol plus tamoxifen, tamoxifen alone, estradiol plus fulvestrant, or fulvestrant alone. Apoptosis was detected by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Protein expression was assessed by western blot analysis. mRNA expression was assessed by real-time reverse transcription-polymerase chain reaction. All statistical tests were two-sided. MCF-7E2 tumor growth was stimulated by estradiol (cross-sectional area at week 13 = 1.06 cm2, 95% confidence interval [CI] = 0.82 to 1.30 cm2; Pestradiol-induced regression to 0.18 cm2 (95% CI = 0.15 to 0.21 cm2; P<.001), and tamoxifen or estradiol plus fulvestrant enhanced tumor growth to 1.00 cm2 (95% CI = 0.88 to 1.22 cm2). Estradiol increased the number of apoptotic cells in tumors by 23% (95% CI = 20% to 26%; P<.001) compared with all other treatments, decreased estrogen receptor alpha(ERalpha) protein expression, increased the expression of Fas mRNA and protein, decreased the expression of HER2/neu mRNA and protein and nuclear factor kappaB (NF-kappaB) protein but did not affect Fas ligand protein expression compared with control. Paradoxically, fulvestrant reversed this effect and stimulated MCF-7TAMLT tumor growth apparently through ERalpha-mediated regulation of Fas, HER2/neu, and NF-kappaB. Physiologic levels of estradiol induced regression of tamoxifen-stimulated breast cancer tumors, apparently by inducing the death receptor Fas and suppressing the antiapoptotic

  20. Peculiarities of different-ligand complexing of rare earths with nitrilotriacetate and adenosine-5'-triphosphate according to the mathematical simulation data

    International Nuclear Information System (INIS)

    Svetlova, I.E.; Dobrynina, N.A.; Smirnova, N.S.; Martynenko, L.I.; Evseev, A.M.

    1988-01-01

    By the method of pH-metric titration using mathematical simulation different-ligand complexing of rare earths with nitrilotriacetate and adenosine-5'-triphosphate is studied. It is shown that the ligands interact with the formation of protonated associates. The composition of different complexes is determined, their stability constants are calculated, their existence regions are found

  1. Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

    Science.gov (United States)

    Omori, Takashi; Idehara, Kenji; Kojima, Hajime; Sozu, Takashi; Arima, Kazunori; Goto, Hirohiko; Hanada, Tomohiko; Ikarashi, Yoshiaki; Inoda, Taketo; Kanazawa, Yukiko; Kosaka, Tadashi; Maki, Eiji; Morimoto, Takashi; Shinoda, Shinsuke; Shinoda, Naoki; Takeyoshi, Masahiro; Tanaka, Masashi; Uratani, Mamoru; Usami, Masahito; Yamanaka, Atsushi; Yoneda, Tomofumi; Yoshimura, Isao; Yuasa, Atsuko

    2008-01-01

    The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

  2. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R

    2007-01-01

    of macrophage-colony stimulating factor (M-CSF) and RANKL. Phalloidin staining in osteoclasts served to study actin ring and podosome formation. RESULTS: pQCT revealed increased bone mass in uPAR-null mice. Mechanical tests showed reduced load-sustaining capability in uPAR KO tibias. uPAR KO osteoblasts showed...... a proliferative advantage with no difference in apoptosis, higher matrix mineralization, and earlier appearance of alkaline phosphatase (ALP). Surface RANKL expression at different stages of differentiation was not altered. AP-1 components, such as JunB and Fra-1, were upregulated in uPAR KO osteoblasts, along...

  3. Artificial oxygen carrier with pharmacologic actions of adenosine-5'-triphosphate, adenosine, and reduced glutathione formulated to treat an array of medical conditions.

    Science.gov (United States)

    Simoni, Jan; Simoni, Grace; Moeller, John F; Feola, Mario; Wesson, Donald E

    2014-08-01

    Effective artificial oxygen carriers may offer a solution to tackling current transfusion medicine challenges such as blood shortages, red blood cell storage lesions, and transmission of emerging pathogens. These products, could provide additional therapeutic benefits besides oxygen delivery for an array of medical conditions. To meet these needs, we developed a hemoglobin (Hb)-based oxygen carrier, HemoTech, which utilizes the concept of pharmacologic cross-linking. It consists of purified bovine Hb cross-linked intramolecularly with open ring adenosine-5'-triphosphate (ATP) and intermolecularly with open ring adenosine, and conjugated with reduced glutathione (GSH). In this composition, ATP prevents Hb dimerization, and adenosine promotes formation of Hb polymers as well as counteracts the vasoconstrictive and pro-inflammatory properties of Hb via stimulation of adenosine receptors. ATP also serves as a regulator of vascular tone through activation of purinergic receptors. GSH blocks Hb's extravasation and glomerular filtration by lowering the isoelectric point, as well as shields heme from nitric oxide and reactive oxygen species. HemoTech and its manufacturing technology have been broadly tested, including viral and prion clearance validation studies and various nonclinical pharmacology, toxicology, genotoxicity, and efficacy tests. The clinical proof-of-concept was carried out in sickle cell anemia subjects. The preclinical and clinical studies indicate that HemoTech works as a physiologic oxygen carrier and has efficacy in treating: (i) acute blood loss anemia by providing a temporary oxygen bridge while stimulating an endogenous erythropoietic response; (ii) sickle cell disease by counteracting vaso-occlusive/inflammatory episodes and anemia; and (iii) ischemic vascular diseases particularly thrombotic and restenotic events. The pharmacologic cross-linking of Hb with ATP, adenosine, and GSH showed usefulness in designing an artificial oxygen carrier for

  4. G protein-coupled receptor 84 controls osteoclastogenesis through inhibition of NF-κB and MAPK signaling pathways.

    Science.gov (United States)

    Park, Ji-Wan; Yoon, Hye-Jin; Kang, Woo Youl; Cho, Seungil; Seong, Sook Jin; Lee, Hae Won; Yoon, Young-Ran; Kim, Hyun-Ju

    2018-02-01

    GPR84, a member of the G protein-coupled receptor family, is found predominantly in immune cells, such as macrophages, and functions as a pivotal modulator of inflammatory responses. In this study, we investigated the role of GPR84 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Our microarray data showed that GPR84 was significantly downregulated in osteoclasts compared to in their precursors, macrophages. The overexpression of GPR84 in bone marrow-derived macrophages suppressed the formation of multinucleated osteoclasts without affecting precursor proliferation. In addition, GPR84 overexpression attenuated the induction of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which are transcription factors that are critical for osteoclastogenesis. Furthermore, knockdown of GPR84 using a small hairpin RNA promoted RANKL-mediated osteoclast differentiation and gene expression of osteoclastogenic markers. Mechanistically, GPR84 overexpression blocked RANKL-stimulated phosphorylation of IκBα and three MAPKs, JNK, ERK, and p38. GPR84 also suppressed NF-κB transcriptional activity mediated by RANKL. Conversely, GPR84 knockdown enhanced RANKL-induced activation of IκBα and the three MAPKs. Collectively, our results revealed that GPR84 functions as a negative regulator of osteoclastogenesis, suggesting that it may be a potential therapeutic target for osteoclast-mediated bone-destructive diseases. © 2017 Wiley Periodicals, Inc.

  5. Repair replication in permeabilized Escherichia coli

    International Nuclear Information System (INIS)

    Masker, W.E.; Simon, T.J.; Hanawalt, P.C.

    1975-01-01

    We have examined the modes of DNA synthesis in Escherichia coli strains made permeable to nucleoside triphosphates by treatment with toluene. In this quasi in vitro system, polymerase-I-deficient mutants exhibit a nonconservative mode of synthesis with properties expected for the resynthesis step of excision-repair. This uv-stimulated DNA synthesis can be performed by either DNA polymerase II or III and it also requires the uvrA gene product. It requires the four deoxynucleoside triphosphates; but, in contrast to the semiconservative mode, the ATP requirement can be partially satisfied by other nucleoside triphosphates. The ATP-dependent recBC nuclease is not involved. The observed uv-stimulated mode of DNA synthesis may be part of an alternate excision-repair mechanism which supplements or complements DNA-polymerase-I-dependent repair in vivo

  6. The enzymatic preparation of [α-32P]nucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP

    International Nuclear Information System (INIS)

    Walseth, T.F.; Johnson, R.A.

    1979-01-01

    A method has been developed for the enzymatic preparation of α- 32 P-labelled ribo- and deoxyribonucleoside triphosphates, cyclic [ 32 P]AMP, and cyclic [ 32 P]GMP of high specific radioactivity and in high yield from 32 Psub(i). The method also enables the preparation of [γ- 32 P]ATP, [γ- 32 P]GTP, [γ- 32 P]ITP, and [γ- 32 P]-dATP of very high specific activity and in high yield. (Auth.)

  7. Hybrid integrated biological-solid-state system powered with adenosine triphosphate

    Science.gov (United States)

    Roseman, Jared M.; Lin, Jianxun; Ramakrishnan, Siddharth; Rosenstein, Jacob K.; Shepard, Kenneth L.

    2015-12-01

    There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na+/K+ adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 106 mm-2) are able to sustain a short-circuit current of 32.6 pA mm-2 and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm-2 from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

  8. Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

    International Nuclear Information System (INIS)

    Ide, H.; Melamede, R.J.; Wallace, S.S.

    1987-01-01

    5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag 2 O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [ 3 H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The result suggests that Escherichia coli DNA polymerase I uses both isomers of DHdTTP as substrates and that the overall efficiency of incorporation is primarily determined by the concentration of the isomers in the nucleotide pool

  9. A-Type Cranberry Proanthocyanidins Inhibit the RANKL-Dependent Differentiation and Function of Human Osteoclasts

    Directory of Open Access Journals (Sweden)

    Amy B. Howell

    2011-03-01

    Full Text Available This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP staining, while the secretion of interleukin-8 (IL-8 and matrix metalloproteinases (MMPs was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

  10. Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase.

    Directory of Open Access Journals (Sweden)

    Ilaria Roato

    Full Text Available BACKGROUND: Phenylketonuria (PKU is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. METHODOLOGY: Peripheral blood mononuclear cell (PBMC cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF and receptor activator of NFκB ligand (RANKL. Flow cytometry was utilized to analyze osteoclast precursors (OCPs and T cell phenotype. Tumour necrosis factor α (TNF-α, RANKL and osteoprotegerin (OPG were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated. PRINCIPAL FINDINGS: Several in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS. This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia. CONCLUSIONS: Our results indicate that PKU spontaneous osteoclastogenesis

  11. Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α

    Directory of Open Access Journals (Sweden)

    Wang L

    2015-07-01

    Full Text Available Ling Wang,1,2,* Xue-Min Qiu,1,2,* Yu-Yan Gui,1,2 Ying-Ping Xu,1,2 Hans-Jürgen Gober,3 Da-Jin Li11Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, 2Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, People’s Republic of China; 3Department of Pharmacy, Wagner Jauregg Hospital and Children’s Hospital, Wagner Jauregg Weg, Linz, Austria*These authors contributed equally to this workIntroduction: Bu-Shen-Ning-Xin decoction (BSNXD is a traditional Chinese medicinal composition that has been used as a remedy for postmenopausal osteoporosis, but the mechanisms affecting bone metabolism are not fully understood.Purpose: We investigated the molecular mechanism and signaling pathway underlying the effect of BSNXD on osteoclastogenesis.Materials and methods: A postmenopausal osteoporosis animal model generated by ovariectomy was administered BSNXD and drug-derived serum was prepared. An enzyme immunoassay was conducted to measure the 17-β-estradiol (E2 concentration in the drug-derived serum. Bone marrow-derived monocyte/macrophage precursor cells were treated with drug-derived serum, and tartrate-resistance acid phosphatase staining was conducted to observe osteoclastogenesis. A bone resorption assay was performed to analyze the effect on osteoclastic resorptive function. Real-time PCR, flow cytometry, Western blotting, transfection, and luciferase assays were conducted to explore the related mechanism.Results: E2 was not elevated in BSNXD-derived serum. BSNXD-derived serum suppressed receptor activation of nuclear factor κB ligand (RANKL-activated osteoclastogenesis in a dose-dependent manner; this effect could be reversed by estrogen receptor α antagonist methyl-piperidino-pyrazole. The serum suppressed RANKL-induced NF-κB transcription and inhibited the accumulation of nuclear factor of activated T-cells, cytoplasmic 1

  12. The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

    International Nuclear Information System (INIS)

    Id Boufker, Hichame; Lagneaux, Laurence; Najar, Mehdi; Piccart, Martine; Ghanem, Ghanem; Body, Jean-Jacques; Journé, Fabrice

    2010-01-01

    The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined. We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]). Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity. Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism

  13. Diphlorethohydroxycarmalol from Ishige okamurae Suppresses Osteoclast Differentiation by Downregulating the NF-κB Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Hye Jung Ihn

    2017-12-01

    Full Text Available Marine algae possess a variety of beneficial effects on human health. In this study, we investigated whether diphlorethohydroxycarmalol (DPHC, isolated from Ishige okamurae, a brown alga, suppresses receptor activator of nuclear factor-κB ligand (RANKL-induced osteoclast differentiation. DPHC significantly suppressed RANKL-induced osteoclast differentiation and macrophage-colony stimulating factor (M-CSF expression in a dose-dependent manner. In addition, it significantly inhibited actin ring formation, the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (TRAP, nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1, cathepsin K (Ctsk, and dendritic cell-specific transmembrane protein (Dcstamp, and osteoclast-induced bone resorption. Analysis of the RANKL-mediated signaling pathway showed that the phosphorylation of both IκB and p65 was specifically inhibited by DPHC. These results suggest that DPHC substantially suppresses osteoclastogenesis by downregulating the RANK-NF-κB signaling pathway. Thus, it holds significant potential for the treatment of skeletal diseases associated with an enhanced osteoclast activity.

  14. Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Huixian [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Shi, Zhenqi [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Qiao, Ping [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Department of Pharmacology, Norman Bethune Medical College, Jilin University, Changchun, Jilin 130021 (China); Li, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); McCoy, Erin M. [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Mao, Ping [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Xu, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Feng, Xu [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Shunqing, E-mail: shqwang_cn@yahoo.com [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China)

    2013-11-01

    Highlights: •IL-3 treatment of bone marrow cells generates a population of hematopoietic cells. •IL-3-dependent hematopoietic cells are capable of differentiating into osteoclasts. •Osteoclasts derived from IL-3-dependent hematopoietic cells are functional. •IL-3 promotes the development of osteoclast progenitors. •IL-3 inhibits the osteoclastogenic process. -- Abstract: Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We found that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the

  15. Enzymatic primer-extension with glycerol-nucleoside triphosphates on DNA templates.

    Directory of Open Access Journals (Sweden)

    Jesse J Chen

    Full Text Available BACKGROUND: Glycerol nucleic acid (GNA has an acyclic phosphoglycerol backbone repeat-unit, but forms stable duplexes based on Watson-Crick base-pairing. Because of its structural simplicity, GNA is of particular interest with respect to the possibility of evolving functional polymers by in vitro selection. Template-dependent GNA synthesis is essential to any GNA-based selection system. PRINCIPAL FINDINGS: In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside triphosphates (gNTPs as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes quantitative primer-extension by the incorporation of two glyceronucleotides, with much less efficient extension up to five glyceronucleotides. Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines. In an attempt to improve pyrimidine incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl substituted pyrimidine nucleobases. This led to more efficient incorporation of gC, but not gT. CONCLUSIONS: We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency.

  16. Hybrid integrated biological-solid-state system powered with adenosine triphosphate.

    Science.gov (United States)

    Roseman, Jared M; Lin, Jianxun; Ramakrishnan, Siddharth; Rosenstein, Jacob K; Shepard, Kenneth L

    2015-12-07

    There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na(+)/K(+) adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 10(6) mm(-2)) are able to sustain a short-circuit current of 32.6 pA mm(-2) and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm(-2) from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

  17. Monitoring of intracellular adenosine triphosphate in CD4(+) T cells to predict the occurrence of cytomegalovirus disease in kidney transplant recipients.

    Science.gov (United States)

    Pérez-Jacoiste Asín, María Asunción; Fernández-Ruiz, Mario; López-Medrano, Francisco; Aquilino, Carolina; González, Esther; Ruiz-Merlo, Tamara; Gutiérrez, Eduardo; San Juan, Rafael; Paz-Artal, Estela; Andrés, Amado; Aguado, José Maria

    2016-10-01

    The measurement of intracellular concentrations of adenosine triphosphate (iATP) in phytohemagglutinin-stimulated CD4(+) T cells constitutes a surrogate marker for post-transplant cell-mediated immunity (CMI). This assay has shown suboptimal accuracy for predicting infection after kidney transplantation (KT). We hypothesize that its predictive capacity depends on the specific contribution of the CMI to host-pathogen interactions. We assessed iATP levels in 100 KT recipients at baseline and months 1, 3, and 6 (363 measurements). No association was found between iATP at month 1 and the risk for overall or bacterial infection, although such association was evident for cytomegalovirus (CMV) disease (multivariate-adjusted hazard ratio [per 50-unit increment]: 0.83; P-value = 0.048). There were no significant differences in mean iATP between stable patients (319.4 ng/ml) and those developing overall (304.1 ng/ml) or bacterial infection (346.9 ng/ml) over the 45 days following monitoring. However, iATP was significantly lower in patients who developed CMV disease (223.5 ng/ml; P-values <0.002). The optimal cutoff (265 ng/ml) for predicting CMV disease in patients not receiving antiviral prophylaxis yielded sensitivity, specificity, positive, and negative predictive values of 85.7%, 68.3%, 15.2%, and 98.6%, respectively. In conclusion, a non-pathogen-specific monitoring of CMI by means of iATP informs the risk of CMV disease in KT recipients. © 2016 Steunstichting ESOT.

  18. Acute effects of dietary fatty acids on osteclastogenesis via RANKL/RANK/OPG system.

    Science.gov (United States)

    Naranjo, M Carmen; Garcia, Indara; Bermudez, Beatriz; Lopez, Sergio; Cardelo, Magdalena P; Abia, Rocio; Muriana, Francisco J G; Montserrat-de la Paz, Sergio

    2016-11-01

    Postprandial state is directly linked with chronic diseases. We hypothesized that dietary fats may have acute effects on health status by modulating osteoclast differentiation and activation in a fatty acid-dependent manner. In healthy subjects, a fat-enriched meal increased plasma levels of the RANKL (receptor activator of nuclear factor κB ligand)/OPG (osteoprotegerin) ratio (SFAs > MUFAs = PUFAs) in the postprandial state. Postprandial TRL-SFAs enhanced tartrate-resistant acid phosphatase (TRAP) activity and the expression of osteoclast marker genes (TRAP, OSCAR, RANK, and CATHK) while downregulated the expression of OPG gene in human monocyte-derived osteoclasts. These effects were not observed with monounsaturated fatty acid (MUFA)-enriched postprandial triglyceride-rich lipoproteins (TRLs). Moreover, postprandial TRL-SFAs increased the release of osteoclastogenic cytokines (TNF-α, IL-1β, and IL-6) meanwhile TRL-MUFAs and TRL-PUFAs increased the release of anti-osteoclastogenic cytokines (IL-4 and IL-10) in the medium of human monocyte-derived osteoclasts. For the first time, we show that postprandial TRLs are metabolic entities with osteoclastogenic activity and that this property is related to the type of dietary fatty acid in the meal. The osteoclastogenic potency was as follows: SFAs > MUFAs = PUFAs. These exciting findings open opportunities for developing nutritional strategies with olive oil as the principal dietary source of MUFAs, notably oleic acid, to prevent development and progression of osteoclast-related diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Niclosamide suppresses RANKL-induced osteoclastogenesis and prevents LPS-induced bone loss

    Energy Technology Data Exchange (ETDEWEB)

    Cheon, Yoon-Hee [Center for Metabolic Function Regulation, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Baek, Jong Min; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); So, Hong-Seob, E-mail: jeanso@wku.ac.kr [Center for Metabolic Function Regulation, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Department of Anatomy, School of Medicine, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    2016-02-05

    Niclosamide (5-chloro-salicyl-(2-chloro-4-nitro) anilide) is an oral anthelmintic drug used for treating intestinal infection of most tapeworms. Recently, niclosamide was shown to have considerable efficacy against some tumor cell lines, including colorectal, prostate, and breast cancers, and acute myelogenous leukemia. Specifically, the drug was identified as a potent inhibitor of signal transducer and activator of transcription 3 (STAT3), which is associated with osteoclast differentiation and function. In this study, we assessed the effect of niclosamide on osteoclastogenesis in vitro and in vivo. Our in vitro study showed that receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast differentiation was inhibited by niclosamide, due to inhibition of serine–threonine protein kinase (Akt) phosphorylation, inhibitor of nuclear factor-kappaB (IκB), and STAT3 serine{sup 727}. Niclosamide decreased the expression of the major transcription factors c-Fos and NFATc1, and thereafter abrogated the mRNA expression of osteoclast-specific genes, including TRAP, OSCAR, αv/β3 integrin (integrin αv, integrin β3), and cathepsin K (CtsK). In an in vivo model, niclosamide prevented lipopolysaccharide-induced bone loss by diminishing osteoclast activity. Taken together, our results show that niclosamide is effective in suppressing osteoclastogenesis and may be considered as a new and safe therapeutic candidate for the clinical treatment of osteoclast-related diseases such as osteoporosis. - Highlights: • We first investigated the anti-osteoclastogenic effects of niclosamide in vitro and in vivo. • Niclosamide impairs the activation of the Akt-IκB-STAT3 ser{sup 727} signaling axis. • Niclosamide acts a negative regulator of actin ring formation during osteoclast differentiation. • Niclosamide suppresses LPS-induced bone loss in vivo. • Niclosamide deserves new evaluation as a potential treatment target in various bone diseases.

  20. Where Do Bone-Targeted Agents RANK in Breast Cancer Treatment?

    Directory of Open Access Journals (Sweden)

    Roger von Moos

    2013-08-01

    Full Text Available Breast cancer cells preferentially metastasise to the skeleton, owing, in part, to the fertile environment provided by bone. Increased bone turnover releases growth factors that promote tumour cell growth. In turn, tumour cells release factors that stimulate further bone turnover, resulting in a vicious cycle of metastasis growth and bone destruction. The RANK-RANK ligand (RANKL pathway plays a key role in this cycle, and inhibition of RANKL using the fully-human monoclonal antibody denosumab, has demonstrated efficacy in delaying skeletal complications associated with bone metastases in three phase 3 trials. Preclinical studies suggest that the RANKL pathway also plays a role in breast cancer tumourigenesis and migration to bone. In a subgroup analysis of the negative Adjuvant Zoledronic Acid to Reduce Recurrence (AZURE trial, the bisphosphonate zoledronic acid showed potential for improving survival in patients who were postmenopausal; however, a prospective study in this patient population is required to validate this observation. Ongoing trials are examining whether adjuvant blockade of the RANKL pathway using denosumab can prevent disease recurrence in patients with high-risk breast cancer. These are building on analogous studies that have shown that denosumab improves bone metastasis-free survival in prostate cancer and suggested that it confers an overall survival benefit in non-small-cell lung cancer.

  1. In vitro comparison of the biological activity of alumina ceramic and titanium particles associated with aseptic loosening

    International Nuclear Information System (INIS)

    Ding Yue; Qin Chuqiang; Xu Jie; Huang Dongsheng; Fu Yuru

    2012-01-01

    Prosthetic wear particles are thought to play a central role in the initiation and development of periprosthetic osteolysis, leading to aseptic loosening of prostheses. This study aimed to compare the biological activity of ceramic and titanium particles that are associated with particle-induced, aseptic joint loosening. Different sizes of alumina-ceramic particles and titanium particles were prepared to stimulate murine macrophage cells RAW 264.7, of which the expressions of tumor necrosis factor alpha (TNF-alpha) and receptor activator of nuclear factor-κB ligand (RANKL) were measured by qPCR and ELISA at various time points. In the presence of all particles, the expression of TNF-alpha increased in a time-dependent manner, whereas the expression of RANKL showed no regular expression patterns. Notably, particles of smaller sizes provoked significantly higher levels of TNF-alpha and RANKL than those of larger sizes. Compared to the titanium particles, the ceramic particles provoked a significantly lower production of TNF-alpha. Thus, the bioactivities of titanium and alumina ceramic particles were inversely proportional to the sizes of the particles, and the expression of RANKL was not parallel to that of TNF-alpha. The successful outcome of ceramic-on-ceramic artificial joint prostheses may be attributed to the low biological activity of ceramic particles, as evidenced here. (paper)

  2. Localization of RNA transcription sites in insect oocytes using microinjections of 5-bromouridine 5'-triphosphate.

    Directory of Open Access Journals (Sweden)

    Dmitry Bogolyubov

    2007-06-01

    Full Text Available In the present study we used 5-bromouridine 5'-triphosphate (BrUTP microinjections to localize the transcription sites in oocytes of insects with different types of the ovarium structure: panoistic, meroistic polytrophic, and meroistic telotrophic. We found that in an insect with panoistic ovaries (Acheta domesticus, oocyte nuclei maintain their transcription activity during the long period of oocyte growth. In insects with meroistic ovaries (Tenebrio molitor and Panorpa communis, early oocyte chromosomes were found to be transcriptionally active, and some transcription activity still persist while the karyosphere, a compact structure formed by all condensed oocyte chromosomes, begins to develop. At the latest stages of karyosphere development, no anti-Br-RNA signal was registered in the karyosphere.

  3. Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

    International Nuclear Information System (INIS)

    Ryu, T.; Davis, J.M.; Schwartz, K.A.

    1990-01-01

    The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding

  4. A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection.

    Science.gov (United States)

    Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia

    2015-01-01

    A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

  5. SIMULTANEOUS ANALYSIS OF AZIDOTHYMIDINE AND ITS MONOPHOSPHATE, DIPHOSPHATE AND TRIPHOSPHATE DERIVATIVES IN BIOLOGICAL-FLUIDS, TISSUE AND CULTURED-CELLS BY A RAPID HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD

    NARCIS (Netherlands)

    MOLEMA, G; JANSEN, RW; Visser, Jan; MEIJER, DKF

    1992-01-01

    A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro

  6. Synthesis of Base-Modified 2 '-Deoxyribonucleoside Triphosphates and Their Use in Enzymatic Synthesis of Modified DNA for Applications in Bioanalysis and Chemical Biology

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal

    2014-01-01

    Roč. 79, č. 21 (2014), s. 9914-9921 ISSN 0022-3263 R&D Projects: GA ČR GBP206/12/G151; GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : cross - coupling reactions * modified nucleoside triphosphates * nucleic acids Subject RIV: CC - Organic Chemistry Impact factor: 4.721, year: 2014

  7. Cell adhesion signaling regulates RANK expression in osteoclast precursors.

    Directory of Open Access Journals (Sweden)

    Ayako Mochizuki

    Full Text Available Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK differentiate into osteoclasts following stimulation with the RANK ligand (RANKL. Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition. BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS and tumor necrosis factor -αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6 in BMMs induced their differentiation into osteoclasts even under the non

  8. Nitrogen and phosphorus co-doped graphene quantum dots: synthesis from adenosine triphosphate, optical properties, and cellular imaging.

    Science.gov (United States)

    Ananthanarayanan, Arundithi; Wang, Yue; Routh, Parimal; Sk, Mahasin Alam; Than, Aung; Lin, Ming; Zhang, Jie; Chen, Jie; Sun, Handong; Chen, Peng

    2015-05-07

    Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of applications, particularly, as superior fluorescent reporters for bio-imaging and optical sensing. Heteroatom doping can endow GQDs with new or improved photoluminescence properties. Here, we demonstrate a simple strategy for the synthesis of nitrogen and phosphorus co-doped GQDs from a single biomolecule precursor (adenosine triphosphate - ATP). Such ATP-GQDs exhibit high fluorescence quantum yield, strong two-photon upconversion, small molecular weight, high photostability, and good biocompatibility. Furthermore, transferrin conjugated ATP-GQDs have been used for imaging and real-time tracking of transferrin receptors in live cells.

  9. Plasma Amino Acids Stimulate Uncoupled Respiration of Muscle Subsarcolemmal Mitochondria in Lean but Not Obese Humans.

    Science.gov (United States)

    Kras, Katon A; Hoffman, Nyssa; Roust, Lori R; Patel, Shivam H; Carroll, Chad C; Katsanos, Christos S

    2017-12-01

    Obesity is associated with mitochondrial dysfunction in skeletal muscle. Increasing the plasma amino acid (AA) concentrations stimulates mitochondrial adenosine triphosphate (ATP) production in lean individuals. To determine whether acute elevation in plasma AAs enhances muscle mitochondrial respiration and ATP production in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in obese adults. Assessment of SS and IMF mitochondrial function during saline (i.e., control) and AA infusions. Eligible participants were healthy lean (body mass index, mass index >30 kg/m2; age 35 ± 3 years; n = 11) subjects. Single trial of saline infusion followed by AA infusion. SS and IMF mitochondria were isolated from muscle biopsies collected at the end of the saline and AA infusions. Mitochondrial respiration and ATP production. AA infusion increased adenosine 5'-diphosphate (ADP)-stimulated respiration and ATP production rates of SS mitochondria in the lean (P lean subjects only (P lean or obese subjects (P > 0.05). Increasing the plasma AA concentrations enhances the capacity for respiration and ATP production of muscle SS, but not IMF, mitochondria in lean individuals, in parallel with increases in uncoupled respiration. However, neither of these parameters increases in muscle SS or IMF mitochondria in obese individuals. Copyright © 2017 Endocrine Society

  10. Ugonin U stimulates NLRP3 inflammasome activation and enhances inflammasome-mediated pathogen clearance

    Directory of Open Access Journals (Sweden)

    Chun-Yu Chen

    2017-04-01

    Full Text Available The NOD-like receptor pyrin domain 3 (NLRP3 inflammasome contains Nod-like receptors, a subclass of pattern recognition receptors, suggesting that this complex has a prominent role in host defenses. Various structurally diverse stimulators activate the NLRP3 inflammasome through different signaling pathways. We previously reported that ugonin U (UgU, a natural flavonoid isolated from Helminthostachys zeylanica (L Hook, directly stimulates phospholipase C (PLC and triggers superoxide release in human neutrophils. In the present study, we showed that UgU induced NLRP3 inflammasome assembly and subsequent caspase-1 and interleukin (IL-1β processing in lipopolysaccharide-primed human monocytes. Moreover, UgU elicited mitochondrial superoxide generation in a dose-dependent manner, and a specific scavenger of mitochondrial reactive oxygen species (ROS diminished UgU-induced IL-1β and caspase-1 activation. UgU induced Ca2+ mobilization, which was inhibited by treatment with inhibitors of PLC or inositol triphosphate receptor (IP3R. Blocking Ca2+ mobilization, PLC, or IP3R diminished UgU-induced IL-1β release, caspase-1 activation, and mitochondrial ROS generation. These data demonstrated that UgU activated the NLPR3 inflammasome activation through Ca2+ mobilization and the production of mitochondrial ROS. We also demonstrated that UgU-dependent NLRP3 inflammasome activation enhanced the bactericidal function of human monocytes. The ability of UgU to stimulate human neutrophils and monocytes, both of which are professional phagocytes, and its capacity to activate the NLRP3 inflammasome, which is a promising molecular target for developing anti-infective medicine, indicate that UgU treatment should be considered as a possible novel therapy for treating infectious diseases.

  11. On the structure of thorium and americium adenosine triphosphate complexes

    International Nuclear Information System (INIS)

    Mostapha, Sarah; Berton, Laurence; Boubals, Nathalie; Zorz, Nicole; Charbonnel, Marie-Christine; Fontaine-Vive, Fabien; Den Auwer, Christophe; Solari, Pier Lorenzo

    2014-01-01

    The actinides are chemical poisons and radiological hazards. One challenge to better appraise their toxicity and develop countermeasures in case of exposure of living organisms is to better assess pathways of contamination. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, nucleotides and in particular adenosine triphosphate nucleotide (ATP) may be considered critical target building blocks for actinides. Combinations of spectroscopic techniques (Fourier transformed Infra Red [FTIR], Electro-spray Ionization Mass Spectrometry [ESI-MS], and Extended X-ray Absorption Fine Structure [EXAFS]) with quantum chemical calculations have been implemented in order to assess the actinides coordination arrangement with ATP. We describe and compare herein the interaction of ATP with thorium and americium; thorium(IV) as a representative of actinide(IV) like plutonium(IV) and americium(III) as a representative of all heavier actinides. In the case of thorium, an insoluble complex is readily formed. In the case of americium, a behavior identical to that described previously for lutetium has been observed with insoluble and soluble complexes. The comparative study of ATP complexation with Th(IV) and Am(III) shows their ability to form insoluble complexes for which a structural model has been proposed by analogy with previously described Lu(III) complexes. (authors)

  12. On the structure of thorium and americium adenosine triphosphate complexes.

    Science.gov (United States)

    Mostapha, Sarah; Fontaine-Vive, Fabien; Berthon, Laurence; Boubals, Nathalie; Zorz, Nicole; Solari, Pier Lorenzo; Charbonnel, Marie Christine; Den Auwer, Christophe

    2014-11-01

    The actinides are chemical poisons and radiological hazards. One challenge to better appraise their toxicity and develop countermeasures in case of exposure of living organisms is to better assess pathways of contamination. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, nucleotides and in particular adenosine triphosphate nucleotide (ATP) may be considered critical target building blocks for actinides. Combinations of spectroscopic techniques (Fourier transformed Infra Red [FTIR], Electrospray Ionization Mass Spectrometry [ESI-MS], and Extended X-ray Absorption Fine Structure [EXAFS]) with quantum chemical calculations have been implemented in order to assess the actinides coordination arrangement with ATP. We describe and compare herein the interaction of ATP with thorium and americium; thorium(IV) as a representative of actinide(IV) like plutonium(IV) and americium(III) as a representative of all heavier actinides. In the case of thorium, an insoluble complex is readily formed. In the case of americium, a behavior identical to that described previously for lutetium has been observed with insoluble and soluble complexes. The comparative study of ATP complexation with Th(IV) and Am(III) shows their ability to form insoluble complexes for which a structural model has been proposed by analogy with previously described Lu(III) complexes.

  13. Effects of a Mikania laevigata extract on bone resorption and RANKL expression during experimental periodontitis in rats

    Directory of Open Access Journals (Sweden)

    Bruno B. Benatti

    2012-06-01

    Full Text Available OBJECTIVES: The Mikania laevigata extract (MLE (popularly known in Brazil as "guaco" possesses anti-inflammatory properties. In the present study we tested the effects of MLE in a periodontitis experimental model in rats. We also investigated possible mechanisms underlying such effects. MATERIAL AND METHODS: Periodontal disease was induced by a ligature placed around the mandibular first molars of each animal. Male Wistar rats were divided into 4 groups: non-ligated animals treated with vehicle; non-ligated animals treated with MLE (10 mg/kg, daily; ligature-induced animals treated with vehicle and ligature-induced animals treated with MLE (10 mg/kg, daily. Thirty days after the induction of periodontal disease, the animals were euthanized and mandibles and gingival tissues removed for further analysis. RESULTS: Morphometric analysis of alveolar bone loss demonstrated that MLE-treated animals presented a decreased alveolar bone loss and a lower expression of the activator of nuclear factor-κB ligand (RANKL measured by immunohistochemistry. Moreover, gingival tissues from the MLE-treated group showed decreased neutrophil migration myeloperoxidase (MPO assay. CONCLUSIONS: These results indicate that MLE may be useful to control bone resorption during progression of experimental periodontitis in rats.

  14. DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption.

    Science.gov (United States)

    Wisitrasameewong, W; Kajiya, M; Movila, A; Rittling, S; Ishii, T; Suzuki, M; Matsuda, S; Mazda, Y; Torruella, M R; Azuma, M M; Egashira, K; Freire, M O; Sasaki, H; Wang, C Y; Han, X; Taubman, M A; Kawai, T

    2017-06-01

    Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP-monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis ( n = 6-7/group) where Pasteurella pneumotropica ( Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis. DC-STAMP was expressed on the cell surface of mature multinuclear OCs, as well as immature mononuclear OCs, in primary cultures of RANKL-stimulated bone marrow cells. Anti-DC-STAMP-mAb suppressed the emergence of large, but not small, multinuclear OCs, suggesting that DC-STAMP is engaged in the late stage of cell fusion. Anti-DC-STAMP-mAb also inhibited pit formation caused by RANKL-stimulated bone marrow cells. Attachment of ligature to a second maxillary molar induced DC-STAMP messenger RNA and protein, along with elevated tartrate-resistant acid phosphatase-positive (TRAP+) OCs and alveolar bone loss. As we expected, systemic administration of anti-DC-STAMP-mAb downregulated the ligature-induced alveolar bone loss. Importantly, local injection of anti-DC-STAMP-mAb also suppressed alveolar bone loss and reduced the total number of multinucleated TRAP+ cells in mice that received ligature attachment. Attachment of ligature induced significantly elevated tumor necrosis factor-α, interleukin-1β, and RANKL in the gingival tissue compared with the control site without ligature ( P < 0.05), which was unaffected by local injection with either anti-DC-STAMP-mAb or control-mAb. Neither in vivo anti- Pp IgG antibody nor in vitro anti- Pp T

  15. Approach to novel functional foods for stress control 4. Regulation of serotonin transporter by food factors.

    Science.gov (United States)

    Ito, Mikiko; Haito, Sakiko; Furumoto, Mari; Kawai, Yoshichika; Terao, Junji; Miyamoto, Ken-ichi

    2005-11-01

    Serotonin transporters (SERTs) are pre-synaptic proteins specialized for the clearance of serotonin following vesicular release at central nervous system (CNS) and enteric nervous system synapses. SERTs are high affinity targets in vivo for antidepressants such as serotonin selective reuptake inhibitors (SSRIs). These include 'medical' psychopharmacological agents such as analgesics and antihistamines, a plant extract called St John's Wort (Hypericum). Osteoclasts are the primary cells responsible for bone resorption. They arise by the differentiation of osteoclast precursors of the monocyte/macrophage lineage. The expression of SERTs was increased in RANKL-induced osteoclast-like cells. Using RANKL stimulation of RAW264.7 cells as a model system for osteoclast differentiation, we studied the direct effects of food factor on serotonin uptake. The SSRIs (fluoxetine and fluvoxamine) inhibited markedly (approximately 95%) in serotonin transport in differentiated osteoclast cells. The major components of St. John's Wort, hyperforin and hypericine were significantly decreased in serotonin transport activity. Thus, a new in vitro model using RANKL-induced osteoclast-like cells may be useful to analyze the regulation of SERT by food factors and SSRIs.

  16. Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Rafiei, Shahrzad; Komarova, Svetlana V

    2013-01-01

    Advanced prostate cancer commonly metastasizes to bone leading to osteoblastic and osteolytic lesions. Although an osteolytic component governed by activation of bone resorbing osteoclasts is prominent in prostate cancer metastasis, the molecular mechanisms of prostate cancer-induced osteoclastogenesis are not well-understood. We studied the effect of soluble mediators released from human prostate carcinoma cells on osteoclast formation from mouse bone marrow and RAW 264.7 monocytes. Soluble factors released from human prostate carcinoma cells significantly increased viability of naïve bone marrow monocytes, as well as osteoclastogenesis from precursors primed with receptor activator of nuclear factor κ-B ligand (RANKL). The prostate cancer-induced osteoclastogenesis was not mediated by RANKL as it was not inhibited by osteoprotegerin (OPG). However inhibition of TGFβ receptor I (TβRI), or macrophage-colony stimulating factor (MCSF) resulted in attenuation of prostate cancer-induced osteoclastogenesis. We characterized the signaling pathways induced in osteoclast precursors by soluble mediators released from human prostate carcinoma cells. Prostate cancer factors increased basal calcium levels and calcium fluctuations, induced nuclear localization of nuclear factor of activated t-cells (NFAT)c1, and activated prolonged phosphorylation of ERK1/2 in RANKL-primed osteoclast precursors. Inhibition of calcium signaling, NFATc1 activation, and ERK1/2 phosphorylation significantly reduced the ability of prostate cancer mediators to stimulate osteoclastogenesis. This study reveals the molecular mechanisms underlying the direct osteoclastogenic effect of prostate cancer derived factors, which may be beneficial in developing novel osteoclast-targeting therapeutic approaches

  17. The effect of experimental gastric dilatation-volvulus on adenosine triphosphate content and conductance of the canine gastric and jejunal mucosa

    OpenAIRE

    Peycke, Laura E.; Hosgood, Giselle; Davidson, Jacqueline R.; Tetens, Joanne; Taylor, H. Wayne

    2005-01-01

    The objective of this study was to determine if experimental gastric dilatation volvulus (GDV) would decrease adenosine triphosphate (ATP) concentration and increase membrane conductance of the canine gastric and jejunal mucosa. Male dogs (n = 15) weighing between 20 and 30 kg were used. Dogs were randomly assigned to 1 of 3 equal groups: Group 1 was control, group 2 was GDV, and group 3 was ischemia. All dogs were anesthetized for 210 min. Group 1 had no manipulation. Group 2 had GDV experim...

  18. Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

    Science.gov (United States)

    Van de Werve, G; Hers, H G

    1979-01-15

    1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

  19. A novel conductometric biosensor based on hexokinase for determination of adenosine triphosphate.

    Science.gov (United States)

    Kucherenko, I S; Kucherenko, D Yu; Soldatkin, O O; Lagarde, F; Dzyadevych, S V; Soldatkin, A P

    2016-04-01

    The paper presents a simple and inexpensive reusable biosensor for determination of the concentration of adenosine-5'-triphosphate (ATP) in aqueous samples. The biosensor is based on a conductometric transducer which contains two pairs of gold interdigitated electrodes. An enzyme hexokinase was immobilized onto one pair of electrodes, and bovine serum albumin-onto another pair (thus, a differential mode of measurement was used). Conditions of hexokinase immobilization on the transducer by cross-linking via glutaraldehyde were optimized. Influence of experimental conditions (concentration of magnesium ions, ionic strength and concentration of the working buffer) on the biosensor work was studied. The reproducibility of biosensor responses and operational stability of the biosensor were checked during one week. Dry storage at -18 °C was shown to be the best conditions to store the biosensor. The biosensor was successfully applied for measurements of ATP concentration in pharmaceutical samples. The proposed biosensor may be used in future for determination of ATP and/or glucose in water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    OpenAIRE

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (?PO3 2??Zr4+?) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). Aft...

  1. Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

    Science.gov (United States)

    Hocek, Michal

    2014-11-07

    The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

  2. Simple, Fast and Selective Detection of Adenosine Triphosphate at Physiological pH Using Unmodified Gold Nanoparticles as Colorimetric Probes and Metal Ions as Cross-Linkers

    Directory of Open Access Journals (Sweden)

    Huan Pang

    2012-11-01

    Full Text Available We report a simple, fast and selective colorimetric assay of adenosine triphosphate (ATP using unmodified gold nanoparticles (AuNPs as probes and metal ions as cross-linkers. ATP can be assembled onto the surface of AuNPs through interaction between the electron-rich nitrogen atoms and the electron-deficient surface of AuNPs. Accordingly, Cu2+ ions induce a change in the color and UV/Vis absorbance of AuNPs by coordinating to the triphosphate groups and a ring nitrogen of ATP. A detection limit of 50 nM was achieved, which is comparable to or lower than that achievable by the currently used electrochemical, spectroscopic or chromatographic methods. The theoretical simplicity and high selectivity reported herein demonstrated that AuNPs-based colorimetric assay could be applied in a wide variety of fields by rationally designing the surface chemistry of AuNPs. In addition, our results indicate that ATP-modified AuNPs are less stable in Cu2+, Cd2+ or Zn2+-containing solutions due to the formation of the corresponding dimeric metal-ATP complexes.

  3. Fludarabine-mediated circumvention of cytarabine resistance is associated with fludarabine triphosphate accumulation in cytarabine-resistant leukemic cells.

    Science.gov (United States)

    Yamamoto, Shuji; Yamauchi, Takahiro; Kawai, Yasukazu; Takemura, Haruyuki; Kishi, Shinji; Yoshida, Akira; Urasaki, Yoshimasa; Iwasaki, Hiromichi; Ueda, Takanori

    2007-02-01

    The combination of cytarabine (ara-C) with fludarabine is a common approach to treating resistant acute myeloid leukemia. Success depends on a fludarabine triphosphate (F-ara-ATP)-mediated increase in the active intracellular metabolite of ara-C, ara-C 5'-triphosphate (ara-CTP). Therapy-resistant leukemia may exhibit ara-C resistance, the mechanisms of which might induce cross-resistance to fludarabine with reduced F-ara-ATP formation. The present study evaluated the effect of combining ara-C and fludarabine on ara-C-resistant leukemic cells in vitro. Two variant cell lines (R1 and R2) were 8-fold and 10-fold more ara-C resistant, respectively, than the parental HL-60 cells. Reduced deoxycytidine kinase activity was demonstrated in R1 and R2 cells, and R2 cells also showed an increase in cytosolic 5'-nucleotidase II activity. Compared with HL-60 cells, R1 and R2 cells produced smaller amounts of ara-CTP. Both variants accumulated less F-ara-ATP than HL-60 cells and showed cross-resistance to fludarabine nucleoside (F-ara-A). R2 cells, however, accumulated much smaller amounts of F-ara-ATP and were more F-ara-A resistant than R1 cells. In HL-60 and R1 cells, F-ara-A pretreatment followed by ara-C incubation produced F-ara-ATP concentrations sufficient for augmenting ara-CTP production, thereby enhancing ara-C cytotoxicity. No potentiation was observed in R2 cells. Nucleotidase might preferentially degrade F-ara-A monophosphate over ara-C monophosphate, leading to reduced F-ara-ATP production and thereby compromising the F-ara-A-mediated potentiation of ara-C cytotoxicity in R2 cells. Thus, F-ara-A-mediated enhancement of ara-C cytotoxicity depended on F-ara-ATP accumulation in ara-C-resistant leukemic cells but ultimately was associated with the mechanism of ara-C resistance.

  4. Simple detection of hepatitis C virus using {sup 125}I-2'-deoxyuridine triphosphate and gamma counter

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Soo Jin; Ahn, S. H.; Chung, W. S.; Woo, K. S.; Lim, S. J.; Choi, C. W.; Lim, S. M. [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    2000-07-01

    Hepatitis C Virus (HCV) is the major cause of post transfusion and sporadic non A, non B hepatitis. Current infection of HCV can be detected by PCR method. Using PCR, it has been possible to detect HCV viremia prior to immunological sero-conversion and to detect fluctuation of viremia in antibody-positive chronic HCV patients undergoing therapy with interferon. In this study, we established the simple method to detect HCV DNA by incorporation of {sup 125}I-deoxyuridine triphosphate(dUTP) into DNA during the PCR, and counted the radioactivity of PCR product by gamma counter. {sup 125}I-2'-deoxyuridine 5'-triphosphate was prepared, and incorporated into DNA during PCR. dUTP was radiolabeled by the iododemercuration of 5-mercuri intermediate. Iododemercuration labeling was completed with 98% yield and the obtained product was incorporated into DNA without further purification. After incorporation, covalently bonded radioiodine substituent was remained stable during PCR procedure HCV positive standard and positive patient sera in immunological assay were centrifuged. HCV RNA is isolated from by GTC(Guanidine Thiocyanate) and phenol/chloroform extraction method and synthesized complementary DNA by using reverse transcriptase. The '1{sup 25}I-dUTP was incorporated into HCV C DNA during PCR. PCR product purified by fiber matrix column and counted by gamma counter. PCR products were electrophoresized, and autoradiography image obtained. Amplified HCV DNA by {sup 125}I-dUTP PCR obtained the band on the gel by electrophoresis and autoradiography at the same position. In patient sera, radioactivity of HCV positive sample was 8 times higher than HCV negative viremia sample. We established HCV detection method using {sup 125}I-dUTP. {sup 125}I-dUTP PCR detection of HCV is convenient and reporducible.

  5. Development of 1-Amino-4-(phenylamino)anthraquinone-2-sulfonate Sodium Derivatives as a New Class of Inhibitors of RANKL-Induced Osteoclastogenesis.

    Science.gov (United States)

    Lee, Chia-Chung; Chen, Chun-Liang; Liu, Fei-Lan; Chiou, Chung-Yu; Chen, Tsung-Chih; Wu, Cheng-Chi; Sun, Wei-Hsin; Chang, Deh-Ming; Huang, Hsu-Shan

    2016-05-01

    A series of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives was synthesized and evaluated for osteoclast inhibition using a TRAP-staining assay. Among them, two compounds, LCCY-13 and LCCY-15, dose-dependently suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. Moreover, the cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds was not a result of their cytotoxicity. Further, the inhibitory activities of compounds LCCY-13 and LCCY-15 were further confirmed by including specific inhibition of NFATc1 expression levels in nuclei using an immunofluorescent analysis. In addition, LCCY-13 and LCCY-15 also significantly attenuated the bone resorption activity of osteoclasts according to a pit formation assay. Thus, a new class of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium compounds might be considered as an essential lead structure for the further development of anti-resorptive agents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Skeletal muscle beta-receptors and isoproterenol-stimulated vasodilation in canine heart failure

    International Nuclear Information System (INIS)

    Frey, M.J.; Lanoce, V.; Molinoff, P.B.; Wilson, J.R.

    1989-01-01

    To investigate whether heart failure alters beta-adrenergic receptors on skeletal muscle and its associated vasculature, the density of beta-adrenergic receptors, isoproterenol-stimulated adenylate cyclase activity, and coupling of the guanine nucleotide-binding regulatory protein were compared in 18 control dogs and 16 dogs with heart failure induced by 5-8 wk of ventricular pacing at 260 beats/min. Hindlimb vascular responses to isoproterenol were compared in eight controls and eight of the dogs with heart failure. In dogs with heart failure, the density of beta-receptors on skeletal muscle was reduced in both gastrocnemius (control: 50 +/- 5; heart failure: 33 +/- 8 fmol/mg of protein) and semitendinosus muscle (control: 43 +/- 9; heart failure: 27 +/- 9 fmol/mg of protein, both P less than 0.05). Receptor coupling to the ternary complex, as determined by isoproterenol competition curves with and without guanosine 5'-triphosphate (GTP), was unchanged. Isoproterenol-stimulated adenylate cyclase activity was significantly decreased in semitendinosus muscle (control: 52.4 +/- 4.6; heart failure: 36.5 +/- 9.5 pmol.mg-1.min-1; P less than 0.05) and tended to be decreased in gastrocnemius muscle (control: 40.1 +/- 8.5; heart failure: 33.5 +/- 4.5 pmol.mg-1.min-1; P = NS). Isoproterenol-induced hindlimb vasodilation was not significantly different in controls and in dogs with heart failure. These findings suggest that heart failure causes downregulation of skeletal muscle beta-adrenergic receptors, probably due to receptor exposure to elevated catecholamine levels, but does not reduce beta-receptor-mediated vasodilation in muscle

  7. Adenosine triphosphate-guided pulmonary vein isolation for atrial fibrillation: the UNmasking Dormant Electrical Reconduction by Adenosine TriPhosphate (UNDER-ATP) trial.

    Science.gov (United States)

    Kobori, Atsushi; Shizuta, Satoshi; Inoue, Koichi; Kaitani, Kazuaki; Morimoto, Takeshi; Nakazawa, Yuko; Ozawa, Tomoya; Kurotobi, Toshiya; Morishima, Itsuro; Miura, Fumiharu; Watanabe, Tetsuya; Masuda, Masaharu; Naito, Masaki; Fujimoto, Hajime; Nishida, Taku; Furukawa, Yoshio; Shirayama, Takeshi; Tanaka, Mariko; Okajima, Katsunori; Yao, Takenori; Egami, Yasuyuki; Satomi, Kazuhiro; Noda, Takashi; Miyamoto, Koji; Haruna, Tetsuya; Kawaji, Tetsuma; Yoshizawa, Takashi; Toyota, Toshiaki; Yahata, Mitsuhiko; Nakai, Kentaro; Sugiyama, Hiroaki; Higashi, Yukei; Ito, Makoto; Horie, Minoru; Kusano, Kengo F; Shimizu, Wataru; Kamakura, Shiro; Kimura, Takeshi

    2015-12-07

    Most of recurrent atrial tachyarrhythmias after pulmonary vein isolation (PVI) for atrial fibrillation (AF) are due to reconnection of PVs. The aim of the present study was to evaluate whether elimination of adenosine triphosphate (ATP)-induced dormant PV conduction by additional energy applications during the first ablation procedure could reduce the incidence of recurrent atrial tachyarrhythmias. We randomly assigned 2113 patients with paroxysmal, persistent, or long-lasting AF to either ATP-guided PVI (1112 patients) or conventional PVI (1001 patients). The primary endpoint was recurrent atrial tachyarrhythmias lasting for >30 s or those requiring repeat ablation, hospital admission, or usage of Vaughan Williams class I or III antiarrhythmic drugs at 1 year with the blanking period of 90 days post ablation. Among patients assigned to ATP-guided PVI, 0.4 mg/kg body weight of ATP provoked dormant PV conduction in 307 patients (27.6%). Additional radiofrequency energy applications successfully eliminated dormant conduction in 302 patients (98.4%). At 1 year, 68.7% of patients in the ATP-guided PVI group and 67.1% of patients in the conventional PVI group were free from the primary endpoint, with no significant difference (adjusted hazard ratio [HR] 0.89; 95% confidence interval [CI] 0.74-1.09; P = 0.25). The results were consistent across all the prespecified subgroups. Also, there was no significant difference in the 1-year event-free rates from repeat ablation for any atrial tachyarrhythmia between the groups (adjusted HR 0.83; 95% CI 0.65-1.08; P = 0.16). In the catheter ablation for AF, we found no significant reduction in the 1-year incidence of recurrent atrial tachyarrhythmias by ATP-guided PVI compared with conventional PVI. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  8. Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts

    International Nuclear Information System (INIS)

    Kaplan, J.H.; Forbush, B. III; Hoffman, J.F.

    1978-01-01

    2-Nitrobenzyl phosphate and 1-(2-nitro)phenylethyl phosphate have been synthesized and demonstrated to be suitable as photolabile sources of inorganic phosphate. The same protecting groups were attached to the terminal phosphate of adenosine 5'-triphosphate. These caged ATP compounds released adenosine 5'-triphosphate on illumination at 340 nm in aqueous solution and P 3 -1-(2-nitro)phenylethyl-ATP gave about a 70 percent yield in under 30 s. The unphotolyzed caged ATP was neither a substrate nor inhibitor of purified renal Na,K-ATPase (EC 3.61.3). Following photolysis in the presence of the enzyme, the liberated ATP was hydrolyzed but at an inhibited rate. The photo-dependent inhibition could be eliminated by prior addition of glutathione or bisulfite to the irradiated solution. Caged ATP was incorporated into resealed human erythrocyte ghosts prepared from red blood cells depleted of internal energy stores. While the NA : K pump was unable to use incorporated caged ATP as a substrate, the ATP liberated by photolysis activated the pump as evidenced by measurements of K-dependent, ouabain-sensitive Na efflux. Thus the caged ATP can be used as a stable source of ATP unmetabolizable by intracellular ATPases until the ATP is released following photolytic irradiation

  9. Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate

    International Nuclear Information System (INIS)

    Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

    2014-01-01

    We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ∼100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm 2  V −1  s −1 . The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM. (paper)

  10. Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate.

    Science.gov (United States)

    Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

    2014-04-25

    We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ∼100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm2 V(-1) s(-1). The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM.

  11. Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

    Science.gov (United States)

    Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

    1977-01-01

    The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

  12. Combined treatment with Dendrobium candidum and black tea extract promotes osteoprotective activity in ovariectomized estrogen deficient rats and osteoclast formation.

    Science.gov (United States)

    Wang, Ming-Yue; Shen, Chang; An, Meng-Fei; Xie, Chuan-Qi; Wu, Xin; Zhu, Qiang-Qiang; Sun, Bin; Huang, Yan-Ping; Zhao, Yun-Li; Wang, Xuan-Jun; Sheng, Jun

    2018-05-01

    Dendrobium candidum (DC) and black tea, are traditional chinese drinks, which contain multiple active ingredients. However, whether or not the combination of these two ingredients can improve osteoporosis remains unknown. This study therefore aimed to examine the effects of the combination of DC and black tea extract (BTE) on osteoporosis. Ovariectomy (OVX)-induced osteoporosis in vivo as well as receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in vitro was selected. Results showed that OVX rats that were treated orally with a DC and BTE combination for 12 weeks maintained their calcium (Ca) and phosphorus (P) homeostasis and exhibited significantly enhanced estradiol (E 2 ) and OPG levels. This combination treatment also simultaneously reduced levels of interleukin (IL)-1β, IL-6 and improved the organ coefficients of the uterus and femur as well as BMD and BMC in OVX rats. In addition, this DC and BTE combination suppressed osteoclast differentiation in the RANKL-stimulated osteoclastogenesis of RAW 264.7 cells and effectively inhibited the expression of osteoclast-associated genes and proteins. The results of this study further highlight the fact that a combination of DC and BTE improved ovariectomy-induced osteoporosis in rats and suppressed RANKL-stimulated osteoclastogenesis in RAW 264.7 cells. This combination also significantly alleviated osteoporosis when compared to the alternative sole treatments above, due to synergistic effects among components. One partial mechanism of this combination might be the inhibition of osteoclast proliferation and the regulation of NFATC1/c-Fos expression. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Regulation by Phloroglucinol of Nrf2/Maf-Mediated Expression of Antioxidant Enzymes and Inhibition of Osteoclastogenesis via the RANKL/RANK Signaling Pathway: In Silico study

    Science.gov (United States)

    Rahim, Agus Hadian; Setiawan, Bambang; Dewi, Firli Rahmah Primula; Noor, Zairin

    2015-01-01

    Introduction: Phloroglucinol is an antioxidant compound with many positive effects on health. The purpose of this study was to determine the role of phloroglucinol in osteoclastogenesis via the RANKL/RANK signaling pathway and the activity of the transcription factor Nrf2. Material and methods: Analysis was performed in silico using the primary method of docking by the use of Hex 8.0 software and Haddock web server. Analysis of interactions was then performed to determine interactions between the ligand and its receptors by using the software LigPlus and LigandScout 3.1. Results: Results indicated that phloroglucinol compound was thought to inhibit osteoclastogenesis via three mechanisms: inhibiting RANKL−RANK interaction, sustaining the RANKL−OPG bond, and increasing the activity of the transcription factor Nrf2. PMID:26483597

  14. Inhibition of osteoclastogenesis by RNA interference targeting RANK

    Directory of Open Access Journals (Sweden)

    Ma Ruofan

    2012-08-01

    Full Text Available Abstract Background Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK ligand (RANKL as well as the macrophage colony-stimulating factor (M-CSF. The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption. Methods Three pairs of short hairpin RNAs (shRNA targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs using the optimal shRNA by targeting RANK. Results Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p Conclusions These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

  15. Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

    DEFF Research Database (Denmark)

    Sørensen, Mette Grøndahl; Henriksen, Kim; Schaller, Sophie

    2007-01-01

    Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone...... resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation...... of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells...

  16. The polymethoxy flavonoid sudachitin suppresses inflammatory bone destruction by directly inhibiting osteoclastogenesis due to reduced ROS production and MAPK activation in osteoclast precursors.

    Directory of Open Access Journals (Sweden)

    Yoko Ohyama

    Full Text Available Inflammatory bone diseases, including rheumatoid arthritis, periodontitis and peri-implantitis, are associated not only with the production of inflammatory cytokines but also with local oxidative status, which is defined by intracellular reactive oxygen species (ROS. Osteoclast differentiation has been reported to be related to increased intracellular ROS levels in osteoclast lineage cells. Sudachitin, which is a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions in mammalian cells. However, the effects of sudachitin on inflammatory bone destruction and osteoclastogenesis remain unknown. In calvaria inflamed by a local lipopolysaccharide (LPS injection, inflammation-induced bone destruction and the accompanying elevated expression of osteoclastogenesis-related genes were reduced by the co-administration of sudachitin and LPS. Moreover, sudachitin inhibited osteoclast formation in cultures of isolated osteoblasts and osteoclast precursors. However, sudachitin rather increased the expression of receptor activator of NF-κB ligand (RANKL, which is an important molecule triggering osteoclast differentiation, and the mRNA ratio of RANKL/osteoprotegerin that is a decoy receptor for RANKL, in the isolated osteoblasts, suggesting the presence of additional target cells. When osteoclast formation was induced from osteoclast precursors derived from bone marrow cells in the presence of soluble RANKL and macrophage colony-stimulating factor, sudachitin inhibited osteoclastogenesis without influencing cell viability. Consistently, the expression of osteoclast differentiation-related molecules including c-fos, NFATc1, cathepsin K and osteoclast fusion proteins such as DC-STAMP and Atp6v0d2 was reduced by sudachitin. In addition, sudachitin decreased activation of MAPKs such as Erk and JNK and the ROS production evoked by RANKL in osteoclast lineage cells. Our findings suggest that sudachitin is a

  17. Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

    2016-01-01

    Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. A label-free fluorescent adenosine triphosphate biosensor via overhanging aptamer-triggered enzyme protection and target recycling amplification.

    Science.gov (United States)

    Wang, Zhaoyin; Zhao, Jian; Dai, Zhihui

    2016-06-20

    Herein, a label-free fluorescent adenosine triphosphate (ATP) aptasensor is fabricated with a DNA hairpin and an overhanging aptamer. In the presence of ATP, the overhanging sequences of the aptamer may form preferred substrates of exo III, and thus trigger the enzyme-assisted amplification, which results in the release of G-rich sequences. Free G-rich sequences subsequently generate an enhanced flourescent signal by binding with thioflavin T. However, if ATP is absent, the overhanging sequence can induce steric hindrance and protect the DNA hairpin against the digestion of exo III, significantly reducing the noise of this biosensor. Accordingly, the signal-to-noise ratio of the sensing system is greatly improved, which ensures the desirable analytical performance of the proposed aptasensor both in pure samples and real samples.

  19. Quantitative assessment of the use of modified nucleoside triphosphates in expression profiling: differential effects on signal intensities and impacts on expression ratios

    Directory of Open Access Journals (Sweden)

    Dorris David

    2002-07-01

    Full Text Available Abstract Background The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. One of the limitations of such powerful analytical tools is the inability to detect certain transcripts in the target sample because of artifacts caused by background noise or poor hybridization kinetics. The use of base-modified analogs of nucleoside triphosphates has been shown to increase complementary duplex stability in other applications, and here we attempted to enhance microarray hybridization signal across a wide range of sequences and expression levels by incorporating these nucleotides into labeled cRNA targets. Results RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences. These results were similar, and additive, to those seen with an increase in the hybridization time. In contrast, 5-methyluridine and 5-methylcytidine decreased signal intensities. Hybridization specificity, as assessed by mismatch controls, was dependent on both target sequence and extent of substitution with the modified nucleotide. Concurrent incorporation of modified and unmodified ATP in a 1:1 ratio resulted in significantly greater numbers of above-threshold ratio calls across tissues, while preserving ratio integrity and reproducibility. Conclusions Incorporation of 2-aminoadenosine triphosphate into cRNA targets is a promising method for increasing signal detection in microarrays. Furthermore, this approach can be optimized to minimize impact on yield of amplified material and to increase the number of expression changes that can be detected.

  20. IL-1β Suppresses the Formation of Osteoclasts by Increasing OPG Production via an Autocrine Mechanism Involving Celecoxib-Related Prostaglandins in Chondrocytes

    Directory of Open Access Journals (Sweden)

    Yusuke Watanabe

    2009-01-01

    Full Text Available Elevated interleukin (IL-1 concentrations in synovial fluid have been implicated in joint bone and cartilage destruction. Previously, we showed that IL-1β stimulated the expression of prostaglandin (PG receptor EP4 via increased PGE2 production. However, the effect of IL-1β on osteoclast formation via chondrocytes is unclear. Therefore, we examined the effect of IL-1β and/or celecoxib on the expression of macrophage colony-stimulating factor (M-CSF, receptor activator of NF-κB ligand (RANKL, and osteoprotegerin (OPG in human chondrocytes, and the indirect effect of IL-1β on osteoclast-like cell formation using RAW264.7 cells. OPG and RANKL expression increased with IL-1β; whereas M-CSF expression decreased. Celecoxib blocked the stimulatory effect of IL-1β. Conditioned medium from IL-1β-treated chondrocytes decreased TRAP staining in RAW264.7 cells. These results suggest that IL-1β suppresses the formation of osteoclast-like cells via increased OPG production and decreased M-CSF production in chondrocytes, and OPG production may increase through an autocrine mechanism involving celecoxib-related PGs.

  1. A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

    Science.gov (United States)

    Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

  2. Effect molybdenum (5, 6) compounds on hydrolysis and reorganization rates of ammonium and potassium triphosphates in aqueous solutions

    International Nuclear Information System (INIS)

    Petrovskaya, L.I.; Prodan, E.A.; Lesnikovich, L.A.

    1992-01-01

    Method of thin-layer chromatography was used to investigate the kinetics of hydrolytic decomposition of triphosphate-anion in aqueous solutions of M 5 P 3 O 10 -(NH 4 ) 6 xMo 7 O 24 -H 2 O systems (M - NH 4 , pH 5.9-6.4; M - K, pH 6.5-6.8) and (NH 4 ) 5 P 3 O 10 -(NH 4 ) 2 MoOCl 5 -H 2 O system (pH 2.5 and 4.5) at 20 deg C and 0.2 mol% concnetration. It is shown that oxoions Mo 5 produce less accelerating effect on hydrolysis, as compared to Mo 6 oxoions, and are able to initiate anion reorganization with formation of tetraphosphate under certain conditions

  3. Pulsed electromagnetic fields preserve bone architecture and mechanical properties and stimulate porous implant osseointegration by promoting bone anabolism in type 1 diabetic rabbits.

    Science.gov (United States)

    Cai, J; Li, W; Sun, T; Li, X; Luo, E; Jing, D

    2018-05-01

    The effects of exogenous pulsed electromagnetic field (PEMF) stimulation on T1DM-associated osteopathy were investigated in alloxan-treated rabbits. We found that PEMF improved bone architecture, mechanical properties, and porous titanium (pTi) osseointegration by promoting bone anabolism through a canonical Wnt/β-catenin signaling-associated mechanism, and revealed the clinical potential of PEMF stimulation for the treatment of T1DM-associated bone complications. Type 1 diabetes mellitus (T1DM) is associated with deteriorated bone architecture and impaired osseous healing potential; nonetheless, effective methods for resisting T1DM-associated osteopenia/osteoporosis and promoting bone defect/fracture healing are still lacking. PEMF, as a safe and noninvasive method, have proven to be effective for promoting osteogenesis, whereas the potential effects of PEMF on T1DM osteopathy remain poorly understood. We herein investigated the effects of PEMF stimulation on bone architecture, mechanical properties, bone turnover, and its potential molecular mechanisms in alloxan-treated diabetic rabbits. We also developed novel nontoxic Ti2448 pTi implants with closer elastic modulus with natural bone and investigated the impacts of PEMF on pTi osseointegration for T1DM bone-defect repair. The deteriorations of cancellous and cortical bone architecture and tissue-level mechanical strength were attenuated by 8-week PEMF stimulation. PEMF also promoted osseointegration and stimulated more adequate bone ingrowths into the pore spaces of pTi in T1DM long-bone defects. Moreover, T1DM-associated reduction of bone formation was significantly attenuated by PEMF, whereas PEMF exerted no impacts on bone resorption. We also found PEMF-induced activation of osteoblastogenesis-related Wnt/β-catenin signaling in T1DM skeletons, but PEMF did not alter osteoclastogenesis-associated RANKL/RANK signaling gene expression. We reveal that PEMF improved bone architecture, mechanical properties, and

  4. Low-intensity laser irradiation at 660 nm stimulates transcription of genes involved in the electron transport chain.

    Science.gov (United States)

    Masha, Roland T; Houreld, Nicolette N; Abrahamse, Heidi

    2013-02-01

    Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

  5. Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast...... cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p......-trifluorometroxyphenylnydrazone), which are known to block the energy-dependent calcium uptake by isolated mitochondria. The mast cell ATP content was reduced during A23187-induced histamine release under anaerobic conditions in the presence of glucose. This indicates an increased utilization of ATP during the release process. 5...

  6. Transcriptional regulation of human RANK ligand gene expression by E2F1

    International Nuclear Information System (INIS)

    Hu Yan; Sun Meng; Nadiminty, Nagalakshmi; Lou Wei; Pinder, Elaine; Gao, Allen C.

    2008-01-01

    Receptor activator of nuclear factor kappa B ligand (RANKL) is a critical osteoclastogenic factor involved in the regulation of bone resorption, immune function, the development of mammary gland and cardiovascular system. To understand the transcriptional regulation of RANKL, we amplified and characterized a 1890 bp 5'-flanking sequence of human RANKL gene (-1782 bp to +108 bp relative to the transcription start site). Using a series of deletion mutations of the 1890 bp RANKL promoter, we identified a 72 bp region (-172 to -100 bp) mediating RANKL basal transcriptional activity. Sequence analysis revealed a putative E2F binding site within this 72 bp region in the human RANKL promoter. Overexpression of E2F1 increased RANKL promoter activity, while down-regulation of E2F1 expression by small interfering RNA decreased RANKL promoter activity. RT-PCR and enzyme linked immunosorbent assays (ELISA) further demonstrated that E2F1 induced the expression of RANKL. Electrophoretic gel mobility shift assays (EMSA) and antibody competition assays confirmed that E2F1 proteins bind to the consensus E2F binding site in the RANKL promoter. Mutation of the E2F consensus binding site in the RANKL promoter profoundly reduced the basal promoter activity and abolished the transcriptional modulation of RANKL by E2F1. These results suggest that E2F1 plays an important role in regulating RANKL transcription through binding to the E2F consensus binding site

  7. Effects of Cinnamoyloxy-mammeisin from Geopropolis on Osteoclast Differentiation and Porphyromonas gingivalis-Induced Periodontitis.

    Science.gov (United States)

    da Cunha, Marcos Guilherme; Ramos-Junior, Erivan Schnaider; Franchin, Marcelo; Taira, Thaise Mayumi; Beutler, John A; Franco, Gilson Cesar Nobre; Ikegaki, Masaharu; de Alencar, Severino Matias; Fukada, Sandra Yasuyo; Rosalen, Pedro Luiz

    2017-06-23

    Bone-loss-related diseases such as rheumatoid arthritis, osteomyelitis, osteoporosis, and periodontitis are associated with high rates of morbidity worldwide. These disorders are characterized by an imbalance between the formation and activity of osteoblasts and osteoclasts, leading to bone loss. In this context, we evaluated the effect of cinnamoyloxy-mammeisin (CNM), an anti-inflammatory coumarin found in Melipona scutellaris geopropolis, on key targets related to bone remodeling. In the present study we investigated the in vitro effects of CNM on osteoclast differentiation and M-CSF+RANKL-induced osteoclastogenic marker expression. Additionally, the interference of CNM treatment on osteoclast activity was evaluated by zymography and resorption area. Finally, we assessed the capacity of the compound to mitigate alveolar bone loss in vivo in experimental murine periodontitis induced by Porphyromonas gingivalis. We observed that treatment with CNM impaired osteoclast differentiation, as evidenced by a reduced number of tartrate-resistant acid-phosphatase-positive multinucleated cells (TRAP+) as well as the expression of osteoclastogenic markers upon M-CSF+RANKL-induced stimulation. Similarly, we observed reduced gelatinolytic and resorption capacity in M-CSF+RANKL-induced cells in vitro. Lastly, CNM attenuated alveolar bone loss in an experimental murine periodontitis model. These findings indicate that CNM may be considered a promising treatment for bone loss diseases.

  8. RANKL inhibition with denosumab does not influence 3-year progression of aortic calcification or incidence of adverse cardiovascular events in postmenopausal women with osteoporosis and high cardiovascular risk.

    Science.gov (United States)

    Samelson, Elizabeth J; Miller, Paul D; Christiansen, Claus; Daizadeh, Nadia S; Grazette, Luanda; Anthony, Mary S; Egbuna, Ogo; Wang, Andrea; Siddhanti, Suresh R; Cheung, Angela M; Franchimont, Nathalie; Kiel, Douglas P

    2014-02-01

    Atherosclerosis and osteoporosis are chronic diseases that progress with age, and studies suggest aortic calcification, an indicator of atherosclerosis, is inversely associated with bone mineral density (BMD). The osteoprotegerin (OPG)/receptor activator of NF-κB (RANK)/RANK ligand (RANKL) system has been proposed as a shared regulatory system for bone and vasculature. Denosumab (DMAb), a monoclonal antibody against RANKL, improved BMD and reduced fracture risk in the Fracture Reduction Evaluation of Denosumab in Osteoporosis Every 6 Months (FREEDOM) trial. We evaluated whether or not treatment with DMAb influenced progression of aortic calcification (AC) and incidence of cardiovascular (CV) adverse events. We included 2363 postmenopausal women with osteoporosis (1142 placebo, 1221 DMAb), selected from 7808 participants in the FREEDOM trial (3906 placebo, 3902 DMAb), at high risk of CV events according to modified Raloxifene Use for the Heart (RUTH) criteria. CV adverse events were reported by participants. AC scores were assessed using a semiquantitative method from lateral spine X-rays. Change in AC score from baseline to 12 (n = 1377), 24 (n = 1231), and 36 months (n = 1045) was calculated as AC score at follow-up minus AC score at baseline. AC progression was defined as change in AC score >0. Baseline characteristics, CV risk factors, and AC scores were similar between treatment groups. Mean age of participants was 74 years (range, 60-90), 88% were white, and 77% had AC score >0 at baseline. Frequency of AC progression over 3 years did not differ between women in placebo (22%) and DMAb (22%) groups (p = 0.98). AC progression did not differ between treatment groups when analyzed by baseline estimated glomerular filtration rate or by baseline AC scores. Frequency of CV adverse events did not differ between placebo (40%) and DMAb (38%) groups (p = 0.26). In conclusion, DMAb treatment had no effect on progression of AC or incidence of CV

  9. In vivo effects of adenosine 5´-triphosphate on rat preneoplastic liver

    Directory of Open Access Journals (Sweden)

    Ana V. Frontini

    2011-04-01

    Full Text Available The utilization of adenosine 5´-triphosphate (ATP infusions to inhibit the growth of some human and animals tumors was based on the anticancer activity observed in in vitro and in vivo experiments, but contradictory results make the use of ATP in clinical practice rather controversial. Moreover, there is no literature regarding the use of ATP infusions to treat hepatocarcinomas. The purpose of this study was to investigate whether ATP prevents in vivo oncogenesis in very-early-stage cancer cells in a well characterized two-stage model of hepatocarcinogenesis in the rat. As we could not preclude the possible effect due to the intrinsic properties of adenosine, a known tumorigenic product of ATP hydrolysis, the effect of the administration of adenosine was also studied. Animals were divided in groups: rats submitted to the two stage preneoplasia initiation/promotion model of hepatocarcinogenesis, rats treated with intraperitoneal ATP or adenosine during the two phases of the model and appropriate control groups. The number and volume of preneoplastic foci per liver identified by the expression of glutathione S-transferase placental type and the number of proliferating nuclear antigen positive cells significantly increased in ATP and adenosine treated groups. Taken together, these results indicate that in this preneoplastic liver model, ATP as well as adenosine disturb the balance between apoptosis and proliferation contributing to malignant transformation.

  10. Particulate matter in cigarette smoke increases ciliary axoneme beating through mechanical stimulation.

    Science.gov (United States)

    Navarrette, Chelsea R; Sisson, Joseph H; Nance, Elizabeth; Allen-Gipson, Diane; Hanes, Justin; Wyatt, Todd A

    2012-06-01

    The lung's ability to trap and clear foreign particles via the mucociliary elevator is an important mechanism for protecting the lung against respirable irritants and microorganisms. Although cigarette smoke (CS) exposure and particulate inhalation are known to alter mucociliary clearance, little is known about how CS and nanoparticles (NPs) modify cilia beating at the cytoskeletal infrastructure, or axonemal, level. We used a cell-free model to introduce cigarette smoke extract (CSE) and NPs with variant size and surface chemistry to isolated axonemes and measured changes in ciliary motility. We hypothesized that CSE would alter cilia beating and that alterations in ciliary beat frequency (CBF) due to particulate matter would be size- and surface chemistry-dependent. Demembranated axonemes were isolated from ciliated bovine tracheas and exposed to adenosine triphosphate (ATP) to initiate motility. CBF was measured in response to 5% CSE, CSE filtrate, and carboxyl-modified (COOH), sulphate (SO(4))-modified (sulfonated), or PEG-coated polystyrene (PS) latex NPs ranging in size from 40 nm to 500 nm. CSE concentrations as low as 5% resulted in rapid, significant stimulation of CBF (pIntroduction of sulphate-modified PS beads ~300 nm in diameter resulted in a similar increase in CBF above baseline ATP levels. Uncharged, PEG-coated beads had no effect on CBF regardless of size. Similarly, COOH-coated particles less than 200 nm in diameter did not alter ciliary motility. However, COOH-coated PS particles larger than 300 nm increased CBF significantly and increased the number of motile points. These data show that NPs, including those found in CSE, mechanically stimulate axonemes in a size- and surface chemistry-dependent manner. Alterations in ciliary motility due to physicochemical properties of NPs may be important for inhalational lung injury and efficient drug delivery of respirable particles.

  11. Adsorption characteristics of 14C-labeled alanine, aspartic acid and adenosine triphosphate by metal-chelating resins

    International Nuclear Information System (INIS)

    Ishiyama, Toshio; Matsunami, Tadao; Shibata, Setsuko; Honda, Yoshihide.

    1987-01-01

    (1) Adsorption properties of 14 C-alanine, 14 C-ATP (adenosine triphosphate) and 14 C-aspartic acid on the metal-chelating resins were determined and found that the Cu(II)-Chelex 100 and Fe(III)-Unicellex UR10, Fe(III)-Chelex 100 chelating resins were highly effective for the adsorption of 14 C-alanine and 14 C-ATP, respectively. (2) Desorption rate of 14 C-ATP from the Fe(III)-Unicellex UR10 and Fe(III)-Chelex 100 resins was somewhat higher than the case of 14 C-alanine, probably because the coordination bonds of Cu-alanine might be stronger than those of Fe-ATP. Thus, 14 C-labeled organic compounds such as 14 C-alanine and 14 C-ATP of a low activity concentration (3.7 mBq/ml) (1 x 10 -7 μCi/ml) in aqueous solution may be measured with liquid scintillation counter after pre-concentration by use of the Fe(III)- and Cu(II)-chelating resin columns. (author)

  12. Cord blood-derived macrophage-lineage cells rapidly stimulate osteoblastic maturation in mesenchymal stem cells in a glycoprotein-130 dependent manner.

    Directory of Open Access Journals (Sweden)

    Tania J Fernandes

    Full Text Available In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM, and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2 actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells

  13. Visual and light scattering spectrometric method for the detection of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles

    Science.gov (United States)

    Liang, Lijiao; Zhen, Shujun; Huang, Chengzhi

    2017-02-01

    A highly selective method was presented for colorimetric determination of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

  14. Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

    International Nuclear Information System (INIS)

    Hamel, E.; Batra, J.K.; Lin, C.M.

    1986-01-01

    Using highly purified calf brain tubulin bearing [8- 14 C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins, the authors have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg 2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly

  15. The clinical value of adenosine triphosphate stress myocardial perfusion tomography for detecting coronary artery disease

    International Nuclear Information System (INIS)

    Yao Zhiming; He Qing; Qu Wanying; Yu Xue; Han Lijun; Yu Zhiguo; Li Wei; Zeng Xuezhai; Zhu Ming; Zhao Hongshan

    2002-01-01

    Objective: To study the clinical value of adenosine triphosphate stress myocardial perfusion tomography imaging (ATP-MPI) in detection of coronary artery disease (CAD). Methods: There were 278 patients underwent ATP-MPI, 51 patients of them also underwent coronary angiography (CAG). Seventy-three patients underwent stress-rest myocardial perfusion tomography imaging with multi-stage submaximal exercise test (ST-MPI) and CAG serving as control group. Results: 1) Side effects: there were 11 different symptoms and atrioventricular conduction block (10 patients), sinoatrial conduction block (2 patients) occurred during ATP stress. Allopathy or interruption of ATP stress did not happen. 2) The sensitivity and specificity of ATP-MPI in detection of CAD were 97.1% and 82.4%, respectively, and those in detection of ≥50% narrowing coronary artery were 91.0% and 94.7%, respectively. 3) In patients without myocardial infarction, the sensitivity and specificity of ATP-MPI in detection of myocardial ischemia were comparable to those of ST-MPI. Conclusion: ATP-MPI is an accurate, safe modality and is comparable to ST-MPI in the detection of CAD

  16. The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.

    Directory of Open Access Journals (Sweden)

    Carolin Massalski

    Full Text Available The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+, Mg(2+ and Mn(2+ were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in

  17. Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona.

    Science.gov (United States)

    Zhang, Deqing; Gaji, Rajshekhar Y; Howe, Daniel K

    2006-09-01

    A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.

  18. A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

    DEFF Research Database (Denmark)

    Henriksen, Kim; Andreassen, Kim V; Thudium, Christian S

    2012-01-01

    Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium wa...... dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.......Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium...... release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed...

  19. Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

    Science.gov (United States)

    Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

    2010-07-01

    Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

  20. Therapeutic effects of radix dipsaci, pyrola herb, and cynomorium songaricum on bone metabolism of ovariectomized rats

    Directory of Open Access Journals (Sweden)

    Liu Meijie

    2012-05-01

    Full Text Available Abstract Background The objective of this study was to evaluate the effects of herbal medicines, such as Radix Dipsaci (RDD, Pyrola Herb (PHD, and Cynomorium songaricum decoction (CSD, on osteoporotic rats induced by ovariectomy (OVX. Methods OVX or sham operations were performed on 69 virgin Wistar rats that were divided into six groups: sham (sham, n = 12, OVX control group (OVX, n = 12, and OVX rats with treatments (diethylstilbestrol, E2, n = 12; RDD, n = 11, PHD, n = 11, and CSD, n = 11. Non-surgical rats served as normal control (NC, n = 12. The treatments began four weeks after surgery and lasted for 12 weeks. Bone mass and bone turnover were analyzed by histomorphometry. Levels of protein expression and mRNA of OPG and RANKL in osteoblasts (OB and bone marrow stromal cells (bMSC were evaluated by immunohistochemistry and in situ hybridization. Results Compared to NC and sham rats, trabecular bone formation was significantly reduced in OVX rats, but restored in E2-treated rats. Treatment with either RDD or PHD enhanced trabecular bone formation remarkably. No significant change of bone formation was observed in CSD-treated rats. OPG expression of protein and mRNA was reduced significantly in OB and bMSC of OVX control rats. RANKL expression of protein and mRNA was increased significantly in OB and bMSC of OVX control rats. These effects were substantially reversed (increased in OPG and decreased in RANKL by treatment with E2, RDD, or PHD in OB and bMSC of OVX rats. No significant changes in either OPG or RANKL expression were observed in OB and bMSC of OVX rats treated with CSD. Conclusions Our study showed that RDD and PHD increased bone formation by stimulating overexpression of OPG and downregulation of RANKL in OB and bMSC. This suggests that RDD and PHD may be used as alternative therapeutic agents for postmenopausal osteoporosis.

  1. Wedelolactone enhances osteoblastogenesis by regulating Wnt/β-catenin signaling pathway but suppresses osteoclastogenesis by NF-κB/c-fos/NFATc1 pathway.

    Science.gov (United States)

    Liu, Yan-Qiu; Hong, Zhi-Lai; Zhan, Li-Bin; Chu, Hui-Ying; Zhang, Xiao-Zhe; Li, Guo-Hui

    2016-08-25

    Bone homeostasis is maintained by formation and destruction of bone, which are two processes tightly coupled and controlled. Targeting both stimulation on bone formation and suppression on bone resorption becomes a promising strategy for treating osteoporosis. In this study, we examined the effect of wedelolactone, a natural product from Ecliptae herba, on osteoblastogenesis as well as osteoclastogenesis. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone stimulated osteoblast differentiation and bone mineralization. At the molecular level, wedelolactone directly inhibited GSK3β activity and enhanced the phosphorylation of GSK3β, thereafter stimulated the nuclear translocation of β-catenin and runx2. The expression of osteoblastogenesis-related marker gene including osteorix, osteocalcin and runx2 increased. At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. Further, wedelolactone blocked NF-kB/p65 phosphorylation and abrogated the NFATc1 nuclear translocation. As a result, osteoclastogenesis-related marker gene expression decreased, including c-src, c-fos, and cathepsin K. In ovariectomized mice, administration of wedelolactone prevented ovariectomy-induced bone loss by enhancing osteoblast activity and inhibiting osteoclast activity. Together, these data demonstrated that wedelolactone facilitated osteoblastogenesis through Wnt/GSK3β/β-catenin signaling pathway and suppressed RANKL-induced osteoclastogenesis through NF-κB/c-fos/NFATc1 pathway. These results suggested that wedelolacone could be a novel dual functional therapeutic agent for osteoporosis.

  2. Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs.

    Directory of Open Access Journals (Sweden)

    Raphael De Souza Vasconcellos

    2014-11-01

    Full Text Available Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection.We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis.In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of

  3. Leishmania infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase-2 is an Apyrase Involved in Macrophage Infection and Expressed in Infected Dogs

    Science.gov (United States)

    Vasconcellos, Raphael De Souza; Mariotini-Moura, Christiane; Gomes, Rodrigo Saar; Serafim, Tiago Donatelli; Firmino, Rafaela de Cássia; Silva e Bastos, Matheus; de Castro, Felipe Freitas; de Oliveira, Claudia Miranda; Borges-Pereira, Lucas; de Souza, Anna Cláudia Alves; de Souza, Ronny Francisco; Gómez, Gabriel Andres Tafur; Pinheiro, Aimara da Costa; Maciel, Talles Eduardo Ferreira; Silva-Júnior, Abelardo; Bressan, Gustavo Costa; Almeida, Márcia Rogéria; Baqui, Munira Muhammad Abdel; Afonso, Luís Carlos Crocco; Fietto, Juliana Lopes Rangel

    2014-01-01

    Background Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. Methodology/Principal Findings We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. Conclusions/Significance In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in

  4. Recombinant Human Endostatin Suppresses Mouse Osteoclast Formation by Inhibiting the NF-κB and MAPKs Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Non eChen

    2016-06-01

    Full Text Available Rheumatoid arthritis is an autoimmune disease characterized by synovial hyperplasia and progressive joint destruction. As reported previously, recombinant human endostatin (rhEndostatin is associated with inhibition of joint bone destruction present in rat adjuvant-induced arthritis; however, the effect of rhEndostatin on bone destruction is not known. This study was designed to assess the inhibitory effect and mechanisms of rhEndostatin on formation and function of osteoclasts in vitro, and to gain insight into the mechanism underlying the inhibitory effect of bone destruction. Bone marrow-derived macrophages isolated from BALB/c mice were stimulated with receptor activator of NF-κB ligand (RANKL and macrophage colony-stimulating factor to establish osteoclast formation. Osteoclast formation was determined by TRAP staining. Cell viability of BMMs affected by rhEndostatin was determined using a MTT assay. Bone resorption was examined with a bone resorption pits assay. The expression of osteoclast-specific markers was analyzed using quantitative real-time PCR. The related signaling pathways were examined using a Luciferase reporter assay and western blot analysis. Indeed, rhEndostatin showed a significant reduction in the number of osteoclast-like cells and early-stage bone resorption. Moreover, molecular analysis demonstrated that rhEndostatin attenuated RANKL-induced NF-κB signaling by inhibiting the phosphorylation of IκBα and NF-κB p65 nuclear translocation. Furthermore, rhEndostatin significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases (MAPKs, such as ERK1/2, JNK, and p38. Hence, we demonstrated for the first time that preventing the formation and function of osteoclasts is an important anti-bone destruction mechanism of rhEndostatin, which might be useful in the prevention and treatment of bone destruction in RA.

  5. Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

    International Nuclear Information System (INIS)

    Hiruma, Yoshiharu; Hirai, Takehiro; Tsuda, Eisuke

    2011-01-01

    Highlights: → Siglec-15 was identified as a gene overexpressed in giant cell tumor. → Siglec-15 mRNA expression increased in association with osteoclast differentiation. → Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D 3 and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

  6. Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Hiruma, Yoshiharu, E-mail: hiruma.yoshiharu.hy@daiichisankyo.co.jp [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Hirai, Takehiro [Translational Medicine and Clinical Pharmacology Department, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Tsuda, Eisuke [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan)

    2011-06-10

    Highlights: {yields} Siglec-15 was identified as a gene overexpressed in giant cell tumor. {yields} Siglec-15 mRNA expression increased in association with osteoclast differentiation. {yields} Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor {kappa}B ligand (RANKL) or tumor necrosis factor (TNF)-{alpha}. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D{sub 3} and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

  7. An Escherichia coli strain deficient for both exonuclease 5 and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Estevenon, A.M.; Kooistra, J.; Sicard, N.

    1995-01-01

    An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity

  8. Conditional deletion of CD98hc inhibits osteoclast development

    Directory of Open Access Journals (Sweden)

    Hideki Tsumura

    2016-03-01

    Full Text Available The CD98 heavy chain (CD98hc regulates virus-induced cell fusion and monocyte fusion, and is involved in amino acid transportation. Here, we examined the role that CD98hc plays in the formation of osteoclasts using CD98hcflox/floxLysM-cre peritoneal macrophages (CD98hc-defect macrophages. Peritoneal macrophages were stimulated with co-cultured with osteoblasts in the presence of 1,25(OH2 vitamin D3, and thereafter stained with tartrate-resistant acid phosphatase staining solution. The multinucleated osteoclast formation was severely impaired in the peritoneal macrophages isolated from the CD98hc-defect mice compared with those from wild-type mice. CD98hc mediates integrin signaling and amino acid transport through the CD98 light chain (CD98lc. In integrin signaling, suppression of the M-CSF-RANKL-induced phosphorylation of ERK, Akt, JNK and p130Cas were observed at the triggering phase in the CD98h-defect peritoneal macrophages. Moreover, we showed that the general control non-derepressible (GCN pathway, which was activated by amino acid starvation, was induced by the CD98hc-defect peritoneal macrophages stimulated with RANKL. These results indicate that CD98 plays two important roles in osteoclast formation through integrin signaling and amino acid transport.

  9. Influence of morphology and topography on potentiometric response of magnesium and calcium sensitive PEDOT films doped with adenosine triphosphate (ATP)

    International Nuclear Information System (INIS)

    Paczosa-Bator, B.; Peltonen, J.; Bobacka, J.; Lewenstam, A.

    2006-01-01

    Poly(3,4-ethylenedioxythiophene) (PEDOT) films doped with adenosine triphosphate (ATP) are used to study the biologically relevant competitive magnesium and calcium ion-exchange at ATP membrane sites. It is shown, by atomic force microscopy (AFM) and scanning electron microscopy (SEM), that the surface topography and morphology of the PEDOT-ATP films determines the quality of their potentiometric response. More smooth and less rough films result in better potentiometric characteristics, particularly in a faster response. The topography/morphology of the PEDOT-ATP films is influenced by conditions during electrodeposition (electrochemical method of deposition, pH, concentration of electrolytes) and post-deposition soaking (including net-time of soaking), as evidenced by X-ray photoelectron spectroscopy (XPS) and energy dispersive analysis of X-rays (EDAX)

  10. Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM

    DEFF Research Database (Denmark)

    Celebioglu, Hasan Ufuk; Olesen, Sita Vaag; Prehn, Kennie

    2017-01-01

    .2-2.1 fold reduced relative abundance comprised elongation factor G, phosphoglycerate kinase, BipAEFTU family GTP-binding protein, ribonucleoside triphosphate reductase, adenylosuccinate synthetase, 30S ribosomal protein S1, and manganese-dependent inorganic pyrophosphatase. Surface proteome of cellobiose...

  11. Heterogeneity of tumor chemosensitivity in ovarian epithelial cancer revealed using the adenosine triphosphate-tumor chemosensitivity assay.

    Science.gov (United States)

    Zhang, Jin; Li, Hongxia

    2015-05-01

    Ovarian cancer has a poor prognosis, primarily due to the heterogeneity in chemosensitivity among patients. In the present study, this heterogeneity was evaluated in ovarian epithelial cancer (OEC) using an in vitro adenosine triphosphate tumor chemosensitivity assay (ATP-TCA). Specimens were collected from 80 patients who underwent cytoreductive surgery. Viable ovarian cancer cells obtained from malignant tissues were tested for sensitivity to paclitaxel (PTX), carboplatin (CBP), topotecan (TPT), gemcitabine (GEM), docetaxel (TXT), etoposide, bleomycin and 4-hydroperoxycyclophosphamide using ATP-TCA. The sensitivity, specificity, positive predictive value and negative predictive value for the clinical chemotherapy sensitivity of OEC were 88.6, 77.8, 83 and 84.8%, respectively. PTX demonstrated the highest sensitivity of all agents tested (82.5% in all specimens, 85.7% in recurrent specimens), followed by CBP (58.8 and 60.7%, respectively). The sensitivities to PTX and docetaxel (PIII) or low-differentiated specimens, respectively. The present study indicated that ATP-TCA is an effective method for guiding the choice of chemotherapy drugs. Notable heterogeneity of chemosensitivity was observed in the OEC specimens.

  12. Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

    Science.gov (United States)

    Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

    2014-03-01

    Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

  13. Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA

    International Nuclear Information System (INIS)

    Evans, R.K.; Haley, B.E.

    1987-01-01

    A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N 3 dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N 3 dUTP is the nitration of dUMP in 98% yield in 5 min at 25 0 C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH 2 dUMP). Diazotization of 5-NH 2 dUMP with HNO 2 followed by the addition NaN 3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N 3 dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N 3 dUTP through a chemical coupling of pyrophosphate to 5-N 3 dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N 3 UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [γ- 32 P]-5-N 3 dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing γ- 32 P-labeled pyrimidine nucleotides was developed. [γ- 32 P]-5-N 3 dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP. The photoactivity of 5-N 3 dUMP is stable to extremes of pH, and [γ- 32 P]-5-N 3 dUTP was an effective photolabeling reagent even in the presence of 10 mM dithiothreitol

  14. Brain Stimulation Therapies

    Science.gov (United States)

    ... Magnetic Seizure Therapy Deep Brain Stimulation Additional Resources Brain Stimulation Therapies Overview Brain stimulation therapies can play ... for a shorter recovery time than ECT Deep Brain Stimulation Deep brain stimulation (DBS) was first developed ...

  15. Computationally Developed Sham Stimulation Protocol for Multichannel Desynchronizing Stimulation

    Directory of Open Access Journals (Sweden)

    Magteld Zeitler

    2018-05-01

    Full Text Available A characteristic pattern of abnormal brain activity is abnormally strong neuronal synchronization, as found in several brain disorders, such as tinnitus, Parkinson's disease, and epilepsy. As observed in several diseases, different therapeutic interventions may induce a placebo effect that may be strong and hinder reliable clinical evaluations. Hence, to distinguish between specific, neuromodulation-induced effects and unspecific, placebo effects, it is important to mimic the therapeutic procedure as precisely as possibly, thereby providing controls that actually lack specific effects. Coordinated Reset (CR stimulation has been developed to specifically counteract abnormally strong synchronization by desynchronization. CR is a spatio-temporally patterned multichannel stimulation which reduces the extent of coincident neuronal activity and aims at an anti-kindling, i.e., an unlearning of both synaptic connectivity and neuronal synchrony. Apart from acute desynchronizing effects, CR may cause sustained, long-lasting desynchronizing effects, as already demonstrated in pre-clinical and clinical proof of concept studies. In this computational study, we set out to computationally develop a sham stimulation protocol for multichannel desynchronizing stimulation. To this end, we compare acute effects and long-lasting effects of six different spatio-temporally patterned stimulation protocols, including three variants of CR, using a no-stimulation condition as additional control. This is to provide an inventory of different stimulation algorithms with similar fundamental stimulation parameters (e.g., mean stimulation rates but qualitatively different acute and/or long-lasting effects. Stimulation protocols sharing basic parameters, but inducing nevertheless completely different or even no acute effects and/or after-effects, might serve as controls to validate the specific effects of particular desynchronizing protocols such as CR. In particular, based on

  16. Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

    Directory of Open Access Journals (Sweden)

    Carmen Clapp

    2011-07-01

    Full Text Available Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK. Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS, as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

  17. Effects of 5 Thio-D-Glucose on cellular adenosine triphosphate levels and deoxyribonucleic acid rejoining in hypoxic and aerobic Chinese hamster cells

    International Nuclear Information System (INIS)

    Nagle, W.A.; Moss, A.J. Jr.; Roberts, H.G. Jr.; Baker, M.L.

    1980-01-01

    Intracellular adenosine triphosphate (ATP) levels were measured in both hypoxic and aerobic cultures of V79 Chinese hamster cells treated with 5-thio-D-glucose (5-SH-D-Glc). This glucose analog, a known inhibitor of D-glucose transport and metabolism, reduced ATP in cell cultures allowed to become hypoxic by cell metabolism, but not in aerobic cultures treated similarly. Cells depleted of ATP were unable to rejoin x-ray induced deoxyribonucleic acid (DNA) strand breaks as measured by the alkaline sucrose gradient sedimentation technique. The inference for radiation therapy is that inhibition of glucose metabolism selectively depletes energy reserves in hypoxic cells, rendering these cells more radiosensitive and leading to a more effective tumor treatment

  18. The potential of chitosan combined with chicken shank collagen as scaffold on bone defect regeneration process in Rattus norvegicus

    Directory of Open Access Journals (Sweden)

    Fitria Rahmitasari

    2016-12-01

    Full Text Available Background: In the field of dentistry, alveolar bone damage can be caused by periodontal disease, traumatic injury due to tooth extraction, cyst enucleation, and tumor surgery. One of the ways to regenerate the bone defect is using graft scaffold. Thus, combination of chitosan and collagen can stimulate osteogenesis. Purpose: The aim of this study was to examine the potential of chitosan combined with chicken shank collagen on bone defect regeneration process. Method: Twelve Rattus norvegicus were prepared as animal models in this research. A bone defect was intentionally created at both of the right and left femoral bones of the models. Next, 24 samples were divided into four groups, namely Group 1 using chitosan – collagen scaffold (50:50, Group 2 using chitosan collagen-scaffold (80:20, Group 3 using chitosan scaffold only, and Control Group using 3% CMC-Na. On 14th day, those animals were sacrificed, and histopathological anatomy examination was conducted to observe osteoclast cells. In addition, immunohistochemistry examination was also performed to observe RANKL expressions. Result: There was a significant difference in RANKL expressions among the groups, except between Group 3 using chitosan scaffold only and control group (p value > 0.05. The highest expression of RANKL was found in Group 1 with chitosan – collagen scaffold (50:50, followed by Group 2 with chitosan-collagen scaffold (80:20. Moreover, there was also a significant difference in osteoclast generation, except between Group 1 using chitosan – collagen scaffold (50:50 and Group 2 using chitosan-collagen scaffold (80:20, p value 0.05. Less osteoclast was found in the groups using chitosan – collagen scaffold (Group 1 and Group 2. Conclusion: Combination of chitosan and chicken shank collagen scaffold can improve regeneration process of bone defect in Rattus novergicus animals through increasing of RANKL expressions, and decreasing of osteoclast.

  19. CaMKII Regulation of Cardiac Ryanodine Receptors and Inositol Triphosphate Receptors

    Directory of Open Access Journals (Sweden)

    Emmanuel eCamors

    2014-05-01

    Full Text Available Ryanodine receptors (RyRs and inositol triphosphate receptors (InsP3Rs are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca2+ signals, triggering muscle contraction and oscillatory Ca2+ waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR, and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and posttranslational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

  20. β-cryptoxanthin regulates bone resorption related-cytokine production in human periodontal ligament cells.

    Science.gov (United States)

    Nishigaki, Masaru; Yamamoto, Toshiro; Ichioka, Hiroaki; Honjo, Ken-Ichi; Yamamoto, Kenta; Oseko, Fumishige; Kita, Masakazu; Mazda, Osam; Kanamura, Narisato

    2013-07-01

    β-cryptoxanthin (β-cry) is a type of carotenoid found in certain fruits and vegetables. Although it has been shown that β-cry inhibits alveolar bone resorption, the molecular mechanisms for this have not yet been clarified. In the present study, we investigated the effects of β-cry on bone resorption related-cytokine production in human periodontal ligament (hPDL) cells. hPDL cells were stimulated with β-cry (1×10(-7)mol/l), mechanical stress (1 or 6MPa), and P. gingivalis. The production of interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by RT-PCR and ELISA. The production of IL-1β, IL-6, IL-8, and TNF-α was not induced in hPDL cells after stimulation with β-cry, although these cytokines were produced after stimulation with P. gingivalis. On the other hand, IL-6 and IL-8 were produced after exposure to 6MPa of mechanical stress. The production of IL-6 and IL-8 was significantly decreased by the addition of β-cry. Furthermore, β-cry up-regulated the production of OPG, but not RANKL. β-cry inhibited the production of IL-6 and IL-8 induced by mechanical stress and periodontopathogenic bacteria in hPDL cells. Moreover, β-cry up-regulated OPG production. These results suggest that β-cry may prevent bone resorption in periodontitis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Aisha, M.D. [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); Nor-Ashikin, M.N.K. [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); DDH, Universiti Teknologi MARA, ShahAlam 40450, Selangor (Malaysia); Sharaniza, A.B.R. [DDH, Universiti Teknologi MARA, ShahAlam 40450, Selangor (Malaysia); Nawawi, H. [Center for Pathology Diagnostic and Research Laboratories, Clinical Training Center, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); I-PPerForM, Universiti Teknologi MARA, Selayang 47000 Selangor (Malaysia); Froemming, G.R.A., E-mail: gabriele@salam.uitm.edu.my [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); I-PPerForM, Universiti Teknologi MARA, Selayang 47000 Selangor (Malaysia)

    2015-09-10

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. - Highlights: • OFS stress transmits anabolic signals to osteoblasts. • Actin and tubulin fibers are rearranged under OFS stress. • OFS stress increases mitochondrial metabolism and proliferation. • Reduced RANKL/OPG ratio in response to OFS inhibits osteoclastogenesis. • OFS stress prevents apoptosis and stimulates ALP and OCN.

  2. Towards a Switched-Capacitor Based Stimulator for Efficient Deep-Brain Stimulation

    Science.gov (United States)

    Vidal, Jose; Ghovanloo, Maysam

    2013-01-01

    We have developed a novel 4-channel prototype stimulation circuit for implantable neurological stimulators (INS). This Switched-Capacitor based Stimulator (SCS) aims to utilize charge storage and charge injection techniques to take advantage of both the efficiency of conventional voltage-controlled stimulators (VCS) and the safety and controllability of current-controlled stimulators (CCS). The discrete SCS prototype offers fine control over stimulation parameters such as voltage, current, pulse width, frequency, and active electrode channel via a LabVIEW graphical user interface (GUI) when connected to a PC through USB. Furthermore, the prototype utilizes a floating current sensor to provide charge-balanced biphasic stimulation and ensure safety. The stimulator was analyzed using an electrode-electrolyte interface (EEI) model as well as with a pair of pacing electrodes in saline. The primary motivation of this research is to test the feasibility and functionality of a safe, effective, and power-efficient switched-capacitor based stimulator for use in Deep Brain Stimulation. PMID:21095987

  3. RANK and RANK ligand expression in primary human osteosarcoma

    Directory of Open Access Journals (Sweden)

    Daniel Branstetter

    2015-09-01

    Our results demonstrate RANKL expression was observed in the tumor element in 68% of human OS using IHC. However, the staining intensity was relatively low and only 37% (29/79 of samples exhibited≥10% RANKL positive tumor cells. RANK expression was not observed in OS tumor cells. In contrast, RANK expression was clearly observed in other cells within OS samples, including the myeloid osteoclast precursor compartment, osteoclasts and in giant osteoclast cells. The intensity and frequency of RANKL and RANK staining in OS samples were substantially less than that observed in GCTB samples. The observation that RANKL is expressed in OS cells themselves suggests that these tumors may mediate an osteoclastic response, and anti-RANKL therapy may potentially be protective against bone pathologies in OS. However, the absence of RANK expression in primary human OS cells suggests that any autocrine RANKL/RANK signaling in human OS tumor cells is not operative, and anti-RANKL therapy would not directly affect the tumor.

  4. Ultrasound stimulation on bone healing. The optimization of stimulation time

    International Nuclear Information System (INIS)

    Rosim, R.C.; Paulin, J.B.P.; Goncalves, R.P.

    1990-01-01

    Previous works in ultrasonic simulation of bone healing dealt with parameters optimization. Albertin (1983) studied the stimulation time and found forty minutes as ideal. However, this stimulation time was the largest one employed and remained some doubt about the most appropriated value. 30, 40, 50 and 60 minutes of stimulation time were selected, while others parameters were held constant with: pulse width in 200 μs, repetition rate in 1000 pulses per second and amplitude in 30 V. Partial incomplete transverse osteotomies were done in the middle third of radio in the right forearm of rabbits. Twenty four animals divided in four subgroups, with 6 animals each were stimulated. The daily stimulation time for each subgroup was 30, 40, 50 and minutes respectively, during 15 consecutive days. The stimulation procedure started 24 hours after surgery. After the stimulation period, radiological, histological and morphometric evaluations were done and greater bone healing was found for the 50 minutes stimulation subgroup, in them new bone was also prominent. (author)

  5. Inositol phosphates influence the membrane bound Ca2+/Mg2+ stimulated ATPase from human erythrocyte membranes

    International Nuclear Information System (INIS)

    Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

    1986-01-01

    The modulation by exogenous inositol phosphates of the membrane Ca 2+ /Mg 2+ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl 2 , 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na 2 ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl 2 and EGTA. The ATPase assay was linear with time at 44 0 C. The inositol phosphates were commercially obtained and were also prepared from 32 P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP 3 ) elevated the Ca 2+ /Mg 2+ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na + /K + -ATPase and a Mg 2+ ATPase were not effected by IP 3 . Ca 2+ /Mg 2+ APTase activity with IP 2 or IP 3 could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP 3 was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP 3 stimulated Ca 2+ /Mg 2+ ATPase

  6. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  7. Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

    International Nuclear Information System (INIS)

    Franco, Gilson C.N.; Kajiya, Mikihito; Nakanishi, Tadashi; Ohta, Kouji; Rosalen, Pedro L.; Groppo, Francisco C.; Ernst, Cory W.O.; Boyesen, Janie L.; Bartlett, John D.; Stashenko, Philip; Taubman, Martin A.; Kawai, Toshihisa

    2011-01-01

    Tetracycline antibiotics, including doxycycli/e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.

  8. Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Franco, Gilson C.N. [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Pharmacology, FOP/UNICAMP, Piracicaba, SP (Brazil); Kajiya, Mikihito [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA (United States); Nakanishi, Tadashi [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Ohta, Kouji [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA (United States); Rosalen, Pedro L.; Groppo, Francisco C. [Department of Pharmacology, FOP/UNICAMP, Piracicaba, SP (Brazil); Ernst, Cory W.O.; Boyesen, Janie L. [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Bartlett, John D.; Stashenko, Philip [Department of Cytokine Biology, Forsyth Institute, Cambridge, MA (United States); Taubman, Martin A. [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Kawai, Toshihisa, E-mail: tkawai@forsyth.org [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA (United States)

    2011-06-10

    Tetracycline antibiotics, including doxycycli/e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.

  9. Nasal solitary chemoreceptor cell responses to bitter and trigeminal stimulants in vitro.

    Science.gov (United States)

    Gulbransen, Brian D; Clapp, Tod R; Finger, Thomas E; Kinnamon, Sue C

    2008-06-01

    Nasal trigeminal chemosensitivity in mice and rats is mediated in part by epithelial solitary chemoreceptor (chemosensory) cells (SCCs), but the exact role of these cells in chemoreception is unclear. Histological evidence suggests that SCCs express elements of the bitter taste transduction pathway including T2R (bitter taste) receptors, the G protein alpha-gustducin, PLCbeta2, and TRPM5, leading to speculation that SCCs are the receptor cells that mediate trigeminal nerve responses to bitter taste receptor ligands. To test this hypothesis, we used calcium imaging to determine whether SCCs respond to classic bitter-tasting or trigeminal stimulants. SCCs from the anterior nasal cavity were isolated from transgenic mice in which green fluorescent protein (GFP) expression was driven by either TRPM5 or gustducin. Isolated cells were exposed to a variety of test stimuli to determine which substances caused an increase in intracellular Ca2+ ([Ca2+]i). GFP-positive cells respond with increased [Ca2+]i to the bitter receptor ligand denatonium and this response is blocked by the PLC inhibitor U73122. In addition, GFP+ cells respond to the neuromodulators adenosine 5'-triphosphate and acetylcholine but only very rarely to other bitter-tasting or trigeminal stimuli. Our results demonstrate that TRPM5- and gustducin-expressing nasal SCCs respond to the T2R agonist denatonium via a PLC-coupled transduction cascade typical of T2Rs in the taste system.

  10. Cloning and bacterial expression of adenosine-5'-triphosphate sulfurylase from the enteric protozoan parasite Entamoeba histolytica.

    Science.gov (United States)

    Nozaki, T; Arase, T; Shigeta, Y; Asai, T; Leustek, T; Takeuchi, T

    1998-12-08

    A gene encoding adenosine-5'-triphosphate sulfurylase (AS) was cloned from the enteric protozoan parasite Entamoeba histolytica by polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the protein from a variety of organisms. The deduced amino acid sequence of E. histolytica AS revealed a calculated molecular mass of 47925 Da and an unusual basic pI of 9.38. The amebic protein sequence showed 23-48% identities with AS from bacteria, yeasts, fungi, plants, and animals with the highest identities being to Synechocystis sp. and Bacillus subtilis (48 and 44%, respectively). Four conserved blocks including putative sulfate-binding and phosphate-binding regions were highly conserved in the E. histolytica AS. The upstream region of the AS gene contained three conserved elements reported for other E. histolytica genes. A recombinant E. histolytica AS revealed enzymatic activity, measured in both the forward and reverse directions. Expression of the E. histolytica AS complemented cysteine auxotrophy of the AS-deficient Escherichia coli strains. Genomic hybridization revealed that the AS gene exists as a single copy gene. In the literature, this is the first description of an AS gene in Protozoa.

  11. Oral sucrose for heel lance increases adenosine triphosphate use and oxidative stress in preterm neonates.

    Science.gov (United States)

    Asmerom, Yayesh; Slater, Laurel; Boskovic, Danilo S; Bahjri, Khaled; Holden, Megan S; Phillips, Raylene; Deming, Douglas; Ashwal, Stephen; Fayard, Elba; Angeles, Danilyn M

    2013-07-01

    To examine the effects of sucrose on pain and biochemical markers of adenosine triphosphate (ATP) degradation and oxidative stress in preterm neonates experiencing a clinically required heel lance. Preterm neonates that met study criteria (n = 131) were randomized into 3 groups: (1) control; (2) heel lance treated with placebo and non-nutritive sucking; and (3) heel lance treated with sucrose and non-nutritive sucking. Plasma markers of ATP degradation (hypoxanthine, xanthine, and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured with the Premature Infant Pain Profile. Data were analyzed by the use of repeated-measures ANOVA and Spearman rho. We found significant increases in plasma hypoxanthine and uric acid over time in neonates who received sucrose. We also found a significant negative correlation between pain scores and plasma allantoin concentration in a subgroup of neonates who received sucrose. A single dose of oral sucrose, given before heel lance, significantly increased ATP use and oxidative stress in premature neonates. Because neonates are given multiple doses of sucrose per day, randomized trials are needed to examine the effects of repeated sucrose administration on ATP degradation, oxidative stress, and cell injury. Copyright © 2013 Mosby, Inc. All rights reserved.

  12. Prolonged Atrioventricular Block and Ventricular Standstill Following Adenosine Triphosphate Injection in a Patient Taking Dipyridamole and Antiarrhythmic Agents: A Case Report

    Directory of Open Access Journals (Sweden)

    Kotaro Oe, MD

    2009-01-01

    Full Text Available An 83-year-old woman was admitted to our hospital because of palpitation. She had hypertension and paroxysmal atrial fibrillation, treated with digoxin and cibenzoline, and took dipyridamole for microalbuminuria. Before admission, she had taken pilsicainide pills in addition. On admission, electrocardiogram showed regular tachycardia with mildly prolonged QRS width. For the purpose of terminating tachycardia, 10 mg of adenosine triphosphate (ATP was rapidly injected. About 20 sec later, atrioventricular block and ventricular standstill occurred. She presented loss of consciousness and convulsion, and chest compression was performed. About 30 sec later, the QRS complex reappeared, and she became alert. Serum concentration of digoxin, cibenzoline and pilsicainide was within therapeutic level, respectively. We should be cautious in using ATP for a patient taking dipyridamole and antiarrhythmic agents.

  13. Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts.

    Science.gov (United States)

    Nishikawa, Keizo; Iwamoto, Yoriko; Ishii, Masaru

    2014-05-01

    The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.

  14. Genetic ablation of CD68 results in mice with increased bone and dysfunctional osteoclasts.

    Directory of Open Access Journals (Sweden)

    Jason W Ashley

    Full Text Available CD68 is a member of the lysosome associated membrane protein (LAMP family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF and the receptor activator of NF-κB ligand (RANKL. We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68(-/- osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68(-/- osteoclasts.

  15. Stimulating at the right time: phase-specific deep brain stimulation.

    Science.gov (United States)

    Cagnan, Hayriye; Pedrosa, David; Little, Simon; Pogosyan, Alek; Cheeran, Binith; Aziz, Tipu; Green, Alexander; Fitzgerald, James; Foltynie, Thomas; Limousin, Patricia; Zrinzo, Ludvic; Hariz, Marwan; Friston, Karl J; Denison, Timothy; Brown, Peter

    2017-01-01

    SEE MOLL AND ENGEL DOI101093/AWW308 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Brain regions dynamically engage and disengage with one another to execute everyday actions from movement to decision making. Pathologies such as Parkinson's disease and tremor emerge when brain regions controlling movement cannot readily decouple, compromising motor function. Here, we propose a novel stimulation strategy that selectively regulates neural synchrony through phase-specific stimulation. We demonstrate for the first time the therapeutic potential of such a stimulation strategy for the treatment of patients with pathological tremor. Symptom suppression is achieved by delivering stimulation to the ventrolateral thalamus, timed according to the patient's tremor rhythm. Sustained locking of deep brain stimulation to a particular phase of tremor afforded clinically significant tremor relief (up to 87% tremor suppression) in selected patients with essential tremor despite delivering less than half the energy of conventional high frequency stimulation. Phase-specific stimulation efficacy depended on the resonant characteristics of the underlying tremor network. Selective regulation of neural synchrony through phase-locked stimulation has the potential to both increase the efficiency of therapy and to minimize stimulation-induced side effects. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain.

  16. Novel Approaches to Breast Cancer Prevention and Inhibition of Metastases

    Science.gov (United States)

    2016-10-01

    Using mouse genetics, we showed that RANKL and its receptor RANK are critical regulators of sex hormone and BRCA1 mutation-driven breast cancer...into breast cancer patients and controls due to self reporting and histological examination. We have tested differences in RANKL, OPG and RANKL/OPG...cohort, we determined RANKL and OPG serum levels of participants from the Bruneck study [28, 29], that allowed us to analyse matched patient samples

  17. [Transcranial magnetic stimulation and motor cortex stimulation in neuropathic pain].

    Science.gov (United States)

    Mylius, V; Ayache, S S; Teepker, M; Kappus, C; Kolodziej, M; Rosenow, F; Nimsky, C; Oertel, W H; Lefaucheur, J P

    2012-12-01

    Non-invasive and invasive cortical stimulation allows the modulation of therapy-refractory neuropathic pain. High-frequency repetitive transcranial magnetic stimulation (rTMS) of the contralateral motor cortex yields therapeutic effects at short-term and predicts the benefits of epidural motor cortex stimulation (MCS). The present article summarizes the findings on application, mechanisms and therapeutic effects of cortical stimulation in neuropathic pain.

  18. Stimulating the Comfort of Textile Electrodes in Wearable Neuromuscular Electrical Stimulation

    Science.gov (United States)

    Zhou, Hui; Lu, Yi; Chen, Wanzhen; Wu, Zhen; Zou, Haiqing; Krundel, Ludovic; Li, Guanglin

    2015-01-01

    Textile electrodes are becoming an attractive means in the facilitation of surface electrical stimulation. However, the stimulation comfort of textile electrodes and the mechanism behind stimulation discomfort is still unknown. In this study, a textile stimulation electrode was developed using conductive fabrics and then its impedance spectroscopy, stimulation thresholds, and stimulation comfort were quantitatively assessed and compared with those of a wet textile electrode and a hydrogel electrode on healthy subjects. The equivalent circuit models and the finite element models of different types of electrode were built based on the measured impedance data of the electrodes to reveal the possible mechanism of electrical stimulation pain. Our results showed that the wet textile electrode could achieve similar stimulation performance as the hydrogel electrode in motor threshold and stimulation comfort. However, the dry textile electrode was found to have very low pain threshold and induced obvious cutaneous painful sensations during stimulation, in comparison to the wet and hydrogel electrodes. Indeed, the finite element modeling results showed that the activation function along the z direction at the depth of dermis epidermis junction of the dry textile electrode was significantly larger than that of the wet and hydrogel electrodes, thus resulting in stronger activation of pain sensing fibers. Future work will be done to make textile electrodes have similar stimulation performance and comfort as hydrogel electrodes. PMID:26193273

  19. Stimulating the Comfort of Textile Electrodes in Wearable Neuromuscular Electrical Stimulation

    Directory of Open Access Journals (Sweden)

    Hui Zhou

    2015-07-01

    Full Text Available Textile electrodes are becoming an attractive means in the facilitation of surface electrical stimulation. However, the stimulation comfort of textile electrodes and the mechanism behind stimulation discomfort is still unknown. In this study, a textile stimulation electrode was developed using conductive fabrics and then its impedance spectroscopy, stimulation thresholds, and stimulation comfort were quantitatively assessed and compared with those of a wet textile electrode and a hydrogel electrode on healthy subjects. The equivalent circuit models and the finite element models of different types of electrode were built based on the measured impedance data of the electrodes to reveal the possible mechanism of electrical stimulation pain. Our results showed that the wet textile electrode could achieve similar stimulation performance as the hydrogel electrode in motor threshold and stimulation comfort. However, the dry textile electrode was found to have very low pain threshold and induced obvious cutaneous painful sensations during stimulation, in comparison to the wet and hydrogel electrodes. Indeed, the finite element modeling results showed that the activation function along the z direction at the depth of dermis epidermis junction of the dry textile electrode was significantly larger than that of the wet and hydrogel electrodes, thus resulting in stronger activation of pain sensing fibers. Future work will be done to make textile electrodes have similar stimulation performance and comfort as hydrogel electrodes.

  20. [Bone Cell Biology Assessed by Microscopic Approach. Response to mechanical stress by osteocyte network].

    Science.gov (United States)

    Komori, Toshihisa

    2015-10-01

    Osteocytes were considered to be involved in the response to mechanical stress from their network structure. However, it was difficult to prove the function because of the lack of animal models for a long time. Recently, the function of osteocytes was clarified using various knockout and transgenic mice. Osteocyte death causes bone remodeling, which is a repair process induced by osteocyte necrosis but not by the loss of the function of live osteocytes. The osteocyte network mildly inhibits bone formation and mildly stimulates bone resorption in physiological condition. In unloaded condition, it strongly inhibits bone formation and strongly stimulates bone resorption, at least in part, through the induction of Sost in osteocytes and Rankl in osteoblasts.

  1. TRAF family member-associated NF-κB activator (TANK) induced by RANKL negatively regulates osteoclasts survival and function.

    Science.gov (United States)

    Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

    2012-01-01

    Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.

  2. Cloning and characterization of a gene encoding Rac/Rop-like monomeric guanosine 5'-triphosphate-binding protein from Scoparia dulcis.

    Science.gov (United States)

    Mitamura, Toshiaki; Shite, Masato; Yamamura, Yoshimi; Kurosaki, Fumiya

    2009-06-01

    A cDNA clone, designated Sd-racrop (969 bp), was isolated from seedlings of Scoparia dulcis. This gene contains an open reading frame encoding the protein of 197 amino acid residues with high homology to Rac/Rop small guanosine 5'-triphosphate-binding proteins from various plant sources. In Southern hybridization analysis, the restriction digests prepared from genomic DNA of S. dulcis showed a main signal together with a few weakly hybridized bands. The transcriptional level of Sd-racrop showed a transient decrease by exposure of the leaf tissues of S. dulcis to the ethylene-generating reagent 2-chloroethylphosphonic acid. However, an appreciable increase in gene expression was reproducibly observed upon treatment of the plant with methyl jasmonate. These results suggest that the Sd-racrop product plays roles in ethylene- and methyl jasmonate-induced responses of S. dulcis accompanying the change in the transcriptional level, however, the cellular events mediated by this protein toward these external stimuli would be regulated by various mechanisms.

  3. Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels.

    Science.gov (United States)

    Zhang, Zhanxia; Sharon, Etery; Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar

    2012-06-05

    The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 μM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

  4. Low-intensity pulsed ultrasound accelerates tooth movement via activation of the BMP-2 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hui Xue

    Full Text Available The present study was designed to determine the underlying mechanism of low-intensity pulsed ultrasound (LIPUS induced alveolar bone remodeling and the role of BMP-2 expression in a rat orthodontic tooth movement model. Orthodontic appliances were placed between the homonymy upper first molars and the upper central incisors in rats under general anesthesia, followed by daily 20-min LIPUS or sham LIPUS treatment beginning at day 0. Tooth movement distances and molecular changes were evaluated at each observation point. In vitro and in vivo studies were conducted to detect HGF (Hepatocyte growth factor/Runx2/BMP-2 signaling pathways and receptor activator of NFκB ligand (RANKL expression by quantitative real time PCR (qRT-PCR, Western blot and immunohistochemistry. At day 3, LIPUS had no effect on the rat orthodontic tooth movement distance and BMP-2-induced alveolar bone remodeling. However, beginning at day 5 and for the following time points, LIPUS significantly increased orthodontic tooth movement distance and BMP-2 signaling pathway and RANKL expression compared with the control group. The qRT-PCR and Western blot data in vitro and in vivo to study BMP-2 expression were consistent with the immunohistochemistry observations. The present study demonstrates that LIPUS promotes alveolar bone remodeling by stimulating the HGF/Runx2/BMP-2 signaling pathway and RANKL expression in a rat orthodontic tooth movement model, and LIPUS increased BMP-2 expression via Runx2 regulation.

  5. F4/80 inhibits osteoclast differentiation via downregulation of nuclear factor of activated T cells, cytoplasmic 1.

    Science.gov (United States)

    Kang, Ju-Hee; Sim, Jung-Sun; Zheng, Ting; Yim, Mijung

    2017-04-01

    Osteoclastogenesis is an essential process in bone metabolism, which can be induced by RANKL stimulation. The F4/80 glycoprotein is a member of the EGF-transmembrane 7 (TM7) family and has been established as a specific cell-surface marker for murine macrophages. This study aimed to identify the role of F4/80 in osteoclastogenesis. Using mouse bone marrow-derived macrophages (BMMs), we observed that the mRNA level of F4/80 was dramatically reduced as these cells differentiated into osteoclasts. Furthermore, osteoclastogenesis was decreased in F4/80 high BMMs compared to F4/80 -/low BMMs. The inhibitory effect of F4/80 was associated with decreased expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). Ectopic overexpression of a constitutively active form of NFATc1 rescued the anti-osteoclastogenic effect of F4/80 completely, suggesting that the anti-osteoclastogenic effect of F4/80 was mainly due to reduction in NFATc1 expression. As an underlying mechanism, we demonstrated that the presence of F4/80 abrogated the effect of RANKL on the phosphorylation of CREB and activated the expression of IFN-β, which are restored by cyclic AMP. Collectively, our results demonstrate that the presence of F4/80 suppresses RANKL-induced osteoclastogenesis by impairing the expression of NFATc1 via CREB and IFN-β. Therefore, F4/80 may hold therapeutic potential for bone destructive diseases.

  6. Tris stimulated ecdysteroid secretion via Ca2+ messenger system in the prothoracic glands of Locusta migratoria

    Czech Academy of Sciences Publication Activity Database

    Neuwirth, Aleš; Kodrík, Dalibor; Birkenbeil, H.; Sehnal, František

    2005-01-01

    Roč. 30, - (2005), s. 270-277 ISSN 0307-6962 Grant - others: Deutsch Gesellschaft fur Luft-und Raumfahrt(DE) TSR-072-97 Institutional research plan: CEZ:AV0Z50070508 Keywords : Calcium channels * ecdysteroids * inositol triphosphate Subject RIV: ED - Physiology Impact factor: 1.221, year: 2005

  7. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

    Science.gov (United States)

    Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2008-11-01

    A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

  8. Biomarkers and Stimulation Algorithms for Adaptive Brain Stimulation

    Directory of Open Access Journals (Sweden)

    Kimberly B. Hoang

    2017-10-01

    Full Text Available The goal of this review is to describe in what ways feedback or adaptive stimulation may be delivered and adjusted based on relevant biomarkers. Specific treatment mechanisms underlying therapeutic brain stimulation remain unclear, in spite of the demonstrated efficacy in a number of nervous system diseases. Brain stimulation appears to exert widespread influence over specific neural networks that are relevant to specific disease entities. In awake patients, activation or suppression of these neural networks can be assessed by either symptom alleviation (i.e., tremor, rigidity, seizures or physiological criteria, which may be predictive of expected symptomatic treatment. Secondary verification of network activation through specific biomarkers that are linked to symptomatic disease improvement may be useful for several reasons. For example, these biomarkers could aid optimal intraoperative localization, possibly improve efficacy or efficiency (i.e., reduced power needs, and provide long-term adaptive automatic adjustment of stimulation parameters. Possible biomarkers for use in portable or implanted devices span from ongoing physiological brain activity, evoked local field potentials (LFPs, and intermittent pathological activity, to wearable devices, biochemical, blood flow, optical, or magnetic resonance imaging (MRI changes, temperature changes, or optogenetic signals. First, however, potential biomarkers must be correlated directly with symptom or disease treatment and network activation. Although numerous biomarkers are under consideration for a variety of stimulation indications the feasibility of these approaches has yet to be fully determined. Particularly, there are critical questions whether the use of adaptive systems can improve efficacy over continuous stimulation, facilitate adjustment of stimulation interventions and improve our understanding of the role of abnormal network function in disease mechanisms.

  9. A distributed current stimulator ASIC for high density neural stimulation.

    Science.gov (United States)

    Jeong Hoan Park; Chaebin Kim; Seung-Hee Ahn; Tae Mok Gwon; Joonsoo Jeong; Sang Beom Jun; Sung June Kim

    2016-08-01

    This paper presents a novel distributed neural stimulator scheme. Instead of a single stimulator ASIC in the package, multiple ASICs are embedded at each electrode site for stimulation with a high density electrode array. This distributed architecture enables the simplification of wiring between electrodes and stimulator ASIC that otherwise could become too complex as the number of electrode increases. The individual ASIC chip is designed to have a shared data bus that independently controls multiple stimulating channels. Therefore, the number of metal lines is determined by the distributed ASICs, not by the channel number. The function of current steering is also implemented within each ASIC in order to increase the effective number of channels via pseudo channel stimulation. Therefore, the chip area can be used more efficiently. The designed chip was fabricated with area of 0.3 mm2 using 0.18 μm BCDMOS process, and the bench-top test was also conducted to validate chip performance.

  10. Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

    Science.gov (United States)

    Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

    2017-06-01

    In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

  11. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    Energy Technology Data Exchange (ETDEWEB)

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  12. Biphasic Elimination of Tenofovir Diphosphate and Nonlinear Pharmacokinetics of Zidovudine Triphosphate in a Microdosing Study

    Science.gov (United States)

    Chen, Jianmeng; Flexner, Charles; Liberman, Rosa G.; Skipper, Paul L.; Louissaint, Nicolette; Tannenbaum, Steven R.; Hendrix, Craig; Fuchs, Edward

    2012-01-01

    Objective Phase 0 studies can provide initial pharmacokinetics (PK) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of two antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. Study Design We administered a microdose (100 μg) of 14C-labeled drug (ZDV or tenofovir disoproxil fumarate (TDF)) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in PBMCs and CD4+ cells were measured by AMS. Results The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 μg to 300 mg), while the intracellular TFV-DP PK were linear over the same dose range. ZDV-TP concentrations were lower in CD4+ cells versus total peripheral blood mononuclear cells (PBMCs), while TFV-DP concentrations were not different in CD4+ cells and PBMCs. Conclusion Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. AMS shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs. PMID:23187888

  13. Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice.

    Science.gov (United States)

    Bayliss, Jill; Delarosa, Sara; Wu, Jianfeng; Peterson, Jonathan R; Eboda, Oluwatobi N; Su, Grace L; Hemmila, Mark; Krebsbach, Paul H; Cederna, Paul S; Wang, Stewart C; Xi, Chuanwu; Levi, Benjamin

    2014-01-01

    Extracellular adenosine triphosphate (ATP), present in thermally injured tissue, modulates the inflammatory response and causes significant tissue damage. The authors hypothesize that neutrophil infiltration and ensuing tissue necrosis would be mitigated by removing ATP-dependent signaling at the burn site. Mice were subjected to 30% TBSA partial-thickness scald burn by dorsal skin immersion in a water bath at 60 or 20°C (nonburn controls). In the treatment arm, an ATP hydrolyzing enzyme, apyrase, was applied directly to the site immediately after injury. Skin was harvested after 24 hours and 5 days for hematoxylin and eosin stain, elastase, and Ki-67 staining. Tumor necrosis factor (TNF)-α and interferon (IFN)-β expression were measured through quantitative real-time polymerase chain reaction. At 24 hours, the amount of neutrophil infiltration was different between the burn and burn + apyrase groups (P burn group at 24 hours and 5 days. TNF-α and IFN-β expression at 24 hours in the apyrase group was lower than in the burn group (P burn site allays the neutrophil response to thermal injury and reduces tissue necrosis. This decrease in inflammation and tissue necrosis is at least partially because of TNF-α and IFN-β signaling. Apyrase could be used as topical inflammatory regulators to quell the injury caused by inflammation.

  14. Transcranial brain stimulation: closing the loop between brain and stimulation

    DEFF Research Database (Denmark)

    Karabanov, Anke; Thielscher, Axel; Siebner, Hartwig Roman

    2016-01-01

    -related and state-related variability. Fluctuations in brain-states can be traced online with functional brain imaging and inform the timing or other settings of transcranial brain stimulation. State-informed open-loop stimulation is aligned to the expression of a predefined brain state, according to prespecified......PURPOSE OF REVIEW: To discuss recent strategies for boosting the efficacy of noninvasive transcranial brain stimulation to improve human brain function. RECENT FINDINGS: Recent research exposed substantial intra- and inter-individual variability in response to plasticity-inducing transcranial brain...... stimulation. Trait-related and state-related determinants contribute to this variability, challenging the standard approach to apply stimulation in a rigid, one-size-fits-all fashion. Several strategies have been identified to reduce variability and maximize the plasticity-inducing effects of noninvasive...

  15. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of health claims related to sugar-free chewing gum with pyro- and triphosphates and reduction of calculus formation (ID 1309) pursuant to Article 13(1) of Regulation (EC) No 1924/2006

    DEFF Research Database (Denmark)

    Tetens, Inge

    calculus/tartar formation, gums health”. The target population is assumed to be the general population. The Panel considers that reduction of calculus formation at sites which are most important for dental health is a beneficial physiological effect. No human studies have been provided from which...... conclusions could be drawn for the scientific substantiation of a claim on the use of sugar-free chewing gum with pyro- and triphosphates and the reduction of calculus formation at sites which are most important for dental health (e.g. gingival margin or between teeth). On the basis of the data presented......, the Panel concludes that a cause and effect relationship has not been established between the use of sugar-free chewing gum with pyro- and triphosphates and reduction of calculus formation at sites which are most important for dental health....

  16. Colorimetric sensor for triphosphates and their application as a viable staining agent for prokaryotes and eukaryotes.

    Science.gov (United States)

    Ghosh, Amrita; Shrivastav, Anupama; Jose, D Amilan; Mishra, Sanjiv K; Chandrakanth, C K; Mishra, Sandhya; Das, Amitava

    2008-07-15

    The chromogenic complex 1 x Zn (where 1 is (E)-4-(4-dimethylamino-phenylazo)-N,N-bispyridin-2-ylmethyl-benzenesulfonamide) showed high affinity toward the phosphate ion in tetrabutylammonium phosphate in acetonitrile solution and could preferentially bind to adenosine triphosphate (ATP) in aqueous solution at physiological pH. This binding caused a visual change in color, whereas no such change was noticed with other related anions (adenosine monophosphate, adenosine diphosphate, pyrophosphate, and phosphate) of biological significance. Thus, 1 x Zn could be used as a staining agent for different biological cells through binding to the ATP, generated in situ by the mitochondria (in eukaryotes). For prokaryotes (bacteria) the cell membrane takes care of the cells' energy conversion, since they lack mitochondria. ATP is produced in their unique cell structure on the cell membrane, which is not found in any eukaryotes. These stained cells could be viewed with normal light microscopy. This reagent could even be used for distinguishing the gram-positive and the gram-negative bacteria (prokaryotes). This dye was found to be nonlipophilic in nature and nontoxic to living microbes (eukaryotes and prokaryotes). Further, stained cells were found to grow in their respective media, and this confirmed the maintenance of viability of the microbes even after staining, unlike with many other dyes available commercially.

  17. HLH-29 regulates ovulation in C. elegans by targeting genes in the inositol triphosphate signaling pathway

    Directory of Open Access Journals (Sweden)

    Ana White

    2012-02-01

    The reproductive cycle in the nematode Caenorhabditis elegans depends in part on the ability of the mature oocyte to ovulate into the spermatheca, fuse with the sperm during fertilization, and then exit the spermatheca as a fertilized egg. This cycle requires the integration of signals between the germ cells and the somatic gonad and relies heavily on the precise control of inositol 1,4,5 triphosphate (IP3levels. The HLH-29 protein, one of five Hairy/Enhancer of Split (HES homologs in C. elegans, was previously shown to affect development of the somatic gonad. Here we show that HLH-29 expression in the adult spermatheca is strongly localized to the distal spermatheca valve and to the spermatheca-uterine valve, and that loss of hlh-29 activity interferes with oocyte entry into and egg exit from the spermatheca. We show that HLH-29 can regulate the transcriptional activity of the IP3 signaling pathway genes ppk-1, ipp-5, and plc-1 and provide evidence that hlh-29 acts in a genetic pathway with each of these genes. We propose that the HES-like protein HLH-29 acts in the spermatheca of larval and adult animals to effectively increase IP3 levels during the reproductive cycle.

  18. Vagal nerve stimulation therapy: what is being stimulated?

    Science.gov (United States)

    Kember, Guy; Ardell, Jeffrey L; Armour, John A; Zamir, Mair

    2014-01-01

    Vagal nerve stimulation in cardiac therapy involves delivering electrical current to the vagal sympathetic complex in patients experiencing heart failure. The therapy has shown promise but the mechanisms by which any benefit accrues is not understood. In this paper we model the response to increased levels of stimulation of individual components of the vagal sympathetic complex as a differential activation of each component in the control of heart rate. The model provides insight beyond what is available in the animal experiment in as much as allowing the simultaneous assessment of neuronal activity throughout the cardiac neural axis. The results indicate that there is sensitivity of the neural network to low level subthreshold stimulation. This leads us to propose that the chronic effects of vagal nerve stimulation therapy lie within the indirect pathways that target intrinsic cardiac local circuit neurons because they have the capacity for plasticity.

  19. Vagal nerve stimulation therapy: what is being stimulated?

    Directory of Open Access Journals (Sweden)

    Guy Kember

    Full Text Available Vagal nerve stimulation in cardiac therapy involves delivering electrical current to the vagal sympathetic complex in patients experiencing heart failure. The therapy has shown promise but the mechanisms by which any benefit accrues is not understood. In this paper we model the response to increased levels of stimulation of individual components of the vagal sympathetic complex as a differential activation of each component in the control of heart rate. The model provides insight beyond what is available in the animal experiment in as much as allowing the simultaneous assessment of neuronal activity throughout the cardiac neural axis. The results indicate that there is sensitivity of the neural network to low level subthreshold stimulation. This leads us to propose that the chronic effects of vagal nerve stimulation therapy lie within the indirect pathways that target intrinsic cardiac local circuit neurons because they have the capacity for plasticity.

  20. Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Hung, Szu-Ying; Shih, Ya-Chen [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); Tseng, Wei-Lung, E-mail: tsengwl@mail.nsysu.edu.tw [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan (China); Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Taiwan (China); Center for Stem Cell Research, Kaohsiung Medical University, Taiwan (China)

    2015-02-01

    Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

  1. Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity

    International Nuclear Information System (INIS)

    Hung, Szu-Ying; Shih, Ya-Chen; Tseng, Wei-Lung

    2015-01-01

    Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

  2. Involvement of purinergic signaling on nitric oxide production by neutrophils stimulated with Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; De Carli, Geraldo Attilio; Bonan, Carla Denise; Tasca, Tiana

    2012-03-01

    Trichomonas vaginalis is a parasite from the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. The neutrophil infiltration has been considered to be primarily responsible for cytological changes observed at infection site, and the chemoattractants can play an important role in this leukocytic recruitment. Nitric oxide (NO) is one of the most widespread mediator compounds, and it is implicated in modulation of immunological mechanisms. Extracellular nucleotides and nucleosides are signaling molecules involved in several processes, including immune responses and control of leukocyte trafficking. Ectonucleoside triphosphate diphosphohydrolase members, ecto-5'-nucleotidase, and adenosine deaminase (ectoADA) have been characterized in T. vaginalis. Herein, we investigated the effects of purinergic system on NO production by neutrophils stimulated with T. vaginalis. The trophozoites were able to induce a high NO synthesis by neutrophils through iNOS pathway. The extracellular nucleotides ATP, ADP, and ATPγS (a non-hydrolyzable ATP analog) showed no significant change in NO secretion. In contrast, adenosine and its degradation product, inosine, promoted a low production of the compound. The immunosuppressive effect of adenosine upon NO release by neutrophils occurred due to adenosine A(2A) receptor activation. The ecto-5'-nucleotidase activity displayed by T. vaginalis was shown to be important in adenosine generation, indicating the efficiency of purinergic cascade. Our data suggest the influence of purinergic signaling, specifically adenosinergic system, on NO production by neutrophils in T. vaginalis infection, contributing to the immunological aspects of disease.

  3. A quantitative analysis of the effects of 2,3-diphosphoglycerate, adenosine triphosphate and inositol hexaphosphate on the oxygen dissociation curve of human haemoglobin.

    Science.gov (United States)

    Goodford, P J; St-Louis, J; Wootton, R

    1978-01-01

    1. Oxygen dissociation curves have been measured for human haemoglobin solutions with different concentrations of the allosteric effectors 2,3-diphosphoglycerate, adenosine triphosphate and inositol hexaphosphate. 2. Each effector produces a concentration dependent right shift of the oxygen dissociation curve, but a point is reached where the shift is maximal and increasing the effector concentration has no further effect. 3. Mathematical models based on the Monod, Wyman & Changeux (1965) treatment of allosteric proteins have been fitted to the data. For each compound the simple two-state model and its extension to take account of subunit inequivalence were shown to be inadequate, and a better fit was obtained by allowing the effector to lower the oxygen affinity of the deoxy conformational state as well as binding preferentially to this conformation. PMID:722582

  4. A novel dual-wavelength laser stimulator to elicit transient and tonic nociceptive stimulation.

    Science.gov (United States)

    Dong, Xiaoxi; Liu, Tianjun; Wang, Han; Yang, Jichun; Chen, Zhuying; Hu, Yong; Li, Yingxin

    2017-07-01

    This study aimed to develop a new laser stimulator to elicit both transient and sustained heat stimulation with a dual-wavelength laser system as a tool for the investigation of both transient and tonic experimental models of pain. The laser stimulator used a 980-nm pulsed laser to generate transient heat stimulation and a 1940-nm continuous-wave (CW) laser to provide sustained heat stimulation. The laser with 980-nm wavelength can elicit transient pain with less thermal injury, while the 1940-nm CW laser can effectively stimulate both superficial and deep nociceptors to elicit tonic pain. A proportional integral-derivative (PID) temperature feedback control system was implemented to ensure constancy of temperature during heat stimulation. The performance of this stimulator was evaluated by in vitro and in vivo animal experiments. In vitro experiments on totally 120 specimens fresh pig skin included transient heat stimulation by 980-nm laser (1.5 J, 10 ms), sustained heat stimulation by 1940-nm laser (50-55 °C temperature control mode or 1.5 W, 5 min continuous power supply), and the combination of transient/sustained heat stimulation by dual lasers (1.5 J, 10 ms, 980-nm pulse laser, and 1940-nm laser with 50-55 °C temperature control mode). Hemoglobin brushing and wind-cooling methods were tested to find better stimulation model. A classic tail-flick latency (TFL) experiment with 20 Wistar rats was used to evaluate the in vivo efficacy of transient and tonic pain stimulation with 15 J, 100 ms 980-nm single laser pulse, and 1.5 W constant 1940-nm laser power. Ideal stimulation parameters to generate transient pain were found to be a 26.6 °C peak temperature rise and 0.67 s pain duration. In our model of tonic pain, 5 min of tonic stimulation produced a temperature change of 53.7 ± 1.3 °C with 1.6 ± 0.2% variation. When the transient and tonic stimulation protocols were combined, no significant difference was observed depending on the order

  5. Sitagliptin, An Anti-diabetic Drug, Suppresses Estrogen Deficiency-Induced OsteoporosisIn Vivo and Inhibits RANKL-Induced Osteoclast Formation and Bone Resorption In Vitro

    Directory of Open Access Journals (Sweden)

    Chuandong Wang

    2017-06-01

    Full Text Available Postmenopausal osteoporosis is a disease characterized by excessive osteoclastic bone resorption. Some anti-diabetic drugs were demonstrated for anti-osteoclastic bone-loss effects. The present study investigated the skeletal effects of chronic administration of sitagliptin, a dipeptidyl peptidase IV (DPP IV inhibitor that is increasingly used for type 2 diabetes treatments, in an estrogen deficiency-induced osteoporosis and elucidated the associated mechanisms. This study indicated that sitagliptin effectively prevented ovariectomy-induced bone loss and reduced osteoclast numbers in vivo. It was also indicated that sitagliptin suppressed receptor activator of nuclear factor-κB ligand (RANKL-mediated osteoclast differentiation, bone resorption, and F-actin ring formation in a manner of dose-dependence. In addition, sitagliptin significantly reduced the expression of osteoclast-specific markers in mouse bone-marrow-derived macrophages, including calcitonin receptor (Calcr, dendrite cell-specific transmembrane protein (Dc-stamp, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1. Further study indicated that sitagliptin inhibited osteoclastogenesis by suppressing AKT and ERK signaling pathways, scavenging ROS activity, and suppressing the Ca2+ oscillation that consequently affects the expression and/or activity of the osteoclast-specific transcription factors, c-Fos and NFATc1. Collectively, these findings suggest that sitagliptin possesses beneficial effects on bone and the suppression of osteoclast number implies that the effect is exerted directly on osteoclastogenesis.

  6. Immunohistochemical detection of receptor activator nuclear κB ligand and osteoprotegerin in odontogenic cysts and tumors.

    Science.gov (United States)

    de Matos, Felipe Rodrigues; de Moraes, Maiara; das Neves Silva, Emília Beatriz; Galvão, Hébel Cavalcanti; de Almeida Freitas, Roseana

    2013-11-01

    The aim of the present study was to compare the immunohistochemical detection of receptor activator nuclear κB ligand (RANKL) and osteoprotegerin (OPG) in radicular cysts (RCs), dentigerous cysts (DCs), solid ameloblastomas (SAs), and keratocystic odontogenic tumors (KOTs). A total of 20 RCs, 20 DCs, 20 KOTs, 14 dental follicles (DFs), and 18 SAs were evaluated by immunohistochemistry using anti-RANKL and anti-OPG antibodies. The analysis was quantitative, and the number of positive cells was counted in 10 microscopic high-power fields (400×). The DFs, KOTs, and SAs showed higher expression of RANKL than did the RCs and DCs in the epithelium (P < .05). The epithelial expression of OPG was higher in the DFs, KOTs, RCs, and DCs than in the SAs (P < .05). The ratio of OPG less than RANKL was more frequent in SAs and OPG greater than RANKL in DCs (P < .05). Our results have shown differences in RANKL and OPG detection in the odontogenic cysts and tumors studied. The higher RANKL and lower OPG detection in SA could play a role in bone resorption, compatible with the tumor's biologic behavior. Copyright © 2013 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  7. Die Rolle von RANK-Ligand und Osteoprotegerin bei Osteoporose

    Directory of Open Access Journals (Sweden)

    Hofbauer LC

    2004-01-01

    Full Text Available Receptor activator of nuclear factor (NF- κB ligand (RANKL, sein zellulärer Rezeptor RANK und der Decoy-Rezeptor Osteoprotegerin (OPG stellen ein essentielles Zytokinsystem für die Zellbiologie von Osteoklasten dar. Verschiedene Untersuchungen belegen die Bedeutung von Störungen des OPG/RANKL/RANK-Systems bei der Pathogenese metabolischer Knochenerkrankungen. In dieser Arbeit werden die wichtigsten Störungen des OPG/RANKL/RANK-Systems bei verschiedenen Osteoporoseformen dargestellt. Östrogenrezeptor- (ER- Agonisten wie 17 β-Östradiol, Raloxifen und Genistein stimulieren die osteoblastäre Produktion von OPG durch Aktivierung von ER- α in vitro, während Lymphozyten von Patientinnen mit Östrogenmangel RANKL überexprimieren. Die parenterale Gabe von OPG vermag den mit Östrogenmangel assoziierten Knochenverlust im Tiermodell und in einer kleineren klinischen Studie zu verhindern. Glukokortikoide und Immunsuppressiva steigern gleichzeitig die RANKL-Expression und hemmen die OPG-Produktion in osteoblastären Zellen in vitro. Glukokortikoide sind auch in vivo imstande, die OPG-Serumspiegel deutlich zu reduzieren. Dagegen hemmen biomechanische Reize in vitro die RANKL-Produktion und steigern die OPG-Produktion. Ein Fehlen dieser biomechanischen Reize bei längerer Immobilisierung kann daher den RANKL/OPG-Quotienten steigern, während die tierexperimentelle Immobilisierungs-Osteoporose durch die parenterale Gabe von OPG gemildert werden kann.

  8. The Relationship Between Vitamin D Levels and Receptor Activator of Nuclear Factor Ligand in Hashimoto’s Thyroiditis

    Directory of Open Access Journals (Sweden)

    Hakan Yavuzer

    2017-12-01

    Full Text Available Aim: In order to analyze the relationship of Hashimoto’s thyroiditis with vitamin D and osteoclastogenic markers, we investigated vitamin D, osteoprotegerin (OPG and receptor activator of nuclear factor ligand (RANKL levels in patients over 60 years with and without Hashimoto’s thyroiditis. Methods: Eighty three female patients (49 with and 34 without Hashimoto’s thyroiditis, who attended the endocrinology and geriatrics departments between May 2013 and October 2013 were included in the study. Results: There was no statistically significant difference in the levels of vitamin D, OPG and RANKL between the groups. Vitamin D was significantly correlated with OPG and RANKL in patients with Hashimoto’s thyroiditis. In addition, a significant relationship was found between OPG and RANKL levels. Of the patients with Hashimoto’s thyroiditis, 33 were autoantibody-positive and 16 were negative. Vitamin D, OPG and RANKL levels were significantly lower in antibody-positive patients than in negative subjects. Conclucion: There were no differences in vitamin D, OPG and RANKL levels between patients with and without Hashimoto’s thyroidits. Autoantibody-positive Hashimoto’s thyroiditis group had statistically significantly lower vitamin D, OPG and RANKL levels. This reverse correlation suggests that autoantibodies may have an effect on osteoclastogenesis.

  9. Use of adenosine triphosphate to audit reprocessing of flexible endoscopes with an elevator mechanism.

    Science.gov (United States)

    Quan, Erik; Mahmood, Rizwan; Naik, Amar; Sargon, Peter; Shastri, Nikhil; Venu, Mukund; Parada, Jorge P; Gupta, Neil

    2018-05-21

    There have been reported outbreaks of carbapenem-resistant Enterobacteriaceae infections linked to endoscopes with elevator mechanisms. Adenosine triphosphate (ATP) testing has been used as a marker for bioburden and monitoring manual cleaning for flexible endoscopes with and without an elevator mechanism. The objective of this study was to determine whether routine ATP testing could identify areas of improvement in cleaning of endoscopes with an elevator mechanism. ATP testing after manual cleaning of TJF-Q180V duodenoscopes and GF-UCT180 linear echoendoscopes (Olympus America Inc, Center Valley, PA) was implemented. Samples were tested from the distal end, the elevator mechanism, and water flushed through the lumen of the biopsy channel. Data were recorded and compared by time point, test point, and reprocessing technician. Overall failure rate was 6.99% (295 out of 4,219). The highest percentage of failed ATP tests (17.05%) was reported in the first quarter of routine testing, with an overall decrease in rates over time. The elevator mechanism and working channel lumen had higher failure rates than the distal end. Quality of manual cleaning between reprocessing technicians showed variation. ATP testing is effective in identifying residual organic material and improving quality of manual cleaning of endoscopes with an elevator mechanism. Cleaning efficacy is influenced by reprocessing technicians and location tested on the endoscope. Close attention to the working channel and elevator mechanism during manual cleaning is warranted. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

  10. MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

    International Nuclear Information System (INIS)

    Guo, Li-Juan; Liao, Lan; Yang, Li; Li, Yu; Jiang, Tie-Jian

    2014-01-01

    MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease. - Highlights: • MiR-125a was significantly down-regulated in osteoclastogenesis of CD14+ PBMCs. • MiR-125a inhibited osteoclast differentiation by targeting TRAF6. • NFATc1 inhibited miR-125a transciption by binding to the promoter of miR-125a. • TRAF6/NFATc1 and miR-125a form a regulatory feedback loop in osteoclastogenesis

  11. Cognitive stimulation in healthy older adults: a cognitive stimulation program using leisure activities compared to a conventional cognitive stimulation program.

    Science.gov (United States)

    Grimaud, Élisabeth; Taconnat, Laurence; Clarys, David

    2017-06-01

    The aim of this study was to compare two methods of cognitive stimulation for the cognitive functions. The first method used an usual approach, the second used leisure activities in order to assess their benefits on cognitive functions (speed of processing; working memory capacity and executive functions) and psychoaffective measures (memory span and self esteem). 67 participants over 60 years old took part in the experiment. They were divided into three groups: 1 group followed a program of conventional cognitive stimulation, 1 group a program of cognitive stimulation using leisure activities and 1 control group. The different measures have been evaluated before and after the training program. Results show that the cognitive stimulation program using leisure activities is as effective on memory span, updating and memory self-perception as the program using conventional cognitive stimulation, and more effective on self-esteem than the conventional program. There is no difference between the two stimulated groups and the control group on speed of processing. Neither of the two cognitive stimulation programs provides a benefit over shifting and inhibition. These results indicate that it seems to be possible to enhance working memory and to observe far transfer benefits over self-perception (self-esteem and memory self-perception) when using leisure activities as a tool for cognitive stimulation.

  12. Relationship between circulating serum osteoprotegerin and total receptor activator of nuclear κ-B ligand levels, triglycerides, and coronary calcification in postmenopausal women.

    Science.gov (United States)

    Poornima, Indu G; Mackey, Rachel H; Buhari, Alhaji M; Cauley, Jane A; Matthews, Karen A; Kuller, Lewis H

    2014-07-01

    This study evaluates the relationship of blood osteoprotegerin (OPG) and receptor activator of nuclear κ-B ligand (RANKL) levels with coronary artery calcium (CAC) and cardiovascular risk factors in two studies of postmenopausal women. OPG, a marker of bone turnover, and its ligand, RANKL, may contribute to cardiovascular disease risk. We tested the hypothesis that serum OPG and RANKL levels were associated with CAC and cardiovascular disease risk factors among postmenopausal women in the Women On the Move through Activity and Nutrition Study (WOMAN Study; n = 86; mean [SD], age 58 [2.9] y) and replicated our findings in the Healthy Women Study (HWS; n = 205; mean [SD] age, 61 [2.3] y). Serum OPG, total RANKL, and CAC were measured at baseline and 48 months in the WOMAN Study and on the eighth postmenopausal visit in the HWS. In the WOMAN Study, higher OPG was associated with higher CAC, and higher total RANKL was associated with lower CAC and triglycerides. In the HWS, higher total RANKL was also associated with lower CAC and triglycerides. In logistic regression models adjusted for body mass index and triglycerides, the odds ratios (95% CIs) for CAC per unit increase in OPG were 1.78 (1.17-2.73) for the WOMAN Study and 1.02 (0.84-1.24) for the HWS, and the odds ratios (95% CIs) for CAC per unit increase in log total RANKL were 0.86 (0.64-1.17) for the WOMAN Study and 0.83 (0.72-0.96) for the HWS. The inverse association of total RANKL with CAC and triglycerides is a new finding and may have important implications given the increasing use of drugs that modify total RANKL and its receptor, receptor activator of nuclear κ-B.

  13. Expression of Msx-1 is suppressed in bisphosphonate associated osteonecrosis related jaw tissue-etiopathology considerations respecting jaw developmental biology-related unique features

    Directory of Open Access Journals (Sweden)

    Schlegel Karl A

    2010-10-01

    Full Text Available Abstract Background Bone-destructive disease treatments include bisphosphonates and antibodies against the osteoclast differentiator, RANKL (aRANKL; however, osteonecrosis of the jaw (ONJ is a frequent side-effect. Current models fail to explain the restriction of bisphosphonate (BP-related and denosumab (anti-RANKL antibody-related ONJ to jaws. Msx-1 is exclusively expressed in craniofacial structures and pivotal to cranial neural crest (CNC-derived periodontal tissue remodeling. We hypothesised that Msx-1 expression might be impaired in bisphosphonate-related ONJ. The study aim was to elucidate Msx-1 and RANKL-associated signal transduction (BMP-2/4, RANKL in ONJ-altered and healthy periodontal tissue. Methods Twenty ONJ and twenty non-BP exposed periodontal samples were processed for RT-PCR and immunohistochemistry. An automated staining-based alkaline phosphatase-anti-alkaline phosphatase method was used to measure the stained cells:total cell-number ratio (labelling index, Bonferroni adjustment. Real-time RT-PCR was performed on ONJ-affected and healthy jaw periodontal samples (n = 20 each to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels. Results Semi-quantitative assessment of the ratio of stained cells showed decreased Msx-1 and RANKL and increased BMP-2/4 (all p Conclusions These results explain the sclerotic and osteopetrotic changes of periodontal tissue following BP application and substantiate clinical findings of BP-related impaired remodeling specific to periodontal tissue. RANKL suppression substantiated the clinical finding of impaired bone remodelling in BP- and aRANKL-induced ONJ-affected bone structures. Msx-1 suppression in ONJ-adjacent periodontal tissue suggested a bisphosphonate-related impairment in cellular differentiation that occurred exclusively jaw remodelling. Further research on developmental biology-related unique features of jaw bone structures will help to elucidate pathologies restricted to

  14. Probing the communication of deoxythymidine triphosphate in HIV-1 reverse transcriptase by communication maps and interaction energy studies.

    Science.gov (United States)

    Gnanasekaran, Ramachandran

    2017-11-08

    We calculate communication maps for HIV-1 Reverse Transcriptase (RT) to elucidate energy transfer pathways between deoxythymidine triphosphate (dTTP) and other parts of the protein. This approach locates energy transport channels from the dTTP to remote regions of the protein via residues and water molecules. We examine the water dynamics near the catalytic site of HIV-1 RT by molecular dynamics (MD) simulations. We find that, within the catalytic site, the relaxation of water molecules is similar to that of the hydration water molecules present in other proteins and the relaxation time scale is fast enough to transport energy and helps in communication between dTTP and other residues in the system. To quantify energy transfer, we also calculate the interaction energies of dTTP, 2Mg 2+ , doxy-guanosine nucleotide (DG22) with their surrounding residues by using the B3LYP-D3 method. The results, from classical vibrational energy diffusivity and QM interaction energy, are complementary to identify the important residues involved in the process of polymerization. The positive and negative interactions by dTTP with different types of residues in the catalytic region make the residues transfer energy through vibrational communication.

  15. A novel fluorescent biosensor for Adenosine Triphosphate detection based on the polydopamine nanospheres integrating with enzymatic recycling amplification.

    Science.gov (United States)

    Ji, Xiaoting; Yi, Bingqing; Xu, Yujuan; Zhao, Yanan; Zhong, Hua; Ding, Caifeng

    2017-07-01

    Based on the protective performance of polydopamine nanospheres (PDANSs) for DNA against nuclease digestion and the specific recognition characteristic of aptamer, we have developed an enzymatic recycling signal amplification method for highly sensitive and selective detection of adenosine triphosphate (ATP). Fluorescence measurements were carried out to verify the DNA polymerase and exonuclease III (Exo III) assisted target recycling process and fluorescence signal amplification. In the absence of the ATP, initially, the signal DNA-PDANSs complex was in the "off" state due to the efficient fluorescence quenching of 6-carboxyfluorescein (FAM) adjacent to the surface of PDANSs. Due to the binding of the aptamer by ATP, it trigger DNA polymerase and Exo III assisted target recycling process by the product of release, the complex would change into the "on" state as a result of the dissociation of the FAM from the surface of PDANSs, thus providing greatly enhanced fluorescence emission intensity. The method allows quantitative detection of ATP in the range of 20-600nM with a detection limit of 8.32nM. This biosensor requires no complex operations, and is a new high efficiency method for ATP detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy.

    Science.gov (United States)

    Ambruso, D R; Hawkins, B; Johnson, D L; Fritzberg, A R; Klingensmith, W C; McCabe, E R

    1986-06-01

    Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.

  17. Mechanism of orientation of stimulating currents in magnetic brain stimulation (abstract)

    Science.gov (United States)

    Ueno, S.; Matsuda, T.

    1991-04-01

    We made a functional map of the human motor cortex related to the hand and foot areas by stimulating the human brain with a focused magnetic pulse. We observed that each functional area in the cortex has an optimum direction for which stimulating currents can produce neural excitation. The present report focuses on the mechanism which is responsible for producing this anisotropic response to brain stimulation. We first obtained a functional map of the brain related to the left ADM (abductor digiti minimi muscles). When the stimulating currents were aligned in the direction from the left to the right hemisphere, clear EMG (electromyographic) responses were obtained only from the left ADM to magnetic stimulation of both hemisphere. When the stimulating currents were aligned in the direction from the right to the left hemisphere, clear EMG signals were obtained only from the right ADM to magnetic stimulation of both hemisphere. The functional maps of the brain were sensitive to changes in the direction of the stimulating currents. To explain the phenomena obtained in the experiments, we developed a model of neural excitation elicited by magnetic stimulation. When eddy currents which are induced by pulsed magnetic fields flow in the direction from soma to the distal part of neural fiber, depolarized area in the distal part are excited, and the membrane excitation propagates along the nerve fiber. In contrast, when the induced currents flow in the direction from the distal part to soma, hyperpolarized parts block or inhibit neural excitation even if the depolarized parts near the soma can be excited. The model explains our observation that the orientation of the induced current vectors reflect both the functional and anatomical organization of the neural fibers in the brain.

  18. Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Hill, Shannon E.; Nguyen, Elaine; Ukachukwu, Chiamaka U.; Freeman, Dana M.; Quirk, Stephen; Lieberman, Raquel L.; Boggon, Titus J.

    2017-07-25

    Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

  19. Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

    Directory of Open Access Journals (Sweden)

    Shannon E Hill

    Full Text Available Dihydroneopterin triphosphate pyrophosphatase (DHNTPase, a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

  20. Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

    International Nuclear Information System (INIS)

    Mahmood, R.

    1985-01-01

    The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz 2 ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz 2 ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF 1 ) with a binding constant of 3.2 x 10 5 M -1 . Bz 2 ATP is also a substrate for the ATPase activity of SF 1 in the presence of different cations. The irradiation of SF 1 with [ 3 H]Bz 2 ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF 1 after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling [ 3 H]Bz 2 ATP is trapped on SF 1 by cross-linking the two reactive thiols, SH 1 and SH 2 , by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified [ 14 C]Bz 2 ATP-SF 1 , after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography

  1. Synovial tissue rank ligand expression and radiographic progression in rheumatoid arthritis: observations from a proof-of-concept randomized clinical trial of cytokine blockade.

    LENUS (Irish Health Repository)

    Rooney, Terence

    2012-02-01

    The objective of the study was to evaluate synovial tissue receptor activator of nuclear factor-kappabeta ligand (RANKL) and osteoprotegerin (OPG) as biomarkers of disease activity, progressive joint damage, and therapeutic response, during cytokine blockade in rheumatoid arthritis (RA). Patients with active RA entered a randomized open-label 12-month study of anakinra 100 mg\\/day, administered as monotherapy or in combination with pegsunercept 800 mug\\/kg twice weekly. Arthroscopic synovial tissue biopsies were obtained at baseline, at 4 weeks and at the final time point. Following immunohistochemical staining, RANKL and OPG expression was quantified using digital image analysis. Radiographic damage was evaluated using the van der Heijde modification of the Sharp scoring system. Twenty-two patients were randomized. Baseline expression of RANKL, but not OPG, correlated significantly with baseline CRP levels (r = 0.61, P < 0.01). While a significant reduction in OPG expression following treatment was observed in clinical responders at the final time point (P < 0.05 vs. baseline), RANKL levels did not change, and the RANKL:OPG ratio remained unaltered, even at the highest levels of clinical response. When potential predictors of radiographic outcome were evaluated, baseline RANKL expression correlated with erosive progression at 1 year (r = 0.71, P < 0.01). Distinct, though related, pathophysiologic processes mediate joint inflammation and destruction in RA. Elevated synovial tissue RANKL expression is associated with progressive joint erosion, and may be independent of the clinical response to targeted therapy. The potential therapeutic importance of modulating RANKL in RA is highlighted, if radiographic arrest is to be achieved.

  2. Effects of caffeine on fractional flow reserve values measured using intravenous adenosine triphosphate.

    Science.gov (United States)

    Nakayama, Masafumi; Chikamori, Taishiro; Uchiyama, Takashi; Kimura, Yo; Hijikata, Nobuhiro; Ito, Ryosuke; Yuhara, Mikio; Sato, Hideaki; Kobori, Yuichi; Yamashina, Akira

    2018-04-01

    We investigated the effects of caffeine intake on fractional flow reserve (FFR) values measured using intravenous adenosine triphosphate (ATP) before cardiac catheterization. Caffeine is a competitive antagonist for adenosine receptors; however, it is unclear whether this antagonism affects FFR values. Patients were evenly randomized into 2 groups preceding the FFR study. In the caffeine group (n = 15), participants were given coffee containing 222 mg of caffeine 2 h before the catheterization. In the non-caffeine group (n = 15), participants were instructed not to take any caffeine-containing drinks or foods for at least 12 h before the catheterization. FFR was performed in patients with more than intermediate coronary stenosis using the intravenous infusion of ATP at 140 μg/kg/min (normal dose) and 170 μg/kg/min (high dose), and the intracoronary infusion of papaverine. FFR was followed for 30 s after maximal hyperemia. In the non-caffeine group, the FFR values measured with ATP infusion were not significantly different from those measured with papaverine infusion. However, in the caffeine group, the FFR values were significantly higher after ATP infusion than after papaverine infusion (P = 0.002 and P = 0.007, at normal and high dose ATP vs. papaverine, respectively). FFR values with ATP infusion were significantly increased 30 s after maximal hyperemia (P = 0.001 and P < 0.001 for normal and high dose ATP, respectively). The stability of the FFR values using papaverine showed no significant difference between the 2 groups. Caffeine intake before the FFR study affected FFR values and their stability. These effects could not be reversed by an increased ATP dose.

  3. [Transcranial magnetic stimulation].

    Science.gov (United States)

    Tormos, J M; Catalá, M D; Pascual-Leone, A

    Transcranial magnetic stimulation (TMS) permits stimulation of the cerebral cortex in humans without requiring open access to the brain and is one of the newest tools available in neuroscience. There are two main types of application: single-pulse TMS and repetitive TMS. The magnetic stimulator is composed of a series of capacitors that store the voltage necessary to generate a stimulus of the sufficient intensity of generate an electric field in the stimulation coil. The safety of TMS is supported by the considerable experience derived from studies involving electrical stimulation of the cortex in animals and humans, and also specific studies on the safety of TMS in humans. In this article we review historical and technical aspects of TMS, describe its adverse effects and how to avoid them, summarize the applications of TMS in the investigation of different cerebral functions, and discuss the possibility of using TMS for the treatment of neuropsychiatric disorders.

  4. Computational analysis of transcranial magnetic stimulation in the presence of deep brain stimulation probes

    Science.gov (United States)

    Syeda, F.; Holloway, K.; El-Gendy, A. A.; Hadimani, R. L.

    2017-05-01

    Transcranial Magnetic Stimulation is an emerging non-invasive treatment for depression, Parkinson's disease, and a variety of other neurological disorders. Many Parkinson's patients receive the treatment known as Deep Brain Stimulation, but often require additional therapy for speech and swallowing impairment. Transcranial Magnetic Stimulation has been explored as a possible treatment by stimulating the mouth motor area of the brain. We have calculated induced electric field, magnetic field, and temperature distributions in the brain using finite element analysis and anatomically realistic heterogeneous head models fitted with Deep Brain Stimulation leads. A Figure of 8 coil, current of 5000 A, and frequency of 2.5 kHz are used as simulation parameters. Results suggest that Deep Brain Stimulation leads cause surrounding tissues to experience slightly increased E-field (Δ Emax =30 V/m), but not exceeding the nominal values induced in brain tissue by Transcranial Magnetic Stimulation without leads (215 V/m). The maximum temperature in the brain tissues surrounding leads did not change significantly from the normal human body temperature of 37 °C. Therefore, we ascertain that Transcranial Magnetic Stimulation in the mouth motor area may stimulate brain tissue surrounding Deep Brain Stimulation leads, but will not cause tissue damage.

  5. Antifouling aptasensor for the detection of adenosine triphosphate in biological media based on mixed self-assembled aptamer and zwitterionic peptide.

    Science.gov (United States)

    Wang, Guixiang; Su, Xiaoli; Xu, Qingjun; Xu, Guiyun; Lin, Jiehua; Luo, Xiliang

    2018-03-15

    Direct detection of targets in complex biological media with conventional biosensors is an enormous challenge due to the nonspecific adsorption and severe biofouling. In this work, a facile strategy for sensitive and low fouling detection of adenosine triphosphate (ATP) is developed through the construction of a mixed self-assembled biosensing interface, which was composed of zwitterionic peptide (antifouling material) and ATP aptamer (bio-recognition element). The peptide and aptamer (both containing thiol groups) were simultaneously self-assembled onto gold electrode surface electrodeposited with gold nanoparticles. The developed aptasensor possessed high selectivity and sensitivity for ATP, and it showed a wide linear response range towards ATP from 0.1pM to 5nM. Owing to the presence of peptide with excellent antifouling property in the biosensing interface, the aptasensor can detect ATP in complex biological media with remarkably reduced biofouling or nonspecific adsorption effect. Moreover, it can directly detect ATP in 1% human whole blood without suffering from any significant interference, indicating its great potential for practical assaying of ATP in biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells

    Science.gov (United States)

    Yoneshima, Yasuto; Abolhassani, Nona; Iyama, Teruaki; Sakumi, Kunihiko; Shiomi, Naoko; Mori, Masahiko; Shiomi, Tadahiro; Noda, Tetsuo; Tsuchimoto, Daisuke; Nakabeppu, Yusaku

    2016-01-01

    Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. PMID:27618981

  7. Enzyme-free and label-free ultrasensitive electrochemical detection of DNA and adenosine triphosphate by dendritic DNA concatamer-based signal amplification.

    Science.gov (United States)

    Liu, Shufeng; Lin, Ying; Liu, Tao; Cheng, Chuanbin; Wei, Wenji; Wang, Li; Li, Feng

    2014-06-15

    Hybridization chain reaction (HCR) strategy has been well developed for the fabrication of various biosensing platforms for signal amplification. Herein, a novel enzyme-free and label-free ultrasensitive electrochemical DNA biosensing platform for the detection of target DNA and adenosine triphosphate (ATP) was firstly proposed, in which three auxiliary DNA probes were ingeniously designed to construct the dendritic DNA concatamer via HCR strategy and used as hexaammineruthenium(III) chloride (RuHex) carrier for signal amplification. With the developed dendritic DNA concatamer-based signal amplification strategy, the DNA biosensor could achieve an ultrasensitive electrochemical detection of DNA and ATP with a superior detection limit as low as 5 aM and 20 fM, respectively, and also demonstrate a high selectivity for DNA and ATP detection. The currently proposed dendritic DNA concatamer opens a promising direction to construct ultrasensitive DNA biosensing platform for biomolecular detection in bioanalysis and clinical biomedicine, which offers the distinct advantages of simplicity and cost efficiency owing to no need of any kind of enzyme, chemical modification or labeling. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Development of Flexible Capacitive Ultrasound Transducers and the Use of Ultrasound for Bone Repair

    Science.gov (United States)

    Wentzell, Scott A.

    Ultrasound is a widely applicable technique for therapy in the biomedical arena. However, conventional ultrasound transducers are not conducive for non-planar surfaces. Therefore, we developed flexible transducers capable of performing ultrasound and evaluated their use in biomedical applications. Flexible capacitive ultrasound transducers based on micrometer-thick dielectric tapes were developed and fabricated. These transducers were able to be made by hand at low-cost while still demonstrating good tolerances in center operating frequency. Intensities of up to 120 mW/cm2 were recorded and operation was dependent upon the applied AC and DC voltages along with the thickness of the dielectric insulation. These capacitive ultrasound transducers were used to stimulate MC3T3-E1 murine osteoblast cells to investigate the effects of low-frequency ultrasound on osteogenic gene expression and anabolic signaling pathways. After stimulation by 94.5 kHz continuous wave ultrasound for 20 minutes, significant increases in the activation of the Wnt signaling pathway and concentration of intracellular calcium were observed. Daily stimulation by ultrasound showed a trend of increased osteogenic gene expression across the phases of matrix deposition, maturation and calcification by osteoblasts. Finally, the heating of osteoblasts for stimulating osteoclastogenic responses was investigated. The application of increased temperatures of 42 and 47 degrees Celsius for 5 minutes showed significant increases in the RANKL/OPG ratio in media conditioned by osteoblasts. However, the altered RANKL/OPG ratio was not able to generate increases in osteoclastogenesis for RAW 264.7 murine macrophage cells culture in the condition media. This was possibly due to high overall osteoprotegerin expression, or unwanted inducement of M1 and M2 macrophage activation in the cell population. The overall work of this thesis demonstrates the development of novel capacitive transducers. These conformable

  9. Ternary Interactions and Energy Transfer between Fluorescein Isothiocyanate, Adenosine Triphosphate, and Graphene Oxide Nanocarriers.

    Science.gov (United States)

    Ratajczak, Katarzyna; Stobiecka, Magdalena

    2017-07-20

    The interactions of fluorescent probes and biomolecules with nanocarriers are of key importance to the emerging targeted drug delivery systems. Graphene oxide nanosheets (GONs) as the nanocarriers offer biocompatibility and robust drug binding capacity. The interactions of GONs with fluorophores lead to strong fluorescence quenching, which may interfere with fluorescence bioimaging and biodetection. Herein, we report on the interactions and energy transfers in a model ternary system: GONs-FITC-ATP, where FITC is a model fluorophore (fluorescein isothiocyanate) and ATP is a common biomolecule (adenosine-5'-triphosphate). We have found that FITC fluorescence is considerably quenched by ATP (the quenching constant K SV = 113 ± 22 M -1 ). The temperature coefficient of K SV is positive (α T = 4.15 M -1 deg -1 ). The detailed analysis of a model for internal self-quenching of FITC indicates that the temperature dependence of the net quenching efficiency η for the FITC-ATP pair is dominated by FITC internal self-quenching modes with their contribution estimated at 79%. The quenching of FITC by GONs is much stronger (K SV = 598 ± 29 M -1 ) than that of FITC-ATP and is associated with the formation of supramolecular assemblies bound with hydrogen bonding and π-π stacking interactions. For the analysis of the complex behavior of the ternary system GONs-FITC-ATP, a model of chemisorption of ATP on GONs, with partial blocking of FITC quenching, has been developed. Our results indicate that ATP acts as a moderator for FITC quenching by GONs. The interactions between ATP, FITC, and GONs have been corroborated using molecular dynamics and quantum mechanical calculations.

  10. Pudendal nerve stimulation and block by a wireless-controlled implantable stimulator in cats.

    Science.gov (United States)

    Yang, Guangning; Wang, Jicheng; Shen, Bing; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2014-07-01

    The study aims to determine the functionality of a wireless-controlled implantable stimulator designed for stimulation and block of the pudendal nerve. In five cats under α-chloralose anesthesia, the stimulator was implanted underneath the skin on the left side in the lower back along the sacral spine. Two tripolar cuff electrodes were implanted bilaterally on the pudendal nerves in addition to one bipolar cuff electrode that was implanted on the left side central to the tripolar cuff electrode. The stimulator provided high-frequency (5-20 kHz) biphasic stimulation waveforms to the two tripolar electrodes and low-frequency (1-100 Hz) rectangular pulses to the bipolar electrode. Bladder and urethral pressures were measured to determine the effects of pudendal nerve stimulation (PNS) or block. The maximal (70-100 cmH2O) urethral pressure generated by 20-Hz PNS applied via the bipolar electrode was completely eliminated by the pudendal nerve block induced by the high-frequency stimulation (6-15 kHz, 6-10 V) applied via the two tripolar electrodes. In a partially filled bladder, 20-30 Hz PNS (2-8 V, 0.2 ms) but not 5 Hz stimulation applied via the bipolar electrode elicited a large sustained bladder contraction (45.9 ± 13.4 to 52.0 ± 22 cmH2O). During cystometry, the 5 Hz PNS significantly (p < 0.05) increased bladder capacity to 176.5 ± 27.1% of control capacity. The wireless-controlled implantable stimulator successfully generated the required waveforms for stimulation and block of pudendal nerve, which will be useful for restoring bladder functions after spinal cord injury. © 2013 International Neuromodulation Society.

  11. Visual and surface plasmon resonance sensor for zirconium based on zirconium-induced aggregation of adenosine triphosphate-stabilized gold nanoparticles.

    Science.gov (United States)

    Qi, Wenjing; Zhao, Jianming; Zhang, Wei; Liu, Zhongyuan; Xu, Min; Anjum, Saima; Majeed, Saadat; Xu, Guobao

    2013-07-17

    Owing to its high affinity with phosphate, Zr(IV) can induce the aggregation of adenosine 5'-triphosphate (ATP)-stabilized AuNPs, leading to the change of surface plasmon resonance (SPR) absorption spectra and color of ATP-stabilized AuNP solutions. Based on these phenomena, visual and SPR sensors for Zr(IV) have been developed for the first time. The A(660 nm)/A(518 nm) values of ATP-stabilized AuNPs in SPR absorption spectra increase linearly with the concentrations of Zr(IV) from 0.5 μM to 100 μM (r=0.9971) with a detection limit of 95 nM. A visual Zr(IV) detection is achieved with a detection limit of 30 μM. The sensor shows excellent selectivity against other metal ions, such as Cu(2+), Fe(3+), Cd(2+), and Pb(2+). The recoveries for the detection of 5 μM, 10 μM, 25 μM and 75 μM Zr(IV) in lake water samples are 96.0%, 97.0%, 95.6% and 102.4%, respectively. The recoveries of the proposed SPR method are comparable with those of ICP-OES method. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

    Science.gov (United States)

    Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

    2016-06-01

    Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. A new brain stimulation method: Noninvasive transcranial magneto–acoustical stimulation

    International Nuclear Information System (INIS)

    Yuan Yi; Chen Yu-Dong; Li Xiao-Li

    2016-01-01

    We investigate transcranial magneto–acoustical stimulation (TMAS) for noninvasive brain neuromodulation in vivo. TMAS as a novel technique uses an ultrasound wave to induce an electric current in the brain tissue in the static magnetic field. It has the advantage of high spatial resolution and penetration depth. The mechanism of TMAS onto a neuron is analyzed by combining the TMAS principle and Hodgkin–Huxley neuron model. The anesthetized rats are stimulated by TMAS, resulting in the local field potentials which are recorded and analyzed. The simulation results show that TMAS can induce neuronal action potential. The experimental results indicate that TMAS can not only increase the amplitude of local field potentials but also enhance the effect of focused ultrasound stimulation on the neuromodulation. In summary, TMAS can accomplish brain neuromodulation, suggesting a potentially powerful noninvasive stimulation method to interfere with brain rhythms for diagnostic and therapeutic purposes. (paper)

  14. Spinal cord stimulation

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007560.htm Spinal cord stimulation To use the sharing features on this page, please enable JavaScript. Spinal cord stimulation is a treatment for pain that uses ...

  15. Stimulating thought: a functional MRI study of transcranial direct current stimulation in schizophrenia.

    Science.gov (United States)

    Orlov, Natasza D; O'Daly, Owen; Tracy, Derek K; Daniju, Yusuf; Hodsoll, John; Valdearenas, Lorena; Rothwell, John; Shergill, Sukhi S

    2017-09-01

    Individuals with schizophrenia typically suffer a range of cognitive deficits, including prominent deficits in working memory and executive function. These difficulties are strongly predictive of functional outcomes, but there is a paucity of effective therapeutic interventions targeting these deficits. Transcranial direct current stimulation is a novel neuromodulatory technique with emerging evidence of potential pro-cognitive effects; however, there is limited understanding of its mechanism. This was a double-blind randomized sham controlled pilot study of transcranial direct current stimulation on a working memory (n-back) and executive function (Stroop) task in 28 individuals with schizophrenia using functional magnetic resonance imaging. Study participants received 30 min of real or sham transcranial direct current stimulation applied to the left frontal cortex. The 'real' and 'sham' groups did not differ in online working memory task performance, but the transcranial direct current stimulation group demonstrated significant improvement in performance at 24 h post-transcranial direct current stimulation. Transcranial direct current stimulation was associated with increased activation in the medial frontal cortex beneath the anode; showing a positive correlation with consolidated working memory performance 24 h post-stimulation. There was reduced activation in the left cerebellum in the transcranial direct current stimulation group, with no change in the middle frontal gyrus or parietal cortices. Improved performance on the executive function task was associated with reduced activity in the anterior cingulate cortex. Transcranial direct current stimulation modulated functional activation in local task-related regions, and in more distal nodes in the network. Transcranial direct current stimulation offers a potential novel approach to altering frontal cortical activity and exerting pro-cognitive effects in schizophrenia. © The Author (2017). Published by Oxford

  16. Expression of Msx-1 is suppressed in bisphosphonate associated osteonecrosis related jaw tissue-etiopathology considerations respecting jaw developmental biology-related unique features.

    Science.gov (United States)

    Wehrhan, Falk; Hyckel, Peter; Ries, Jutta; Stockmann, Phillip; Nkenke, Emeka; Schlegel, Karl A; Neukam, Friedrich W; Amann, Kerstin

    2010-10-13

    Bone-destructive disease treatments include bisphosphonates and antibodies against the osteoclast differentiator, RANKL (aRANKL); however, osteonecrosis of the jaw (ONJ) is a frequent side-effect. Current models fail to explain the restriction of bisphosphonate (BP)-related and denosumab (anti-RANKL antibody)-related ONJ to jaws. Msx-1 is exclusively expressed in craniofacial structures and pivotal to cranial neural crest (CNC)-derived periodontal tissue remodeling. We hypothesised that Msx-1 expression might be impaired in bisphosphonate-related ONJ. The study aim was to elucidate Msx-1 and RANKL-associated signal transduction (BMP-2/4, RANKL) in ONJ-altered and healthy periodontal tissue. Twenty ONJ and twenty non-BP exposed periodontal samples were processed for RT-PCR and immunohistochemistry. An automated staining-based alkaline phosphatase-anti-alkaline phosphatase method was used to measure the stained cells:total cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed on ONJ-affected and healthy jaw periodontal samples (n = 20 each) to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels. Semi-quantitative assessment of the ratio of stained cells showed decreased Msx-1 and RANKL and increased BMP-2/4 (all p Msx-1 (p Msx-1 suppression in ONJ-adjacent periodontal tissue suggested a bisphosphonate-related impairment in cellular differentiation that occurred exclusively jaw remodelling. Further research on developmental biology-related unique features of jaw bone structures will help to elucidate pathologies restricted to maxillofacial tissue.

  17. Spectroscopic study of the interaction between adenosine disodium triphosphate and gatifloxacin-Al3+ complex and its analytical application.

    Science.gov (United States)

    Kamruzzaman, Mohammad; Faruqui, A Nayeem; Hossain, Mohammed Ifteker; Lee, Sang Hak

    2015-11-01

    A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)-aluminium (III) (Al(3+) ) complex and ATP and studied using UV-visible and fluorescence spectroscopy. Weak luminescence spectra of Al(3+) were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al(3+) ion. It was observed that the FL emission spectrum of GFLX-Al(3+) was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10(-10) - 1.0 × 10(-8) mol L(-1) with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10(-11) mol L(-1) for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10(-8) mol L(-1) ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Damage to adenosine-triphosphate induced by monochromatic X rays around the K shell absorption edge of phosphorus

    International Nuclear Information System (INIS)

    Watanabe, Ritsuko; Ishikawa, Mitsuo; Takakura, Kaoru; Kobayashi, Katsumi

    1992-01-01

    Adenosine-triphosphate (ATP) is well known to have an important role in the energy metabolism in biological systems. The purpose of this study is to clarify the radiation effects on ATP specific to inner shell ionization. ATP, in concentrated aqueous solution, was irradiated with monochromatic X rays having energies of the resonance absorption peak of the phosphorus K shell, 2.153 keV, and slightly below and above the peak, 2.145 keV and 2.160 keV, selected from synchrotron radiation. Adenine, Adenosine 5'monophosphate (5'AMP) and Adenosine 5'diphosphate (5'ADP) were obtained as radioproducts by the method of high performance liquid chromatography (HPLC). G values of these products were calculated on the basis of the absorbed energy. When the ATP solution of 0.282 mol/l was irradiated with 2.160 keV X rays which can ionize the K shell of phosphorus, G values of Adenine, 5'AMP and 5'ADP were estimated to be 1.4, 0.40 and 0.46, respectively. These values were respectively 1.3, 2.9 and 3.8 times higher than those obtained upon irradiation with 2.146 keV X rays which cannot ionize the K shell of phosphorus. These energy dependent enhancements may reflect the difference in energy absorption processes, especially the Auger cascade in phosphorus may be suspected to play an important role in these enhancements

  19. Kinetic, spectroscopic and chemical modification study of iron release from transferrin; iron(III) complexation to adenosine triphosphate

    International Nuclear Information System (INIS)

    Thompson, C.P.

    1985-01-01

    Amino acids other than those that serve as ligands have been found to influence the chemical properties of transferrin iron. The catalytic ability of pyrophosphate to mediate transferrin iron release to a terminal acceptor is largely quenched by modification non-liganded histine groups on the protein. The first order rate constants of iron release for several partially histidine modified protein samples were measured. A statistical method was employed to establish that one non-liganded histidine per metal binding domain was responsible for the reduction in rate constant. These results imply that the iron mediated chelator, pyrophosphate, binds directly to a histidine residue on the protein during the iron release process. EPR spectroscopic results are consistent with this interpretation. Kinetic and amino acid sequence studies of ovotransferrin and lactoferrin, in addition to human serum transferrin, have allowed the tentative assignment of His-207 in the N-terminal domain and His-535 in the C-terminal domain as the groups responsible for the reduction in rate of iron release. The above concepts have been extended to lysine modified transferrin. Complexation of iron(II) to adenosine triphosphate (ATP) was also studied to gain insight into the nature of iron-ATP species present at physiological pH. 31 P NMR spectra are observed when ATP is presented in large excess

  20. Clinical characteristics in patients showing ischemic electrocardiographic changes during adenosine triphosphate loading single-photon emission computed tomography

    International Nuclear Information System (INIS)

    Ohtaki, Yuka; Chikamori, Taishiro; Hida, Satoshi; Tanaka, Hirokazu; Igarashi, Yuko; Hatano, Tsuguhisa; Usui, Yasuhiro; Miyagi, Manabu; Yamashina, Akira

    2010-01-01

    Although ischemic electrocardiographic (ECG) changes during dipyridamole or adenosine infusion have been reported as a marker for severe coronary artery disease (CAD), few studies have focused on ST-segment changes with adenosine triphosphate (ATP)-loading myocardial single-photon emission computed tomography (SPECT). Between January 2003 and August 2008, 4650 consecutive patients underwent ATP-loading SPECT. After 1412 patients with left bundle branch block, pacemaker rhythm, or previous coronary revascularization were excluded, 16 out of 3238 patients (0.5%) showed ischemic ST-segment depression during ATP-loading myocardial SPECT. They were aged 67±11 years; 10 were men and 6 women. Of these patients, 8 demonstrated perfusion abnormalities, whereas the remaining 8 showed normal myocardial perfusion imaging. In 6 of the 8 patients with abnormal SPECT, coronary angiography was performed, revealing left main trunk disease in 1 patient, 3-vessel disease in 4, 1-vessel disease with proximal left ascending artery occlusion in 1, and an insignificant lesion in 1. By contrast, no major cardiac event was observed in the 8 patients with normal SPECT during follow-up for an average of 2 years. The prevalence of ischemic ST-segment changes during ATP loading is very rare. However, this finding should be taken into account since almost half of the patients, particularly those with perfusion abnormalities, may have severe CAD which requires coronary revascularization. (author)

  1. Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

    Science.gov (United States)

    Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

    2015-03-03

    A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

  2. Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

    Science.gov (United States)

    Bushon, R.N.; Brady, A.M.; Likirdopulos, C.A.; Cireddu, J.V.

    2009-01-01

    Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

  3. The Codacs™ direct acoustic cochlear implant actuator: exploring alternative stimulation sites and their stimulation efficiency.

    Science.gov (United States)

    Grossöhmichen, Martin; Salcher, Rolf; Kreipe, Hans-Heinrich; Lenarz, Thomas; Maier, Hannes

    2015-01-01

    This work assesses the efficiency of the Codacs system actuator (Cochlear Ltd., Sydney Australia) in different inner ear stimulation modalities. Originally the actuator was intended for direct perilymph stimulation after stapedotomy using a piston prosthesis. A possible alternative application is the stimulation of middle ear structures or the round window (RW). Here the perilymph stimulation with a K-piston through a stapes footplate (SFP) fenestration (N = 10) as well as stimulation of the stapes head (SH) with a Bell prosthesis (N = 9), SFP stimulation with an Omega/Aerial prosthesis (N = 8) and reverse RW stimulation (N = 10) were performed in cadaveric human temporal bones (TBs). Codacs actuator output is expressed as equivalent sound pressure level (eq. SPL) using RW and SFP displacement responses, measured by Laser Doppler velocimetry as reference. The axial actuator coupling force in stimulation of stapes and RW was adjusted to ~5 mN. The Bell prosthesis and Omega/Aerial prosthesis stimulation generated similar mean eq. SPLs (Bell: 127.5-141.8 eq. dB SPL; Omega/Aerial: 123.6-143.9 eq. dB SPL), being significantly more efficient than K-piston perilymph stimulation (108.6-131.6 eq. dB SPL) and RW stimulation (108.3-128.2 eq. dB SPL). Our results demonstrate that SH, SFP and RW are adequate alternative stimulation sites for the Codacs actuator using coupling prostheses and an axial coupling force of ~5 mN. Based on the eq. SPLs, all investigated methods were adequate for in vivo hearing aid applications, provided that experimental conditions including constant coupling force will be implemented.

  4. Role of CXC chemokine ligand 13 in oral squamous cell carcinoma associated osteolysis in athymic mice.

    Science.gov (United States)

    Pandruvada, Subramanya N M; Yuvaraj, Sambandam; Liu, Xiang; Sundaram, Kumaran; Shanmugarajan, Srinivasan; Ries, William L; Norris, James S; London, Steven D; Reddy, Sakamuri V

    2010-05-15

    Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion; however, the molecular mechanisms of tumor osteolysis are unclear. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in OSCC cells (SCC1, SCC12 and SCC14a). OSCC cell-conditioned media (20%) induced osteoclast differentiation which was inhibited by OPG in peripheral blood monocyte cultures indicating that OSCC cells produce soluble RANKL. Recombinant hCXCL13 (10 ng/ml) significantly enhanced RANKL-stimulated osteoclast differentiation in these cultures. Trans-well migration assay identified that CXCL13 induces chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti-CXCR5 receptor antibody. Zymogram analysis of conditioned media from OSCC cells revealed matrix metalloproteinase-9 (MMP-9) activity. Interestingly, CXCL13 treatment to OSCC cells induced CXCR5 and MMP-9 expression suggesting an autocrine regulatory function in OSCC cells. To examine the OSCC tumor cell bone invasion/osteolysis, we established an in vivo model for OSCC by subcutaneous injection of OSCC cells onto the surface of calvaria in NCr-nu/nu athymic mice, which developed tumors in 4-5 weeks. muCT analysis revealed numerous osteolytic lesions in calvaria from OSCC tumor-bearing mice. Histochemical staining of calvarial sections from these mice revealed a significant increase in the numbers of TRAP-positive osteoclasts at the tumor-bone interface. Immunohistochemical analysis confirmed CXCL13 and MMP-9 expression in tumor cells. Thus, our data implicate a functional role for CXCL13 in bone invasion and may be a potential therapeutic target to prevent osteolysis associated with OSCC tumors in vivo.

  5. In vitro magnetic stimulation: a simple stimulation device to deliver defined low intensity electromagnetic fields

    Directory of Open Access Journals (Sweden)

    Stephanie Grehl

    2016-11-01

    Full Text Available Non-invasive electromagnetic field brain stimulation (NIBS appears to benefit human neurological and psychiatric conditions, although the optimal stimulation parameters and underlying mechanisms remain unclear. Although in vitro studies have begun to elucidate cellular mechanisms, stimulation is delivered by a range of coils (from commercially available human stimulation coils to laboratory-built circuits so that the electromagnetic fields induced within the tissue to produce the reported effects are ill-defined.Here we develop a simple in vitro stimulation device with plug-and-play features that allow delivery of a range of stimulation parameters. We chose to test low intensity repetitive magnetic stimulation (LI-rMS delivered at 3 frequencies to hindbrain explant cultures containing the olivocerebellar pathway. We used computational modelling to define the parameters of a stimulation circuit and coil that deliver a unidirectional homogeneous magnetic field of known intensity and direction, and therefore a predictable electric field, to the target. We built the coil to be compatible with culture requirements: stimulation within an incubator; a flat surface allowing consistent position and magnetic field direction; location outside the culture plate to maintain sterility and no heating or vibration. Measurements at the explant confirmed the induced magnetic field was homogenous and matched the simulation results. To validate our system we investigated biological effects following LI-rMS at 1 Hz, 10 Hz and biomimetic high frequency (BHFS, which we have previously shown induces neural circuit reorganisation. We found that gene expression was modified by LI-rMS in a frequency-related manner. Four hours after a single 10-minute stimulation session, the number of c-fos positive cells increased, indicating that our stimulation activated the tissue. Also, after 14 days of LI-rMS, the expression of genes normally present in the tissue was differentially

  6. The Codacs™ direct acoustic cochlear implant actuator: exploring alternative stimulation sites and their stimulation efficiency.

    Directory of Open Access Journals (Sweden)

    Martin Grossöhmichen

    Full Text Available This work assesses the efficiency of the Codacs system actuator (Cochlear Ltd., Sydney Australia in different inner ear stimulation modalities. Originally the actuator was intended for direct perilymph stimulation after stapedotomy using a piston prosthesis. A possible alternative application is the stimulation of middle ear structures or the round window (RW. Here the perilymph stimulation with a K-piston through a stapes footplate (SFP fenestration (N = 10 as well as stimulation of the stapes head (SH with a Bell prosthesis (N = 9, SFP stimulation with an Omega/Aerial prosthesis (N = 8 and reverse RW stimulation (N = 10 were performed in cadaveric human temporal bones (TBs. Codacs actuator output is expressed as equivalent sound pressure level (eq. SPL using RW and SFP displacement responses, measured by Laser Doppler velocimetry as reference. The axial actuator coupling force in stimulation of stapes and RW was adjusted to ~5 mN. The Bell prosthesis and Omega/Aerial prosthesis stimulation generated similar mean eq. SPLs (Bell: 127.5-141.8 eq. dB SPL; Omega/Aerial: 123.6-143.9 eq. dB SPL, being significantly more efficient than K-piston perilymph stimulation (108.6-131.6 eq. dB SPL and RW stimulation (108.3-128.2 eq. dB SPL. Our results demonstrate that SH, SFP and RW are adequate alternative stimulation sites for the Codacs actuator using coupling prostheses and an axial coupling force of ~5 mN. Based on the eq. SPLs, all investigated methods were adequate for in vivo hearing aid applications, provided that experimental conditions including constant coupling force will be implemented.

  7. Msx-1 is suppressed in bisphosphonate-exposed jaw bone analysis of bone turnover-related cell signalling after bisphosphonate treatment.

    Science.gov (United States)

    Wehrhan, F; Hyckel, P; Amann, K; Ries, J; Stockmann, P; Schlegel, Ka; Neukam, Fw; Nkenke, E

    2011-05-01

    Bone-destructive disease treatments include bisphosphonates and antibodies against receptor activator for nuclear factor κB ligand (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Aetiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonate-related ONJ. This study compared the expression of Msx-1, Bone Morphogenetic Protein (BMP)-2 and RANKL, in ONJ-affected and healthy jaw bone. An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n = 20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL and GAPDH mRNA levels. Labelling indices were significantly lower for Msx-1 (P Msx-1, 22-fold lower (P Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonate-associated osteopetrosis and impaired bone remodelling in BP- and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explanation of the exclusivity of ONJ in jaw bone. Functional analyses of Msx-1- RANKL interaction during bone remodelling should be performed in the future. © 2011 John Wiley & Sons A/S.

  8. [The expression and significance of receptor activator of nuclear factor kappaB ligand and osteoprotegerin in periapical cyst and periapical granuloma].

    Science.gov (United States)

    Zhang, Meihua; Yu, Yunzhi; Miao, Yu

    2012-08-01

    To investigate the expression of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) in periapical cyst and periapical granuloma by comparison with the expression in the normal periodontal tissue as control, and to identify their functional mechanism in the bone destruction of periapical cyst and granuloma. 20 periapical cyst tissues (cyst group), 20 periapical granuloma tissues (granuloma group), and 20 normal periodontal tissues (control group) were collected respectively. Immunohistochemical technology was performed to detect the expression of RANKL and OPG in above three groups. In cyst group, granuloma group and control group, the expression of RANKL were 75.00 +/- 7.54, 68.40 +/- 6.74 and 29.40 +/- 2.46, respectively. The expression of OPG were 38.10 +/- 7.09, 47.65 +/- 13.85 and 58.60 +/- 5.88, respectively. The differences among the three groups were statistically significant (Pcysts group were negatively correlated (r=-0.56, P=0.01) and were not correlated with granuloma and control group (P>0.05). RANKL and OPG play roles in the bone absorption of periapical disease. In periapical disease, abnormal expression of RANKL and OPG are detected, RANKL significantly increase, OPG decrease, bone absorption accelerate and osteolytic lesion are observed. In periapical cyst, the bone absorption is more active compared with periapical granuloma.

  9. Higher success rate with transcranial electrical stimulation of motor-evoked potentials using constant-voltage stimulation compared with constant-current stimulation in patients undergoing spinal surgery.

    Science.gov (United States)

    Shigematsu, Hideki; Kawaguchi, Masahiko; Hayashi, Hironobu; Takatani, Tsunenori; Iwata, Eiichiro; Tanaka, Masato; Okuda, Akinori; Morimoto, Yasuhiko; Masuda, Keisuke; Tanaka, Yuu; Tanaka, Yasuhito

    2017-10-01

    During spine surgery, the spinal cord is electrophysiologically monitored via transcranial electrical stimulation of motor-evoked potentials (TES-MEPs) to prevent injury. Transcranial electrical stimulation of motor-evoked potential involves the use of either constant-current or constant-voltage stimulation; however, there are few comparative data available regarding their ability to adequately elicit compound motor action potentials. We hypothesized that the success rates of TES-MEP recordings would be similar between constant-current and constant-voltage stimulations in patients undergoing spine surgery. The objective of this study was to compare the success rates of TES-MEP recordings between constant-current and constant-voltage stimulation. This is a prospective, within-subject study. Data from 100 patients undergoing spinal surgery at the cervical, thoracic, or lumbar level were analyzed. The success rates of the TES-MEP recordings from each muscle were examined. Transcranial electrical stimulation with constant-current and constant-voltage stimulations at the C3 and C4 electrode positions (international "10-20" system) was applied to each patient. Compound muscle action potentials were bilaterally recorded from the abductor pollicis brevis (APB), deltoid (Del), abductor hallucis (AH), tibialis anterior (TA), gastrocnemius (GC), and quadriceps (Quad) muscles. The success rates of the TES-MEP recordings from the right Del, right APB, bilateral Quad, right TA, right GC, and bilateral AH muscles were significantly higher using constant-voltage stimulation than those using constant-current stimulation. The overall success rates with constant-voltage and constant-current stimulations were 86.3% and 68.8%, respectively (risk ratio 1.25 [95% confidence interval: 1.20-1.31]). The success rates of TES-MEP recordings were higher using constant-voltage stimulation compared with constant-current stimulation in patients undergoing spinal surgery. Copyright © 2017

  10. Paracrine stimulation of P2X7 receptor by ATP activates a proliferative pathway in ovarian carcinoma cells.

    Science.gov (United States)

    Vázquez-Cuevas, Francisco G; Martínez-Ramírez, Angélica S; Robles-Martínez, Leticia; Garay, Edith; García-Carrancá, Alejandro; Pérez-Montiel, Delia; Castañeda-García, Carolina; Arellano, Rogelio O

    2014-11-01

    P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability. © 2014 Wiley Periodicals, Inc.

  11. Music acupuncture stimulation method.

    Science.gov (United States)

    Brătilă, F; Moldovan, C

    2007-01-01

    Harmonic Medicine is the model using the theory that the body rhythms synchronize to an outer rhythm applied for therapeutic purpose, can restores the energy balance in acupuncture channels and organs and the condition of well-being. The purpose of this scientific work was to demonstrate the role played by harmonic sounds in the stimulation of the Lung (LU) Meridian (Shoutaiyin Feijing) and of the Kidney (KI) Meridian (Zushaoyin Shenjing). It was used an original method that included: measurement and electronic sound stimulation of the Meridian Entry Point, measurement of Meridian Exit Point, computer data processing, bio feed-back adjustment of the music stimulation parameters. After data processing, it was found that the sound stimulation of the Lung Meridian Frequency is optimal between 122 Hz and 128 Hz, with an average of 124 Hz (87% of the subjects) and for Kidney Meridian from 118 Hz to 121 Hz, with an average of 120 Hz (67% of the subjects). The acupuncture stimulation was more intense for female subjects (> 7%) than for the male ones. We preliminarily consider that an informational resonance phenomenon can be developed between the acupuncture music stimulation frequency and the cellular dipole frequency, being a really "resonant frequency signature" of an acupoint. The harmonic generation and the electronic excitation or low-excitation status of an acupuncture point may be considered as a resonance mechanism. By this kind of acupunctural stimulation, a symphony may act and play a healer role.

  12. Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors.

    Science.gov (United States)

    Hassan, Hammad Ali; Rani, Sadaf; Fatima, Tabeer; Kiani, Farooq Ahmad; Fischer, Stefan

    2017-11-01

    Hydrolysis of phosphate groups is a crucial reaction in living cells. It involves the breaking of two strong bonds, i.e. the O a H bond of the attacking water molecule, and the PO l bond of the substrate (O a and O l stand for attacking and leaving oxygen atoms). Mechanism of the hydrolysis reaction can proceed either by a concurrent or a sequential mechanism. In the concurrent mechanism, the breaking of O a H and PO l bonds occurs simultaneously, whereas in the sequential mechanism, the O a H and PO l bonds break at different stages of the reaction. To understand how protonation affects the mechanism of hydrolysis of phosphate monoester, we have studied the mechanism of hydrolysis of protonated and deprotonated phosphate monoester at M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory (where ZPE stands for zero point energy). Our calculations show that in both protonated and deprotonated cases, the breaking of the water O a H bond occurs before the breaking of the PO l bond. Because the two events are not separated by a stable intermediate, the mechanism can be categorized as semi-concurrent. The overall energy barrier is 41kcalmol -1 in the unprotonated case. Most (5/6th) of this is due to the initial breaking of the water O a H bond. This component is lowered from 34 to 25kcalmol -1 by adding one proton to the phosphate. The rest of the overall energy barrier comes from the subsequent breaking of the PO l bond and is not sensitive to protonation. This is consistent with previous findings about the effect of triphosphate protonation on the hydrolysis, where the equivalent protonation (on the γ-phosphate) was seen to lower the barrier of breaking the water O a H bond and to have little effect on the PO l bond breaking. Hydrolysis pathways of phosphate monoester with initial breaking of the PO l bond could not be found here. This is because the leaving group in phosphate monoester cannot be protonated, unlike in triphosphate hydrolysis, where protonation of the

  13. Low intensity transcranial electric stimulation

    DEFF Research Database (Denmark)

    Antal, Andrea; Alekseichuk, I; Bikson, M

    2017-01-01

    Low intensity transcranial electrical stimulation (TES) in humans, encompassing transcranial direct current (tDCS), transcutaneous spinal Direct Current Stimulation (tsDCS), transcranial alternating current (tACS), and transcranial random noise (tRNS) stimulation or their combinations, appears...

  14. Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

    Science.gov (United States)

    Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

    2014-07-15

    A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

  15. Adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided versus empirical chemotherapy in unresectable non-small cell lung cancer.

    Science.gov (United States)

    Moon, Yong Wha; Sohn, Joo Hyuk; Kim, Yong Tai; Chang, Hyun; Jeong, Jae Heon; Lee, Young Joo; Chang, Joon; Kim, Se Kyu; Jung, Minkyu; Hong, Soojung; Choi, Sung Ho; Kim, Joo-Hang

    2009-10-01

    We retrospectively compared adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided and empirical chemotherapies for unresectable non-small cell lung cancer (NSCLC) in this case-control study. Unresectable NSCLC patients receiving ATP-CRA-guided platinum-based doublets as first-line therapy were enrolled as cases (n=27; 14 platinum-sensitive and 13 platinum-resistant patients). Performance status, stage, and chemotherapeutic regimen-matched patients receiving empirical chemotherapy were selected from the retrospective database as controls (n=93) in a case to control ratio of approximately 1:3. Response rate and survival (progression-free; overall) in both groups were not significantly different. However, the platinum-sensitive subgroup by ATP-CRA showed a higher response rate than the empirical group (71 versus 38%; p=0.023) with a trend toward longer progression-free survival (8.7 versus 4.8 months for platinum-sensitive versus empirical; p=0.223) and overall survival (not reached versus 12.6 months for platinum-sensitive versus empirical for p=0.134). ATP-CRA may be helpful in selecting platinum-responsive patients in unresectable NSCLC. We consider that nonplatinum doublets in platinum-resistant patients by ATP-CRA may be a more adapted approach than platinum-based doublets in future clinical trials.

  16. A feasibility study of adenosine triphosphate-based chemotherapy response assay (ATP-CRA) as a chemosensitivity test for lung cancer.

    Science.gov (United States)

    Kang, Shin Myung; Park, Moo Suk; Chang, Joon; Kim, Se Kyu; Kim, Haeryoung; Shin, Dong-Hwan; Chung, Kyung Young; Kim, Dae Joon; Sohn, Joo Hyuk; Choi, Sung Ho; Kim, Jeongmi; Yoon, Eun Jin; Kim, Joo-Hang

    2005-08-01

    A chemosensitivity test can reflect the differences in responses of individual cancer patients to chemotherapeutic agents. The adenosine triphosphate-based chemotherapy response assay (ATP-CRA) is an accurate method, which does not require a large amount of tissue specimen. So far, no studies have evaluated the utility of the ATP-CRA in Korea. Therefore, we investigated the clinical usefulness of the ATP-CRA in 53 patients with lung cancer. Tumor tissues were obtained from bronchoscopic biopsies or surgical resections. The validity of ATP-CRA was assessed focusing on the success rate, experimental error level (intraassay mean coefficient of variation [CV]) and reproducibility. The overall success rate of ATP-CRA was 90.6% (48/53). Normal cells were effectively eliminated from the tumor tissues with the use of ficoll gradient centrifugation and immunomagnetic separation, which was confirmed using loss of heterozygosity analysis of the 3p deletion. The mean CV of ATP assays was 10.5+/-4.6%. The reproducibility of ATP assays was 94+/-3.8%. The results of the ATP assays were reported to physicians within 7 days of specimen collection. More than 6 anticancer drugs were tested on the tumor specimens obtained from bronchoscopic biopsies. The ATP-CRA is a stable, accurate and potentially practical chemosensitivity test in patients with lung cancer.

  17. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

    Science.gov (United States)

    Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

    2014-11-01

    The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

  18. Approximating transcranial magnetic stimulation with electric stimulation in mouse: a simulation study.

    Science.gov (United States)

    Barnes, Walter L; Lee, Won Hee; Peterchev, Angel V

    2014-01-01

    Rodent models are valuable for preclinical examination of novel therapeutic techniques, including transcranial magnetic stimulation (TMS). However, comparison of TMS effects in rodents and humans is confounded by inaccurate scaling of the spatial extent of the induced electric field in rodents. The electric field is substantially less focal in rodent models of TMS due to the technical restrictions of making very small coils that can handle the currents required for TMS. We examine the electric field distributions generated by various electrode configurations of electric stimulation in an inhomogeneous high-resolution finite element mouse model, and show that the electric field distributions produced by human TMS can be approximated by electric stimulation in mouse. Based on these results and the limits of magnetic stimulation in mice, we argue that the most practical and accurate way to model focal TMS in mice is electric stimulation through either cortical surface electrodes or electrodes implanted halfway through the mouse cranium. This approach could allow much more accurate approximation of the human TMS electric field focality and strength than that offered by TMS in mouse, enabling, for example, focal targeting of specific cortical regions, which is common in human TMS paradigms.

  19. Interaction of transcranial magnetic stimulation and electrical transmastoid stimulation in human subjects

    DEFF Research Database (Denmark)

    Taylor, Janet L; Petersen, Nicolas Caesar; Butler, Jane E

    2002-01-01

    Transcranial magnetic stimulation activates corticospinal neurones directly and transsynaptically and hence, activates motoneurones and results in a response in the muscle. Transmastoid stimulation results in a similar muscle response through activation of axons in the spinal cord. This study...... was designed to determine whether the two stimuli activate the same descending axons. Responses to transcranial magnetic stimuli paired with electrical transmastoid stimuli were examined in biceps brachii in human subjects. Twelve interstimulus intervals (ISIs) from -6 ms (magnet before transmastoid) to 5 ms......-wave, facilitation still occurred at ISIs of -6 and -5 ms and depression of the paired response at ISIs of 0, 1, 4 and 5 ms. The interaction of the response to transmastoid stimulation with the multiple descending volleys elicited by magnetic stimulation of the cortex is complex. However, depression of the response...

  20. Feldspar, Infrared Stimulated Luminescence

    DEFF Research Database (Denmark)

    Jain, Mayank

    2014-01-01

    This entry primarily concerns the characteristics and the origins of infrared-stimulated luminescence in feldspars.......This entry primarily concerns the characteristics and the origins of infrared-stimulated luminescence in feldspars....

  1. [Immunoexpression and clinical significance of interleukin-21 and receptor activator of nuclear factor κB ligand in human periapical granulomas and radicular cysts].

    Science.gov (United States)

    Hu, Juhua; Li, Qian; Wang, Yanqing; Li, Song

    2015-06-01

    This study aimed to detect the immunoexpression of interleukin-21 (IL-21) and receptor activator. of nuclear factor KB ligand (RANKL) in periapical granulomas (PGs) and radicular cysts (RCs). The interaction of IL-21 with RANKL and its role in periapical pathogenesis were also speculated. A total of 32 PGs and 23 RCs were selected as experimental samples. Lesion size and occurrence of tenderness were recorded. Up to 10 healthy gingival tissues were collected as normal control samples. All tissues were subjected to immunohistocheincal analysis with anti-human IL-21 and RANKL polyclonal antibodies. The correlations of IL-21 with RANKL, lesion size, and the occurrence of tenderness of the PGs and RCs were evaluated. IL-21-positive cells were detected in all periapical lesion tissues but not in normal tissues. In the cyst group and granuloma group, the corresponding expression levels of IL-21 were 59.92±6.57 and 36.80± 6.81, whereas those of RANKL were 68.81±18.59 and 36.12±14.87, respectively. Moreover, t-test revealed a significantly higher expression of IL-21 and RANKL in RCs than in PGs (P<0.05). IL-21 and RANKL were positively correlated in both PGs and RCs (P<0.05). Furthermore, IL-21 was correlated with lesion size (P<0.05). This study demonstrated that IL-21 is potentially involved in the pathogenesis of apical periodontitis lesions. A role in the exacerbation of chronic inflammation, as well as in bone resorption, is suspected. Further studies are required to elucidate the specific functions of IL-21 in periradicular inflammatory processes.

  2. Noninvasive Transcranial Brain Stimulation and Pain

    OpenAIRE

    Rosen, Allyson C.; Ramkumar, Mukund; Nguyen, Tam; Hoeft, Fumiko

    2009-01-01

    Transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) are two noninvasive brain stimulation techniques that can modulate activity in specific regions of the cortex. At this point, their use in brain stimulation is primarily investigational; however, there is clear evidence that these tools can reduce pain and modify neurophysiologic correlates of the pain experience. TMS has also been used to predict response to surgically implanted stimulation for the tre...

  3. Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride

    International Nuclear Information System (INIS)

    Quirk, S.M.; Reichert, L.E. Jr.

    1988-01-01

    Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-[2-3H] inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total [3H]inositol phosphates [[3H]inositol mono-, di-, and triphosphates (IP, IP2, and IP3)] in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%

  4. Melatonin-Stimulated Triacylglycerol Breakdown and Energy Turnover under Salinity Stress Contributes to the Maintenance of Plasma Membrane H+-ATPase Activity and K+/Na+ Homeostasis in Sweet Potato.

    Science.gov (United States)

    Yu, Yicheng; Wang, Aimin; Li, Xiang; Kou, Meng; Wang, Wenjun; Chen, Xianyang; Xu, Tao; Zhu, Mingku; Ma, Daifu; Li, Zongyun; Sun, Jian

    2018-01-01

    Melatonin (MT) is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [ Ipomoea batatas (L.) Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K + /Na + homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K + /Na + homeostasis in sweet potato seedlings as indicated by the low reduced K + content in tissues and low accumulation of Na + content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K + efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM) H + -ATPase activity and intracellular adenosine triphosphate (ATP) level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG) accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA) β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H + -ATPase activity, and K + /Na + homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H + -ATPase activity and K + /Na + homeostasis in sweet potato.

  5. Age-associated metabolic and morphologic changes in mitochondria of individual mouse and hamster oocytes.

    Directory of Open Access Journals (Sweden)

    Fatma Simsek-Duran

    Full Text Available BACKGROUND: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus and mouse (Mus musculus ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. METHODOLOGY/PRINCIPAL FINDINGS: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. CONCLUSIONS/SIGNIFICANCE: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

  6. Growth hormone stimulation test

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003377.htm Growth hormone stimulation test To use the sharing features on this page, please enable JavaScript. The growth hormone (GH) stimulation test measures the ability of ...

  7. Role of hemolysis in red cell adenosine triphosphate release in simulated exercise conditions in vitro.

    Science.gov (United States)

    Mairbäurl, Heimo; Ruppe, Florian A; Bärtsch, Peter

    2013-10-01

    Specific adenosine triphosphate (ATP) release from red blood cells has been discussed as a possible mediator controlling microcirculation in states of decreased tissue oxygen. Because intravascular hemolysis might also contribute to plasma ATP, we tested in vitro which portion of ATP release is due to hemolysis in typical exercise-induced strains to the red blood cells (shear stress, deoxygenation, and lactic acidosis). Human erythrocytes were suspended in dextran-containing media (hematocrit 10%) and were exposed to shear stress in a rotating Couette viscometer at 37°C. Desaturation (oxygen saturation of hemoglobin ∼20%) was achieved by tonometry with N2 before shear stress exposure. Cells not exposed to shear stress were used as controls. Na lactate (15 mM), lactic acid (15 mM, pH 7.0), and HCl (pH 7.0) were added to simulate exercise-induced lactic acidosis. After incubation, extracellular hemoglobin was measured to quantify hemolysis. ATP was measured with the luciferase assay. Shear stress increased extracellular ATP in a stress-related and time-dependent manner. Hypoxia induced a ∼10-fold increase in extracellular ATP in nonsheared cells and shear stress-exposed cells. Lactic acid had no significant effect on ATP release and hemolysis. In normoxic cells, approximately 20%-50% of extracellular ATP was due to hemolysis. This proportion decreased to less than 10% in hypoxic cells. Our results indicate that when exposing red blood cells to typical strains they encounter when passing through capillaries of exercising skeletal muscle, ATP release from red blood cells is caused mainly by deoxygenation and shear stress, whereas lactic acidosis had only a minor effect. Hemolysis effects were decreased when hemoglobin was deoxygenated. Together, by specific release and hemolysis, extracellular ATP reaches values that have been shown to cause local vasodilatation.

  8. Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Haruhiko [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Nakashima, Tomoki [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, PRESTO, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Mikihito [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Izawa, Naohiro; Yasui, Tetsuro [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Aburatani, Hiroyuki [Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8904 (Japan); Tanaka, Sakae [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Takayanagi, Hiroshi, E-mail: takayana@m.u-tokyo.ac.jp [Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2014-12-12

    Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion.

  9. Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

    International Nuclear Information System (INIS)

    Nakamura, Haruhiko; Nakashima, Tomoki; Hayashi, Mikihito; Izawa, Naohiro; Yasui, Tetsuro; Aburatani, Hiroyuki; Tanaka, Sakae; Takayanagi, Hiroshi

    2014-01-01

    Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion

  10. Effects of loxoprofen on the apical root resorption during orthodontic tooth movement in rats.

    Science.gov (United States)

    Yamamoto, Taeko; Kaku, Masato; Sumi, Hiromi; Yashima, Yuka; Izumino, Jin; Tanimoto, Kotaro

    2018-01-01

    Studies have revealed that severe apical root resorption during tooth movement is caused by the noninfective inflammatory reaction of apical root tissues. We hypothesized that loxoprofen can suppress apical root resorption during tooth movement. Cyclic tensile force (CTF) of 10 kPa was applied to the human pulp cells for 48 hours by the Flexcell Strain Unit. Loxoprofen (10 and 100 μM) was added to the culture cells, and expression of cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1β, receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and macrophage colony-stimulating factor (M-CSF) were examined. To determine the effects of loxoprofen sodium on apical root reabsorption during tooth movement, the upper first molars of 7-week-old rats were subjected to mesial movement by 10g force for 30 days with or without the oral administration of loxoprofen. Gene expression and protein concentration of COX-1, COX-2, IL-1β, TNF-α, RANKL and M-CSF were significantly higher in the CTF group than in the control group. However, these levels were decreased by loxoprofen administration. After orthodontic tooth movement, the expression of IL-1β, TNF-α, RANKL and M-CSF decreased in the loxoprofen group than in the control group by immunohistochemical staining. In comparison to control group, less number of odontoclasts and a decrease in the amount of apical root resorption was observed in the loxoprofen group. Many osteoclasts became visible on the pressure side of the alveolar bone in the both groups, and the amount of tooth movement did not show a significant difference. These findings demonstrate that severe apical root resorption may be suppressed by loxoprofen administration, without a disturbance of tooth movement.

  11. Apyrase Elicits Host Antimicrobial Responses and Resolves Infection in Burns.

    Science.gov (United States)

    Bayliss, Jill M; Levi, Benjamin; Wu, Jianfeng; Wang, Stewart C; Su, Grace L; Xi, Chuanwu

    The authors previously reported that adenosine triphosphate (ATP) stimulates biofilm formation and removal of the ATP could reduce biofilm formation. The main objective of this study was to evaluate the effects of the ATP-hydrolyzing enzyme, apyrase, on control of Acinetabacter baumannii infection in the burn wound as well as to assess host skin antimicrobial responses. The authors found that apyrase stimulated nitric oxide formation at the wound site and reduced CD55 expression, thereby inducing the assembly of membrane attack complexes. Apyrase treatment nearly eradicated multidrug-resistant A. baumannii from burn wounds in the absence of antibiotics. Apyrase may be an effective therapy against antibiotic-resistant bacterial infections in burns.

  12. Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage.

    Science.gov (United States)

    de Korte, Dirk; Kleine, Mya; Korsten, Herbert G H; Verhoeven, Arthur J

    2008-06-01

    Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.

  13. Avoiding Internal Capsule Stimulation With a New Eight-Channel Steering Deep Brain Stimulation Lead.

    Science.gov (United States)

    van Dijk, Kees J; Verhagen, Rens; Bour, Lo J; Heida, Ciska; Veltink, Peter H

    2017-10-15

    Novel deep brain stimulation (DBS) lead designs are currently entering the market, which are hypothesized to provide a way to steer the stimulation field away from neural populations responsible for side effects and towards populations responsible for beneficial effects. The objective of this study is to assess the performances of a new eight channel steering-DBS lead and compare this with a conventional cylindrical contact (CC) lead. The two leads were evaluated in a finite element electric field model combined with multicompartment neuron and axon models, representing the internal capsule (IC) fibers and subthalamic nucleus (STN) cells. We defined the optimal stimulation setting as the configuration that activated the highest percentage of STN cells, without activating any IC fibers. With this criterion, we compared monopolar stimulation using a single contact of the steering-DBS lead and CC lead, on three locations and four orientations of the lead. In addition, we performed a current steering test case by dividing the current over two contacts with the steering-DBS lead in its worst-case orientation. In most cases, the steering-DBS lead is able to stimulate a significantly higher percentage of STN cells compared to the CC lead using single contact stimulation or using a two contact current steering protocol when there is approximately a 1 mm displacement of the CC lead. The results also show that correct placement and orientation of the lead in the target remains an important aspect in achieving the optimal stimulation outcome. Currently, clinical trials are set up in Europe with a similar design as the steering-DBS lead. Our results illustrate the importance of the orientation of the new steering-DBS lead in avoiding side effects induced by stimulation of IC fibers. Therefore, in clinical trials sufficient attention should be paid to implanting the steering DBS-lead in the most effective orientation. © 2017 International Neuromodulation Society.

  14. Melatonin-Stimulated Triacylglycerol Breakdown and Energy Turnover under Salinity Stress Contributes to the Maintenance of Plasma Membrane H+–ATPase Activity and K+/Na+ Homeostasis in Sweet Potato

    Directory of Open Access Journals (Sweden)

    Yicheng Yu

    2018-02-01

    Full Text Available Melatonin (MT is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [Ipomoea batatas (L. Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K+/Na+ homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K+/Na+ homeostasis in sweet potato seedlings as indicated by the low reduced K+ content in tissues and low accumulation of Na+ content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K+ efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM H+–ATPase activity and intracellular adenosine triphosphate (ATP level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H+–ATPase activity, and K+/Na+ homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H+–ATPase activity and K+/Na+ homeostasis in sweet potato.

  15. Regulation of DNA repair processes in mammalian cell

    International Nuclear Information System (INIS)

    Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

    1992-01-01

    A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

  16. Evaluation of intradural stimulation efficiency and selectivity in a computational model of spinal cord stimulation.

    Directory of Open Access Journals (Sweden)

    Bryan Howell

    Full Text Available Spinal cord stimulation (SCS is an alternative or adjunct therapy to treat chronic pain, a prevalent and clinically challenging condition. Although SCS has substantial clinical success, the therapy is still prone to failures, including lead breakage, lead migration, and poor pain relief. The goal of this study was to develop a computational model of SCS and use the model to compare activation of neural elements during intradural and extradural electrode placement. We constructed five patient-specific models of SCS. Stimulation thresholds predicted by the model were compared to stimulation thresholds measured intraoperatively, and we used these models to quantify the efficiency and selectivity of intradural and extradural SCS. Intradural placement dramatically increased stimulation efficiency and reduced the power required to stimulate the dorsal columns by more than 90%. Intradural placement also increased selectivity, allowing activation of a greater proportion of dorsal column fibers before spread of activation to dorsal root fibers, as well as more selective activation of individual dermatomes at different lateral deviations from the midline. Further, the results suggest that current electrode designs used for extradural SCS are not optimal for intradural SCS, and a novel azimuthal tripolar design increased stimulation selectivity, even beyond that achieved with an intradural paddle array. Increased stimulation efficiency is expected to increase the battery life of implantable pulse generators, increase the recharge interval of rechargeable implantable pulse generators, and potentially reduce stimulator volume. The greater selectivity of intradural stimulation may improve the success rate of SCS by mitigating the sensitivity of pain relief to malpositioning of the electrode. The outcome of this effort is a better quantitative understanding of how intradural electrode placement can potentially increase the selectivity and efficiency of SCS

  17. Ghrelin attenuates vascular calcification in diabetic patients with amputation.

    Science.gov (United States)

    Xu, Suining; Ye, Fei; Li, Lihua; Yan, Jinchuan; Bao, Zhengyang; Sun, Zhen; Xu, Liangjie; Zhu, Jie; Wang, Zhongqun

    2017-07-01

    Vascular calcification is established to be a critical factor in diabetes mellitus, which causes cardiovascular and amputation complication of diabetic patients. OPG/RANKL/RANK axis serves as a regulatory role in vascular calcification. Ghrelin, an endogenous ligand of growth hormone secretagogue receptor (GHSR), has been reported to exhibit potent cardiovascular protective effects. However, the role of ghrelin in the regulation of diabetic vascular calcification is still elusive. Here, we reported the role of ghrelin and its relationship with OPG/RANKL/RANK system in patients with diabetic foot amputation. In vivo and in vitro investigations were performed. Sixty type 2 diabetic patients with foot amputation were enrolled in vivo investigation, and they were divided into three groups through Doppler ultrasound: mild stenosis group (n=20), moderate stenosis group (n=20), and severe stenosis/occlusion group (n=20). Morphological analysis results showed diffused calcium depositions in the anterior tibial artery of diabetic amputees. Compared with the mild and moderate stenosis group, the severe stenosis/occlusion group had more spotty calcium depositions in atherosclerotic plaques. Western blot analysis indicated the expressions of osteoprotegerin (OPG) and ghrelin were downregulated, while the expression of receptor activator of nuclear factor kappa B ligand (RANKL) was upregulated with the vascular stenosis aggravation. Pearson correlation analysis revealed a negative correlation between calcium content and ghrelin levels (r=-0.58, Pghrelin levels and sRANKL levels (r=-0.57, Pghrelin levels (r=0.63, PGhrelin blunted calcification in a dose-dependent manner. In addition, ghrelin upregulated OPG expression and downregulated RANKL expression in VSMC calcification when anti-OPG antibody and RANKL were performed. Collectively, we therefore conclude serum ghrelin level may be a predictor of diabetic vascular calcification. The possible mechanism may be related with OPG/RANKL

  18. Receptor activator NFkappaB-ligand and osteoprotegerin protein expression in human periapical cysts and granulomas.

    Science.gov (United States)

    Menezes, Renato; Bramante, Clóvis Monteiro; da Silva Paiva, Katiúcia Batista; Letra, Ariadne; Carneiro, Everdan; Fernando Zambuzzi, Willian; Granjeiro, José Mauro

    2006-09-01

    The purpose of this study was to determine the expression of receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG) associated with bone destruction in periapical cysts and granulomas. Forty human dental chronic periapical lesions were collected after periapical surgery. The lesions collected were fixed in 10% buffered formalin and histologically processed. At least 2 sections of each specimen were stained with hematoxylin and eosin for microscopic diagnosis. After that, 10 human periapical granulomas and 10 cysts were selected for immunohistochemical analysis for RANKL, OPG, and CD68+. Polymorphonuclear neutrophils, macrophages, endothelial cells, and lymphocytes were stained for RANKL and OPG in both lesions. Epithelial cells were also stained for RANKL and OPG in periapical cysts. Quantitative analysis was conducted and the results were expressed as a ratio of the number of immunostained cells over the total number of cells in the field (n = 100). The ratio of RANKL+/total cells was higher than OPG+/total cells in periapical granulomas (0.553 +/- 0.153 and 0.483 +/- 0.189, respectively; P cysts (0.519 +/- 0.09 and 0.339 +/- 0.117, respectively; P cysts. However, the ratio RANKL+/OPG+ in granulomas (1.336 +/- 0.723) and cysts (1.404 +/- 0.385) was not significantly different. The ratio of CD68+/total cells was significantly higher in granulomas (0.381 +/- 0.040) than in cysts (0.307 +/- 0.068) (P cysts and granulomas, strongly suggesting the involvement of these gene products in the development of periapical lesions.

  19. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    Science.gov (United States)

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  20. Development of Femtosecond Stimulated Raman Spectroscopy: Stimulated Raman Gain via Elimination of Cross Phase Modulation

    International Nuclear Information System (INIS)

    Jin, Seung Min; Lee, Young Jong; Yu, Jong Wan; Kim, Seong Keun

    2004-01-01

    We have developed a new femtosecond probe technique by using stimulated Raman spectroscopy. The cross phase modulation in femtosecond time scale associated with off-resonant interaction was shown to be eliminated by integrating the transient gain/loss signal over the time delay between the Raman pump pulse and the continuum pulse. The stimulated Raman gain of neat cyclohexane was obtained to demonstrate the feasibility of the technique. Spectral and temporal widths of stimulated Raman spectra were controlled by using a narrow band pass filter. Femtosecond stimulated Raman spectroscopy was proposed as a highly useful probe in time-resolved vibrational spectroscopy

  1. Discovery of novel benzopyranyl tetracycles that act as inhibitors of osteoclastogenesis induced by receptor activator of NF-κB ligand.

    Science.gov (United States)

    Zhu, Mingyan; Kim, Myung Hee; Lee, Sanghee; Bae, Su Jung; Kim, Seong Hwan; Park, Seung Bum

    2010-12-23

    A novel benzopyran-fused molecular framework 7ai was discovered as a specific inhibitor of RANKL-induced osteoclastogenesis using a cell-based TRAP activity assay from drug-like small-molecule libraries constructed by diversity-oriented synthesis. Its inhibitory activity was confirmed by in vitro evaluations including specific inhibition of RANKL-induced ERK phosphorylation and NF-κB transcriptional activation. 7ai can serve as a specific small-molecule modulator for mechanistic studies of RANKL-induced osteoclast differentiation as well as a potential lead for the development of antiresorptive drugs.

  2. Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

    International Nuclear Information System (INIS)

    Hunting, D.J.; Dresler, S.L.

    1985-01-01

    We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

  3. Noninvasive transcranial brain stimulation and pain.

    Science.gov (United States)

    Rosen, Allyson C; Ramkumar, Mukund; Nguyen, Tam; Hoeft, Fumiko

    2009-02-01

    Transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) are two noninvasive brain stimulation techniques that can modulate activity in specific regions of the cortex. At this point, their use in brain stimulation is primarily investigational; however, there is clear evidence that these tools can reduce pain and modify neurophysiologic correlates of the pain experience. TMS has also been used to predict response to surgically implanted stimulation for the treatment of chronic pain. Furthermore, TMS and tDCS can be applied with other techniques, such as event-related potentials and pharmacologic manipulation, to illuminate the underlying physiologic mechanisms of normal and pathological pain. This review presents a description and overview of the uses of two major brain stimulation techniques and a listing of useful references for further study.

  4. Higher-order power harmonics of pulsed electrical stimulation modulates corticospinal contribution of peripheral nerve stimulation.

    Science.gov (United States)

    Chen, Chiun-Fan; Bikson, Marom; Chou, Li-Wei; Shan, Chunlei; Khadka, Niranjan; Chen, Wen-Shiang; Fregni, Felipe

    2017-03-03

    It is well established that electrical-stimulation frequency is crucial to determining the scale of induced neuromodulation, particularly when attempting to modulate corticospinal excitability. However, the modulatory effects of stimulation frequency are not only determined by its absolute value but also by other parameters such as power at harmonics. The stimulus pulse shape further influences parameters such as excitation threshold and fiber selectivity. The explicit role of the power in these harmonics in determining the outcome of stimulation has not previously been analyzed. In this study, we adopted an animal model of peripheral electrical stimulation that includes an amplitude-adapted pulse train which induces force enhancements with a corticospinal contribution. We report that the electrical-stimulation-induced force enhancements were correlated with the amplitude of stimulation power harmonics during the amplitude-adapted pulse train. In an exploratory analysis, different levels of correlation were observed between force enhancement and power harmonics of 20-80 Hz (r = 0.4247, p = 0.0243), 100-180 Hz (r = 0.5894, p = 0.0001), 200-280 Hz (r = 0.7002, p harmonics. This is a pilot, but important first demonstration that power at high order harmonics in the frequency spectrum of electrical stimulation pulses may contribute to neuromodulation, thus warrant explicit attention in therapy design and analysis.

  5. Staphylococcal enterotoxin C2 promotes osteogenesis and suppresses osteoclastogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Fu, Wei-ming; Zhu, Xiao; Wang, Hua; Wei-Mao Wang; Chen, Ju-yu; Liang, Yan; Zhang, Jin-fang; Kung, Hsiang-fu

    2014-03-10

    As a super-antigen, staphylococcal enterotoxin C2 (SEC2) stimulates the release of massive inflammatory cytokines such as interferon-gamma (IFN-γ), interleukin-1 (IL-1) and interleukin-2 (IL-2) which are documented to implicate osteoblast differentiation. In the present study, SEC2 was found to significantly improve the osteoblast differentiation by up-regulating BMP2 and Runx2/Cbfa1 expression. Interferon (IFN)-inducible gene IFI16, a co-activator of Runx2/Cbfa1, was also activated by SEC2 in the osteoblast differentiation. In addition, exogenous introduction of SEC2 stimulated OPG expression and suppressed RANKL, suggesting suppression of osteoclastogenesis in hMSCs. Therefore, our results displayed that SEC2 plays an important role in the commitment of MSC to the osteoblast and it might be a potential new therapeutic candidate for bone regeneration. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Electrical stimulation in exercise training

    Science.gov (United States)

    Kroll, Walter

    1994-01-01

    Electrical stimulation has a long history of use in medicine dating back to 46 A.D. when the Roman physician Largus found the electrical discharge of torpedo fishes useful in the treatment of pain produced by headache and gout. A rival Greek physician, Dioscorides, discounted the value of the torpedo fish for headache relief but did recommend its use in the treatment of hemorrhoids. In 1745, the Leyden jar and various sized electrostatic generators were used to treat angina pectoris, epilepsy, hemiplegia, kidney stones, and sciatica. Benjamin Franklin used an electrical device to treat successfully a young woman suffering from convulsive fits. In the late 1800's battery powered hydroelectric baths were used to treat chronic inflammation of the uterus while electrified athletic supporters were advertised for the treatment of male problems. Fortunately, such an amusing early history of the simple beginnings of electrical stimulation did not prevent eventual development of a variety of useful therapeutic and rehabilitative applications of electrical stimulation. Over the centuries electrical stimulation has survived as a modality in the treatment of various medical disorders with its primary application being in the rehabilitation area. Recently, a surge of new interest in electrical stimulation has been kindled by the work of a Russian sport scientist who reported remarkable muscle strength and endurance improvements in elite athletes. Yakov Kots reported his research on electric stimulation and strength improvements in 1977 at a Canadian-Soviet Exchange Symposium held at Concordia University in Montreal. Since then an explosion of new studies has been seen in both sport science and in medicine. Based upon the reported works of Kots and the present surge of new investigations, one could be misled as to the origin of electrical stimulation as a technique to increase muscle strength. As a matter of fact, electric stimulation has been used as a technique to improve

  7. Electrocutaneous stimulation system for Braille reading.

    Science.gov (United States)

    Echenique, Ana Maria; Graffigna, Juan Pablo; Mut, Vicente

    2010-01-01

    This work is an assistive technology for people with visual disabilities and aims to facilitate access to written information in order to achieve better social inclusion and integration into work and educational activities. Two methods of electrical stimulation (by current and voltage) of the mechanoreceptors was tested to obtain tactile sensations on the fingertip. Current and voltage stimulation were tested in a Braille cell and line prototype, respectively. These prototypes are evaluated in 33 blind and visually impaired subjects. The result of experimentation with both methods showed that electrical stimulation causes sensations of touch defined in the fingertip. Better results in the Braille characters reading were obtained with current stimulation (85% accuracy). However this form of stimulation causes uncomfortable sensations. The latter feeling was minimized with the method of voltage stimulation, but with low efficiency (50% accuracy) in terms of identification of the characters. We concluded that electrical stimulation is a promising method for the development of a simple and unexpensive Braille reading system for blind people. We observed that voltage stimulation is preferred by the users. However, more experimental tests must be carry out in order to find the optimum values of the stimulus parameters and increase the accuracy the Braille characters reading.

  8. A fully implantable rodent neural stimulator

    Science.gov (United States)

    Perry, D. W. J.; Grayden, D. B.; Shepherd, R. K.; Fallon, J. B.

    2012-02-01

    The ability to electrically stimulate neural and other excitable tissues in behaving experimental animals is invaluable for both the development of neural prostheses and basic neurological research. We developed a fully implantable neural stimulator that is able to deliver two channels of intra-cochlear electrical stimulation in the rat. It is powered via a novel omni-directional inductive link and includes an on-board microcontroller with integrated radio link, programmable current sources and switching circuitry to generate charge-balanced biphasic stimulation. We tested the implant in vivo and were able to elicit both neural and behavioural responses. The implants continued to function for up to five months in vivo. While targeted to cochlear stimulation, with appropriate electrode arrays the stimulator is well suited to stimulating other neurons within the peripheral or central nervous systems. Moreover, it includes significant on-board data acquisition and processing capabilities, which could potentially make it a useful platform for telemetry applications, where there is a need to chronically monitor physiological variables in unrestrained animals.

  9. Vertex Stimulation as a Control Site for Transcranial Magnetic Stimulation: A Concurrent TMS/fMRI Study.

    Science.gov (United States)

    Jung, JeYoung; Bungert, Andreas; Bowtell, Richard; Jackson, Stephen R

    2016-01-01

    A common control condition for transcranial magnetic stimulation (TMS) studies is to apply stimulation at the vertex. An assumption of vertex stimulation is that it has relatively little influence over on-going brain processes involved in most experimental tasks, however there has been little attempt to measure neural changes linked to vertex TMS. Here we directly test this assumption by using a concurrent TMS/fMRI paradigm in which we investigate fMRI blood-oxygenation-level-dependent (BOLD) signal changes across the whole brain linked to vertex stimulation. Thirty-two healthy participants to part in this study. Twenty-one were stimulated at the vertex, at 120% of resting motor threshold (RMT), with short bursts of 1 Hz TMS, while functional magnetic resonance imaging (fMRI) BOLD images were acquired. As a control condition, we delivered TMS pulses over the left primary motor cortex using identical parameters to 11 other participants. Vertex stimulation did not evoke increased BOLD activation at the stimulated site. By contrast we observed widespread BOLD deactivations across the brain, including regions within the default mode network (DMN). To examine the effects of vertex stimulation a functional connectivity analysis was conducted. The results demonstrated that stimulating the vertex with suprathreshold TMS reduced neural activity in brain regions related to the DMN but did not influence the functional connectivity of this network. Our findings provide brain imaging evidence in support of the use of vertex simulation as a control condition in TMS but confirm that vertex TMS induces regional widespread decreases in BOLD activation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Noninvasive Stimulation of the Human Brain

    DEFF Research Database (Denmark)

    Di Lazzaro, Vincenzo; Rothwell, John; Capogna, Marco

    2017-01-01

    Noninvasive brain stimulation methods, such as transcranial electric stimulation and transcranial magnetic stimulation are widely used tools for both basic research and clinical applications. However, the cortical circuits underlying their effects are poorly defined. Here we review the current...

  11. Reducing interaction in simultaneous paired stimulation with CI.

    Directory of Open Access Journals (Sweden)

    Dirk Vellinga

    Full Text Available In this study simultaneous paired stimulation of electrodes in cochlear implants is investigated by psychophysical experiments in 8 post-lingually deaf subjects (and one extra subject who only participated in part of the experiments. Simultaneous and sequential monopolar stimulation modes are used as references and are compared to channel interaction compensation, partial tripolar stimulation and a novel sequential stimulation strategy named phased array compensation. Psychophysical experiments are performed to investigate both the loudness integration during paired stimulation at the main electrodes as well as the interaction with the electrode contact located halfway between the stimulating pair. The study shows that simultaneous monopolar stimulation has more loudness integration on the main electrodes and more interaction in between the electrodes than sequential stimulation. Channel interaction compensation works to reduce the loudness integration at the main electrodes, but does not reduce the interaction in between the electrodes caused by paired stimulation. Partial tripolar stimulation uses much more current to reach the needed loudness, but shows the same interaction in between the electrodes as sequential monopolar stimulation. In phased array compensation we have used the individual impedance matrix of each subject to calculate the current needed on each electrode to exactly match the stimulation voltage along the array to that of sequential stimulation. The results show that the interaction in between the electrodes is the same as monopolar stimulation. The strategy uses less current than partial tripolar stimulation, but more than monopolar stimulation. In conclusion, the paper shows that paired stimulation is possible if the interaction is compensated.

  12. Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation

    NARCIS (Netherlands)

    Feng, Qian; Langereis, Martijn A; Olagnier, David; Chiang, Cindy; van de Winkel, Roel; van Essen, Peter; Zoll, Jan; Hiscott, John; van Kuppeveld, Frank J M

    2014-01-01

    Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by

  13. Knochenstoffwechsel bei malignen Erkrankungen

    Directory of Open Access Journals (Sweden)

    Keck A-V

    2003-01-01

    Full Text Available Bei malignen Erkrankungen, wie dem Mammakarzinom, dem Prostatakarzinom, dem Bronchialkarzinom oder dem multiplen Myelom ist der Knochen eines der am häufigsten von Metastasen betroffenen Organe. Das klinische Beschwerdebild ist durch das Auftreten pathologischer Frakturen, Schmerzen und Hyperkalzämien gekennzeichnet. Eine erhöhte Knochenresorptionsrate und Verlust an Knochenmasse im Sinne von osteoporoseartigen Veränderungen bedingt durch eine inadäquat überschießende Osteoklastendifferenzierung und -aktivierung sind ursächlich dafür verantwortlich. Durch jüngst publizierte Ergebnisse diverser Studien und Untersuchungen konnten die genauen Mechanismen der Rekrutierung und Aktivierung von Osteoklasten weiter aufgeklärt werden. Receptor activator of nuclear factor-kappa-B ligand (RANKL, ein Mitglied der Tumor necrosis factor (TNF Superfamilie konnte als entscheidender Faktor in der Osteoklastengenese identifiziert werden. Die Interaktion von RANKL mit seinem Rezeptor RANK (receptor activator of nuclear factor-kappa-B induziert die Entwicklung der osteoklastären Reihe aus dem hämatopoetischen Kompartment und fördert weiters die Differenzierung zu Osteoklastenvorstufen und die Aktivierung reifer Osteoklasten, ebenso wird eine verzögert einsetzende Apoptose bewirkt. Neben RANK und RANKL ist Osteoprotegerin (OPG als dritte Komponente in der Regulation der Knochenresorption involviert. Als Mitglied der TNF-Superfamilie bindet OPG an RANKL und blockiert somit alle stimulierenden Effekte von RANK auf die Osteoklastenentwicklung und neutralisiert so alle biologischen Effekte von RANKL. Diverse Hormone und Zytokine üben durch die Modifikation der Ratio von RANKL zu OPG einen regulatorischen Effekt auf die Knochenresorption aus. Diese ist bei osteolytischen Knochenmetastasen, beim multiplen Myelom und bei der malignen Hyperkalzämie erhöht. Das Gleichgewicht knochenaufbauender wie -abbauender Mechanismen scheint somit durch die Ratio von

  14. The safety of transcranial magnetic stimulation with deep brain stimulation instruments

    OpenAIRE

    Shimojima, Yoshio; Morita, Hiroshi; Nishikawa, Noriko; Kodaira, Minori; Hashimoto, Takao; Ikeda, Shu-ichi

    2010-01-01

    Objectives: Transcranial magnetic stimulation (TMS) has been employed in patients with an implanted deep brain Stimulation (DBS) device. We investigated the safety of TMS using Simulation models with an implanted DBS device. Methods: The DBS lead was inserted into plastic phantoms filled with dilute gelatin showing impedance similar to that of human brain. TMS was performed with three different types of magnetic coil. During TMS (I) electrode movement, (2) temperature change around the lead, ...

  15. Optical stimulator for vision-based sensors

    DEFF Research Database (Denmark)

    Rössler, Dirk; Pedersen, David Arge Klevang; Benn, Mathias

    2014-01-01

    We have developed an optical stimulator system for vision-based sensors. The stimulator is an efficient tool for stimulating a camera during on-ground testing with scenes representative of spacecraft flights. Such scenes include starry sky, planetary objects, and other spacecraft. The optical...

  16. Mimicking muscle activity with electrical stimulation

    Science.gov (United States)

    Johnson, Lise A.; Fuglevand, Andrew J.

    2011-02-01

    Functional electrical stimulation is a rehabilitation technology that can restore some degree of motor function in individuals who have sustained a spinal cord injury or stroke. One way to identify the spatio-temporal patterns of muscle stimulation needed to elicit complex upper limb movements is to use electromyographic (EMG) activity recorded from able-bodied subjects as a template for electrical stimulation. However, this requires a transfer function to convert the recorded (or predicted) EMG signals into an appropriate pattern of electrical stimulation. Here we develop a generalized transfer function that maps EMG activity into a stimulation pattern that modulates muscle output by varying both the pulse frequency and the pulse amplitude. We show that the stimulation patterns produced by this transfer function mimic the active state measured by EMG insofar as they reproduce with good fidelity the complex patterns of joint torque and joint displacement.

  17. Denosumab for bone diseases: translating bone biology into targeted therapy.

    Science.gov (United States)

    Tsourdi, Elena; Rachner, Tilman D; Rauner, Martina; Hamann, Christine; Hofbauer, Lorenz C

    2011-12-01

    Signalling of receptor activator of nuclear factor-κB (RANK) ligand (RANKL) through RANK is a critical pathway to regulate the differentiation and activity of osteoclasts and, hence, a master regulator of bone resorption. Increased RANKL activity has been demonstrated in diseases characterised by excessive bone loss such as osteoporosis, rheumatoid arthritis and osteolytic bone metastases. The development and approval of denosumab, a fully MAB against RANKL, has heralded a new era in the treatment of bone diseases by providing a potent, targeted and reversible inhibitor of bone resorption. This article summarises the molecular and cellular biology of the RANKL/RANK system and critically reviews preclinical and clinical studies that have established denosumab as a promising novel therapy for metabolic and malignant bone diseases. We will discuss the potential indications for denosumab along with a critical review of safety and analyse its potential within the concert of established therapies.

  18. Twiddler's syndrome in spinal cord stimulation.

    Science.gov (United States)

    Al-Mahfoudh, Rafid; Chan, Yuen; Chong, Hsu Pheen; Farah, Jibril Osman

    2016-01-01

    The aims are to present a case series of Twiddler's syndrome in spinal cord stimulators with analysis of the possible mechanism of this syndrome and discuss how this phenomenon can be prevented. Data were collected retrospectively between 2007 and 2013 for all patients presenting with failure of spinal cord stimulators. The diagnostic criterion for Twiddler's syndrome is radiological evidence of twisting of wires in the presence of failure of spinal cord stimulation. Our unit implants on average 110 spinal cord stimulators a year. Over the 5-year study period, all consecutive cases of spinal cord stimulation failure were studied. Three patients with Twiddler's syndrome were identified. Presentation ranged from 4 to 228 weeks after implantation. Imaging revealed repeated rotations and twisting of the wires of the spinal cord stimulators leading to hardware failure. To the best of our knowledge this is the first reported series of Twiddler's syndrome with implantable pulse generators (IPGs) for spinal cord stimulation. Hardware failure is not uncommon in spinal cord stimulation. Awareness and identification of Twiddler's syndrome may help prevent its occurrence and further revisions. This may be achieved by implanting the IPG in the lumbar region subcutaneously above the belt line. Psychological intervention may have a preventative role for those who are deemed at high risk of Twiddler's syndrome from initial psychological screening.

  19. Adenosine Triphosphate (ATP Is a Candidate Signaling Molecule in the Mitochondria-to-Nucleus Retrograde Response Pathway

    Directory of Open Access Journals (Sweden)

    Zhengchang Liu

    2013-03-01

    Full Text Available Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to and inactivates Mks1, allowing for activation of Rtg1/3 and the RTG pathway. When the pathway is inactive, Mks1 has dissociated from Rtg2 and bound to Bmh1/2, preventing activation of Rtg1/3. What signals association or disassociation of Mks1 and Rtg2 is unknown. Here, we show that ATP at physiological concentrations dissociates Mks1 from Rtg2 in a highly cooperative fashion. We report that ATP-mediated dissociation of Mks1 from Rtg2 is conserved in two other fungal species, K. lactis and K. waltii. Activation of Rtg1/3 upregulates expression of genes encoding enzymes catalyzing the first three reactions of the Krebs cycle, which is coupled to ATP synthesis through oxidative phosphorylation. Therefore, we propose that the retrograde response is an ATP homeostasis pathway coupling ATP production with ATP-mediated repression of the retrograde response by releasing Mks1 from Rtg2.

  20. Evaluation of different types of rooting stimulators

    Directory of Open Access Journals (Sweden)

    Petr Salaš

    2012-01-01

    Full Text Available This paper focuses on the assessment of selected stimulators, especially from Rhizopon product line, which are used for rooting and root system enhancement in various ornamental woody species. Two available methods of cuttings stimulation were selected from the available range of rooting stimulators: stimulation by long-term immersion in solutions or treatment of cuttings with powder stimulators. The experiment involved stimulators with two active components, currently the most commonly used phytohormones for this purpose – IBA and NAA – that were applied in different concentrations. The experiment took place in three propagation terms with twelve coniferous and deciduous shrub varieties. The results of the experiment show the different reactions of the individual species as well as varieties on the respective term of propagation and used form of stimulator.

  1. Step-wise stimulated martensitic transformations

    International Nuclear Information System (INIS)

    Airoldi, G.; Riva, G.

    1991-01-01

    NiTi alloys, widely known both for their shape memory properties and for unusual pseudoelastic behaviour, are now on the forefront attention for step-wise induced memory processes, thermal or stress stimulated. Literature results related to step-wise stimulated martensite (direct transformation) are examined and contrasted with step-wise thermal stimulated parent phase (reverse transformation). Hypothesis are given to explain the key characters of both transformations, a thermodynamic model from first principles being till now lacking

  2. The Underlying Mechanism of Preventing Facial Nerve Stimulation by Triphasic Pulse Stimulation in Cochlear Implant Users Assessed With Objective Measure.

    Science.gov (United States)

    Bahmer, Andreas; Baumann, Uwe

    2016-10-01

    Triphasic pulse stimulation prevents from facial nerve stimulation (FNS) because of a different electromyographic input-output function compared with biphasic pulse stimulation. FNS is sometimes observed in cochlear implant users as an unwanted side effect of electrical stimulation of the auditory nerve. The common stimulation applied in current cochlear implant consists of biphasic pulse patterns. Two common clinical remedies to prevent unpleasant FNS caused by activation of certain electrodes are to expand their pulse phase duration or simply deactivate them. Unfortunately, in some patients these methods do not provide sufficient FNS prevention. In these patients triphasic pulse can prevent from FNS. The underlying mechanism is yet unclear. Electromyographic (EMG) recordings of muscles innervated by the facial nerve (musculi orbicularis ori and oculi) were applied to quantitatively assess the effects on FNS. Triphasic and biphasic fitting maps were compared in four subjects with severe FNS. Based on the recordings, a model is presented which intends to explain the beneficial effects of triphasic pulse application. Triphasic stimulation provided by fitting of an OPUS 2 speech processor device. For three patients, EMG was successfully recorded depending on stimulation level up to uncomfortable and intolerable FNS stimulation as upper boarder. The obtained EMG recordings demonstrated high individual variability. However, a difference between the input-output function for biphasic and triphasic pulse stimulation was visually observable. Compared with standard biphasic stimulation, triphasic pulses require higher stimulation levels to elicit an equal amount of FNS, as reflected by EMG amplitudes. In addition, we assume a steeper slope of the input-output function for biphasic pulse stimulation compared with triphasic pulse stimulation. Triphasic pulse stimulation prevents from FNS because of a smaller gradient of EMG input-output function compared with biphasic pulse

  3. Transcranial alternating current stimulation with sawtooth waves: simultaneous stimulation and EEG recording

    Directory of Open Access Journals (Sweden)

    James eDowsett

    2016-03-01

    Full Text Available Transcranial alternating current stimulation (tACS has until now mostly been administered as an alternating sinusoidal wave. Despite modern tACS stimulators being able to deliver alternating current with any arbitrary shape there has been no systematic exploration into the relative benefits of different waveforms. As tACS is a relatively new technique there is a huge parameter space of unexplored possibilities which may prove superior or complimentary to the traditional sinusoidal waveform. Here we begin to address this with an investigation into the effects of sawtooth wave tACS on individual alpha power. Evidence from animal models suggests that the gradient and direction of an electric current should be important factors for the subsequent neural firing rate; we compared positive and negative ramp sawtooth waves to test this. An additional advantage of sawtooth waves is that the resulting artefact in the electroencephalogram (EEG recording is significantly simpler to remove than a sine wave; accordingly we were able to observe alpha oscillations both during and after stimulation.We found that positive ramp sawtooth, but not negative ramp sawtooth, significantly enhanced alpha power during stimulation relative to sham (p<0.01. In addition we tested for an after-effect of both sawtooth and sinusoidal stimulation on alpha power but in this case did not find any significant effect. This preliminary study paves the way for further investigations into the effect of the gradient and direction of the current in tACS which could significantly improve the usefulness of this technique.

  4. Targeting Integrin-β1 Impedes Cytokine-Induced Osteoclast ...

    African Journals Online (AJOL)

    but not in RANKL pathway. Given that, inflammatory cytokine secretions such as TNF-α are progressively implicated in pathological osteolysis, targeting this pathway may .... RANKL or TNF-alpha treated culture systems ... universal PCR Master Mix (Life Technologies,. USA). ... and developed using Super Signal West Dura.

  5. Bone resorptive activity in symptomatic and asymptomatic apical lesions of endodontic origin.

    Science.gov (United States)

    Salinas-Muñoz, M; Garrido-Flores, M; Baeza, M; Huamán-Chipana, P; García-Sesnich, J; Bologna, R; Vernal, R; Hernández, M

    2017-11-01

    The aim of this study is to assess the levels and diagnostic accuracy of a set of bone resorption biomarkers, including TRAP-5, RANKL, and OPG in symptomatic and asymptomatic apical lesions and controls. Apical tissues from symptomatic and asymptomatic apical periodontitis patients and periodontal ligaments from healthy teeth extracted for orthodontic reasons were processed for tissue homogenization and the levels of TRAP-5, RANKL, and OPG were determined by multiplex assay. Marker levels were analyzed by Kruskal Wallis test, and diagnostic accuracy was analyzed with ROC curves. Higher levels of RANKL, OPG, and RANKL/OPG ratio were determined in both types of apical lesions compared to healthy periodontal ligament, whereas higher TRAP-5 levels were found only in symptomatic apical lesions (p apical lesions versus healthy controls (AUC = 0.69, p asymptomatic apical periodontitis (AUC = 0.71, p Apical lesions showed higher RANKL and OPG levels than healthy tissues. TRAP-5 levels were the highest in symptomatic apical lesions, suggesting that these represent a progressive state, and showed diagnostic potential. Clinically symptomatic apical periodontitis might represent biologically progressive apical lesions based on TRAP5 levels. TRAP5 has diagnostic potential to identify these lesions, representing a candidate prognostic biomarker.

  6. Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing

    OpenAIRE

    Kim, Jin S.; Nanfara, Michael T.; Chodavarapu, Sundari; Jin, Kyeong S.; Babu, Vignesh?M.?P.; Ghazy, Mohamed A.; Chung, Scisung; Kaguni, Jon M.; Sutton, Mark D.; Cho, Yunje

    2017-01-01

    Abstract Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda?sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda?? clam...

  7. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2

    International Nuclear Information System (INIS)

    Burch, R.M.; Axelrod, J.

    1987-01-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E 2 (PGE 2 ) synthesis. The EC 50 values for stimulation of PGE 2 synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-[γ-thio]triphosphate stimulated PGE 2 synthesis and InsP formation, and guanosine-5'-[β-thio]diphosphate inhibited both PGE 2 synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE 2 synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE 2 synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE 2 synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE 2 synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with [ 3 H] choline, the phospholipase A 2 products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A 2 and that phospholipase A 2 is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis

  8. Effects of contraction mode and stimulation frequency on electrical stimulation-induced skeletal muscle hypertrophy.

    Science.gov (United States)

    Ashida, Yuki; Himori, Koichi; Tatebayashi, Daisuke; Yamada, Ryotaro; Ogasawara, Riki; Yamada, Takashi

    2018-02-01

    We compared the skeletal muscle hypertrophy resulting from isometric (Iso) or eccentric (Ecc) electrical stimulation (ES) training with different stimulation frequencies. Male Wistar rats were assigned to the Iso and Ecc groups. These were divided into three further subgroups that were stimulated at 10 Hz (Iso-10 and Ecc-10), 30 Hz (Iso-30 and Ecc-30), or 100 Hz (Iso-100 and Ecc-100). In experiment 1, the left plantarflexor muscles were stimulated every other day for 3 wk. In experiment 2, mammalian target of rapamycin complex 1 (mTORC1) signaling was investigated 6 h after one bout of ES. The contralateral right muscle served as a control (non-ES). Ecc contractions comprised forced dorsiflexion combined with ES. The peak torque and torque-time integral during ES were higher in the Ecc group than that in the Iso group in all stimulation frequencies examined. The gastrocnemius muscle weight normalized to body weight in ES side was increased compared with the non-ES side by 6, 7, and 17% in the Ecc-30, Iso-100, and Ecc-100 groups, respectively, with a greater gain in Ecc-100 than the Ecc-30 and Iso-100 groups. The p70S6K (Thr389) phosphorylation level was higher in the Ecc-30 and -100 than in the Iso-30 and -100 groups, respectively. The peak torque and torque-time integral were highly correlated with the magnitude of increase in muscle mass and the phosphorylation of p70S6K. These data suggest that ES-induced muscle hypertrophy and mTORC1 activity are determined by loading intensity and volume during muscle contraction independent of the contraction mode. NEW & NOTEWORTHY Eccentric contraction and high-frequency stimulation (HFS) are regarded as an effective way to increase muscle mass by electrical stimulation (ES) training. However, little is known about whether muscle hypertrophy is affected by contraction mode and stimulation frequency in ES training. Here, we provide the evidence that muscle hypertrophy and mammalian target of rapamycin complex 1 activity are

  9. Avoiding Internal Capsule Stimulation With a New Eight-Channel Steering Deep Brain Stimulation Lead

    NARCIS (Netherlands)

    van Dijk, Kees J.; Verhagen, Rens; Bour, Lo J.; Heida, Ciska; Veltink, Peter H.

    2017-01-01

    Objective: Novel deep brain stimulation (DBS) lead designs are currently entering the market, which are hypothesized to provide a way to steer the stimulation field away from neural populations responsible for side effects and towards populations responsible for beneficial effects. The objective of

  10. Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes.

    Science.gov (United States)

    Gooi, J H; Pompolo, S; Karsdal, M A; Kulkarni, N H; Kalajzic, I; McAhren, S H M; Han, B; Onyia, J E; Ho, P W M; Gillespie, M T; Walsh, N C; Chia, L Y; Quinn, J M W; Martin, T J; Sims, N A

    2010-06-01

    The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously

  11. Brain stimulation in posttraumatic stress disorder

    Directory of Open Access Journals (Sweden)

    Vladan Novakovic

    2011-10-01

    Full Text Available Posttraumatic stress disorder (PTSD is a complex, heterogeneous disorder that develops following trauma and often includes perceptual, cognitive, affective, physiological, and psychological features. PTSD is characterized by hyperarousal, intrusive thoughts, exaggerated startle response, flashbacks, nightmares, sleep disturbances, emotional numbness, and persistent avoidance of trauma-associated stimuli. The efficacy of available treatments for PTSD may result in part from relief of associated depressive and anxiety-related symptoms in addition to treatment of core symptoms that derive from reexperiencing, numbing, and hyperarousal. Diverse, heterogeneous mechanisms of action and the ability to act broadly or very locally may enable brain stimulation devices to address PTSD core symptoms in more targeted ways. To achieve this goal, specific theoretical bases derived from novel, well-designed research protocols will be necessary. Brain stimulation devices include both long-used and new electrical and magnetic devices. Electroconvulsive therapy (ECT and Cranial electrotherapy stimulation (CES have both been in use for decades; transcranial magnetic stimulation (TMS, magnetic seizure therapy (MST, deep brain stimulation (DBS, transcranial Direct Current Stimulation (tDCS, and vagus nerve stimulation (VNS have been developed recently, over approximately the past twenty years. The efficacy of brain stimulation has been demonstrated as a treatment for psychiatric and neurological disorders such as anxiety (CES, depression (ECT, CES, rTMS, VNS, DBS, obsessive-compulsive disorder (OCD (DBS, essential tremor, dystonia (DBS, epilepsy (DBS, VNS, Parkinson Disease (DBS, pain (CES, and insomnia (CES. To date, limited data on brain stimulation for PTSD offer only modest guidance. ECT has shown some efficacy in reducing comorbid depression in PTSD patients but has not been demonstrated to improve most core PTSD symptoms. CES and VNS have shown some efficacy in

  12. A Programmable High-Voltage Compliance Neural Stimulator for Deep Brain Stimulation in Vivo

    Directory of Open Access Journals (Sweden)

    Cihun-Siyong Alex Gong

    2015-05-01

    Full Text Available Deep brain stimulation (DBS is one of the most effective therapies for movement and other disorders. The DBS neurosurgical procedure involves the implantation of a DBS device and a battery-operated neurotransmitter, which delivers electrical impulses to treatment targets through implanted electrodes. The DBS modulates the neuronal activities in the brain nucleus for improving physiological responses as long as an electric discharge above the stimulation threshold can be achieved. In an effort to improve the performance of an implanted DBS device, the device size, implementation cost, and power efficiency are among the most important DBS device design aspects. This study aims to present preliminary research results of an efficient stimulator, with emphasis on conversion efficiency. The prototype stimulator features high-voltage compliance, implemented with only a standard semiconductor process, without the use of extra masks in the foundry through our proposed circuit structure. The results of animal experiments, including evaluation of evoked responses induced by thalamic electrical stimuli with our fabricated chip, were shown to demonstrate the proof of concept of our design.

  13. Colony-stimulating factor (CSF) radioimmunoassay: detection of a CSF subclass stimulating macrophage production

    International Nuclear Information System (INIS)

    Stanley, E.R.

    1979-01-01

    Colony-stimulating factors (CSFs) stimulate the differentiation of immature precursor cells to mature granulocytes and macrophages. Purified 125 I-labeled murine L cell CSF has been used to develop a radioimmunoassay (RIA) that detects a subclass of CSFs that stimulates macrophage production. Murine CSF preparations that contain this subclass of CSF compete for all of the CSF binding sites on anti-L cell CSF antibody. With the exception of mouse serum, which can contain inhibitors of the bioassay, there is complete correspondence between activities determined by RIA and those determined by bioassay. The RIA is slightly more sensitive than the bioassay, detecting approximately 0.3 fmol of purified L cell CSF. It can also detect this subclass of CSF in chickens, rats, and humans. In the mouse, the subclass is distinguished from other CSFs by a murine cell bioassay dose-response curve in which 90% of the response occurs over a 10-fold (rather than a 100-fold) increase in concentration, by stimulating the formations of colonies contaning a high proportion of mononuclear (rather than granulocytic) cells, and by certain physical characteristics

  14. growth stimulant

    African Journals Online (AJOL)

    Effects of timing and duration of supplementation of LIVFIT VET ® (growth stimulant) as substitute for fish meal on the growth performance, haematology and clinical enzymes concentration of growing pigs.

  15. Tonic aortic depressor nerve stimulation does not impede baroreflex dynamic characteristics concomitantly mediated by the stimulated nerve.

    Science.gov (United States)

    Kawada, Toru; Turner, Michael J; Shimizu, Shuji; Kamiya, Atsunori; Shishido, Toshiaki; Sugimachi, Masaru

    2018-03-01

    Although electrical activation of the carotid sinus baroreflex (baroreflex activation therapy) is being explored as a device therapy for resistant hypertension, possible effects on baroreflex dynamic characteristics of interaction between electrical stimulation and pressure inputs are not fully elucidated. To examine whether the electrical stimulation of the baroreceptor afferent nerve impedes normal short-term arterial pressure (AP) regulation mediated by the stimulated nerve, we electrically stimulated the right aortic depressor nerve (ADN) while estimating the baroreflex dynamic characteristics by imposing pressure inputs to the isolated baroreceptor region of the right ADN in nine anesthetized rats. A Gaussian white noise signal with a mean of 120 mmHg and standard deviation of 20 mmHg was used for the pressure perturbation. A tonic ADN stimulation (2 or 5 Hz, 10 V, 0.1-ms pulse width) decreased mean sympathetic nerve activity (367.0 ± 70.9 vs. 247.3 ± 47.2 arbitrary units, P ADN stimulation did not affect the slope of dynamic gain in the neural arc transfer function from pressure perturbation to sympathetic nerve activity (16.9 ± 1.0 vs. 14.7 ± 1.6 dB/decade, not significant). These results indicate that electrical stimulation of the baroreceptor afferent nerve does not significantly impede the dynamic characteristics of the arterial baroreflex concomitantly mediated by the stimulated nerve. Short-term AP regulation by the arterial baroreflex may be preserved during the baroreflex activation therapy.

  16. Avoiding Internal Capsule Stimulation With a New Eight-Channel Steering Deep Brain Stimulation Lead

    NARCIS (Netherlands)

    van Dijk, Kees J.; Verhagen, Rens; Bour, Lo J.; Heida, Ciska; Veltink, Peter H.

    2017-01-01

    Novel deep brain stimulation (DBS) lead designs are currently entering the market, which are hypothesized to provide a way to steer the stimulation field away from neural populations responsible for side effects and towards populations responsible for beneficial effects. The objective of this study

  17. Tactile stimulation interventions: influence of stimulation parameters on sensorimotor behavior and neurophysiological correlates in healthy and clinical samples.

    Science.gov (United States)

    Parianen Lesemann, Franca H; Reuter, Eva-Maria; Godde, Ben

    2015-04-01

    The pure exposure to extensive tactile stimulation, without the requirement of attention or active training, has been revealed to enhance sensorimotor functioning presumably due to an induction of plasticity in the somatosensory cortex. The induced effects, including increased tactile acuity and manual dexterity have repeatedly been observed in basic as well as clinical research. However, results vary greatly in respect to the strength and direction of the effects on the behavioral and on the brain level. Multiple evidences show that differences in the stimulation protocols (e.g., two vs. multiple stimulation sites) and parameters (e.g., duration, frequency, and amplitude) might contribute to this variability of effects. Nevertheless, stimulation protocols have not been comprehensively compared yet. Identifying favorable parameters for tactile stimulation interventions is especially important because of its possible application as a treatment option for patients suffering from sensory loss, maladaptive plasticity, or certain forms of motor impairment. This review aims to compare the effects of different tactile stimulation protocols and to assess possible implications for tactile interventions. Our goal is to identify ways of optimizing stimulation protocols to improve sensorimotor performance. To this end, we reviewed research on tactile stimulation in the healthy population, with a focus on the effectiveness of the applied parameters regarding psychophysiological measures. We discuss the association of stimulation-induced changes on the behavioral level with alterations in neural representations and response characteristics. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Effects of stimulation parameters and electrode location on thresholds for epidural stimulation of cat motor cortex

    Science.gov (United States)

    Wongsarnpigoon, Amorn; Grill, Warren M.

    2011-12-01

    Epidural electrical stimulation (ECS) of the motor cortex is a developing therapy for neurological disorders. Both placement and programming of ECS systems may affect the therapeutic outcome, but the treatment parameters that will maximize therapeutic outcomes and minimize side effects are not known. We delivered ECS to the motor cortex of anesthetized cats and investigated the effects of electrode placement and stimulation parameters on thresholds for evoking motor responses in the contralateral forelimb. Thresholds were inversely related to stimulation frequency and the number of pulses per stimulus train. Thresholds were lower over the forelimb representation in motor cortex (primary site) than surrounding sites (secondary sites), and thresholds at sites 4 mm away. Electrode location and montage influenced the effects of polarity on thresholds: monopolar anodic and cathodic thresholds were not significantly different over the primary site, cathodic thresholds were significantly lower than anodic thresholds over secondary sites and bipolar thresholds were significantly lower with the anode over the primary site than with the cathode over the primary site. A majority of bipolar thresholds were either between or equal to the respective monopolar thresholds, but several bipolar thresholds were greater than or less than the monopolar thresholds of both the anode and cathode. During bipolar stimulation, thresholds were influenced by both electric field superposition and indirect, synaptically mediated interactions. These results demonstrate the influence of stimulation parameters and electrode location during cortical stimulation, and these effects should be considered during the programming of systems for therapeutic cortical stimulation.

  19. Pharmacological interventions for adolescents and adults with ADHD: stimulant and nonstimulant medications and misuse of prescription stimulants

    Directory of Open Access Journals (Sweden)

    Weyandt LL

    2014-09-01

    Full Text Available Lisa L Weyandt, Danielle R Oster, Marisa E Marraccini, Bergljot Gyda Gudmundsdottir, Bailey A Munro, Brynheld Martinez Zavras, Ben Kuhar Department of Psychology, University of Rhode Island, Kingston, RI, USA Abstract: Attention-deficit/hyperactivity disorder (ADHD is a neurodevelopmental disorder characterized by symptoms of inattention, hyperactivity, and impulsivity that cause functional impairment. Recent research indicates that symptoms persist into adulthood in the majority of cases, with prevalence estimates of approximately 5% in the school age population and 2.5%–4% in the adult population. Although students with ADHD are at greater risk for academic underachievement and psychosocial problems, increasing numbers of students with ADHD are graduating from high school and pursuing higher education. Stimulant medications are considered the first line of pharmacotherapy for individuals with ADHD, including college students. Although preliminary evidence indicates that prescription stimulants are safe and effective for college students with ADHD when used as prescribed, very few controlled studies have been conducted concerning the efficacy of prescription stimulants with college students. In addition, misuse of prescription stimulants has become a serious problem on college campuses across the US and has been recently documented in other countries as well. The purpose of the present systematic review was to investigate the efficacy of prescription stimulants for adolescents and young adults with ADHD and the nonmedical use and misuse of prescription stimulants. Results revealed that both prostimulant and stimulant medications, including lisdexamfetamine dimesylate, methylphenidate, amphetamines, and mixed-amphetamine salts, are effective at reducing ADHD symptoms in adolescents and adults with ADHD. Findings also suggest that individuals with ADHD may have higher rates of stimulant misuse than individuals without the disorder, and

  20. Frequency shifts in stimulated Raman scattering

    International Nuclear Information System (INIS)

    Zinth, W.; Kaiser, W.

    1980-01-01

    The nonresonant contributions to the nonlinear susceptibility chisup(()3) produce a frequency chirp during stimulated Raman scattering. In the case of transient stimulated Raman scattering, the spectrum of the generated Stokes pulse is found at higher frequencies than expected from spontaneous Raman data. The frequency difference can be calculated from the theory of stimulated Raman scattering. (orig.)

  1. Bone status in patients with epilepsy: relationship to markers of bone remodeling

    Directory of Open Access Journals (Sweden)

    Sherifa Ahmed Hamed

    2014-08-01

    Full Text Available Patients with epilepsy and treated with antiepileptic drugs (AEDs may develop metabolic bone disease, however, the exact pathogenesis of bone loss with AEDs is still unclear. Included were 75 adults with epilepsy (mean age: 31.90±5.62years; duration of treatment with AEDs: 10.57±3.55years and 40 matched healthy controls. Bone mineral content (BMC and densities (BMD of the femoral neck and lumbar spine were measured using dual energy X-ray absorptiometry (DEXA. Blood samples were analyzed for calcium, magnesium, phosphate, alkaline phosphatase (ALP, 25-hydroxy vitamin D (25OHD, soluble receptor activator of nuclear factor kB ligand (sRANKL, osteoprotegerin (OPG and OPG/RANKL ratio (markers of bone remodeling. Compared to controls, patients had lower BMD, BMC, Z-score and T-score at the femoral neck and lumbar spine (all p<0.001. 72% and 29.33% of patients had osteoporosis of the lumbar spine and femoral neck. Patients had significantly lower serum calcium, 25(OHD and OPG and higher ALP, sRANKL levels and sRANKL/OPG (all p<0.001. 52% of patients had hypocalcemia, 93% had hypovitaminosis D, 31% had high levels of sRANKL and 49% had low levels of OPG. No differences were identified between DEXA and laboratory results in relation to the type, dose or serum levels of AEDs. BMD at the femoral neck and lumbar spine were found to be correlated with the duration of illness (p=0.043; p=0.010, duration of treatment with AEDs (p<0.001; p=0.012 and serum levels of 25(OHD (p<0.042; p=0.010, sRANKLs (p=0.005; p=0.01 and OPG (p<0.006; p<0.01. In linear regression analysis and after adjusting for gender, age, weight, duration and number of AEDs, we observed an association between BMD, 25(OHD (p=0.04 and sRANKL (p=0.03 concentrations. We conclude that AEDs may compromise bone health through disturbance of mineral metabolism and acceleration of bone turnover mechanisms.

  2. Optimal number of stimulation contacts for coordinated reset neuromodulation

    Science.gov (United States)

    Lysyansky, Borys; Popovych, Oleksandr V.; Tass, Peter A.

    2013-01-01

    In this computational study we investigate coordinated reset (CR) neuromodulation designed for an effective control of synchronization by multi-site stimulation of neuronal target populations. This method was suggested to effectively counteract pathological neuronal synchrony characteristic for several neurological disorders. We study how many stimulation sites are required for optimal CR-induced desynchronization. We found that a moderate increase of the number of stimulation sites may significantly prolong the post-stimulation desynchronized transient after the stimulation is completely switched off. This can, in turn, reduce the amount of the administered stimulation current for the intermittent ON–OFF CR stimulation protocol, where time intervals with stimulation ON are recurrently followed by time intervals with stimulation OFF. In addition, we found that the optimal number of stimulation sites essentially depends on how strongly the administered current decays within the neuronal tissue with increasing distance from the stimulation site. In particular, for a broad spatial stimulation profile, i.e., for a weak spatial decay rate of the stimulation current, CR stimulation can optimally be delivered via a small number of stimulation sites. Our findings may contribute to an optimization of therapeutic applications of CR neuromodulation. PMID:23885239

  3. Evaluation of various somatosensory stimulations for functional MRI

    International Nuclear Information System (INIS)

    Hara, Kazushi; Nakasato, Nobukazu; Mizoi, Kazuo; Yoshimoto, Takashi; Shimizu, Hiroaki.

    1997-01-01

    The aim of this functional magnetic resonance imaging (fMRI) study was to test detectability of activated area using various somatosensory stimulations. The following stimulations were performed in normal volunteers: regular or irregular electrical median nerve stimulation (n=5, each), tactile stimulation to the palm and fingers (n=8), pain stimulation to the index finger (n=5) or to the palm and fingers (n=5). fMRI was acquired with a spoiled gradient echo sequence at 1.5 T. Detectability of activated area was the highest when the pain stimulation was applied to the palm and fingers (80%). A successful rate for the tactile stimulation was 25%, and the other stimulations failed to demonstrate any activation. When successful, the highest signal activation on fMRI was seen on a sulcus, which presumably arose from a vein. The sulcus was defined as the central sulcus by somatosensory evoked field using a median nerve stimulation. Our study indicates that the pain stimulation to the palm and fingers may be a choice for the sensory fMRI. (author)

  4. Economics of nuclear gas stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Frank, G W [Austral Oil Company Incorporated, Houston, TX (United States); Coffer, H F; Luetkehans, G R [CER Geonuclear Corporation, Las Vegas, NV (United States)

    1970-05-01

    Nuclear stimulation of the Mesaverde Formation in the Piceance Basin appears to be the only available method that can release the contained gas economically. In the Rulison Field alone estimates show six to eight trillion cubic feet of gas may be made available by nuclear means, and possibly one hundred trillion cubic feet could be released in the Piceance Basin. Several problems remain to be solved before this tremendous gas reserve can be tapped. Among these are (1) rates of production following nuclear stimulation; (2) costs of nuclear stimulation; (3) radioactivity of the chimney gas; and (4) development of the ideal type of device to carry out the stimulations. Each of these problems is discussed in detail with possible solutions suggested. First and foremost is the rate at which gas can be delivered following nuclear stimulation. Calculations have been made for expected production behavior following a 5-kiloton device and a 40-kiloton device with different permeabilities. These are shown, along with conventional production history. The calculations show that rates of production will be sufficient if costs can be controlled. Costs of nuclear stimulation must be drastically reduced for a commercial process. Project Rulison will cost approximately $3.7 million, excluding lease costs, preliminary tests, and well costs. At such prices, nothing can possibly be commercial; however, these costs can come down in a logical step-wise fashion. Radiation contamination of the gas remains a problem. Three possible solutions to this problem are included. (author)

  5. Economics of nuclear gas stimulation

    International Nuclear Information System (INIS)

    Frank, G.W.; Coffer, H.F.; Luetkehans, G.R.

    1970-01-01

    Nuclear stimulation of the Mesaverde Formation in the Piceance Basin appears to be the only available method that can release the contained gas economically. In the Rulison Field alone estimates show six to eight trillion cubic feet of gas may be made available by nuclear means, and possibly one hundred trillion cubic feet could be released in the Piceance Basin. Several problems remain to be solved before this tremendous gas reserve can be tapped. Among these are (1) rates of production following nuclear stimulation; (2) costs of nuclear stimulation; (3) radioactivity of the chimney gas; and (4) development of the ideal type of device to carry out the stimulations. Each of these problems is discussed in detail with possible solutions suggested. First and foremost is the rate at which gas can be delivered following nuclear stimulation. Calculations have been made for expected production behavior following a 5-kiloton device and a 40-kiloton device with different permeabilities. These are shown, along with conventional production history. The calculations show that rates of production will be sufficient if costs can be controlled. Costs of nuclear stimulation must be drastically reduced for a commercial process. Project Rulison will cost approximately $3.7 million, excluding lease costs, preliminary tests, and well costs. At such prices, nothing can possibly be commercial; however, these costs can come down in a logical step-wise fashion. Radiation contamination of the gas remains a problem. Three possible solutions to this problem are included. (author)

  6. Protective protein/cathepsin A down-regulates osteoclastogenesis by associating with and degrading NF-kappaB p50/p65.

    Science.gov (United States)

    Masuhara, Masaaki; Sato, Takuya; Hada, Naoto; Hakeda, Yoshiyuki

    2009-01-01

    Disruption of the cooperative function balance between osteoblasts and osteoclasts causes various bone disorders, some of which are attributed to abnormal osteoclast recruitment. Osteoclast differentiation is dependent on the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) as well as the macrophage colony-stimulating factor. The osteoclast formation induced by cytokines requires activation of NF-kappaB, AP-1 and nuclear factor of activated T cells c1. However, osteoclasts are not the only cell types that express these transcription factors, suggesting that some unknown molecules specific for osteoclasts may associate with the transcription factors. Here, we explored the possibility of molecules binding directly to NF-kappaB and cloned protective protein/cathepsin A (PPCA) by yeast two-hybrid screening using a cDNA library of osteoclast precursors. Forced expression of PPCA with p50/p65 in HEK293 cells decreased both the level of p50/p65 proteins and the transcriptional activity. Abundant PPCA was detected in the lysosomes of the transfected HEK293 cells, but a small amount of this enzyme was also present in the cytosolic fraction. In addition, over-expression of PPCA caused the disappearance of p50/p65 in both the lysosomal and cytosolic fractions. PPCA was expressed throughout osteoclastogenesis, and the expression was slightly up-regulated by RANKL signaling. Knockdown of PPCA in osteoclast precursors with PPCA siRNA stimulated binding of nuclear proteins to oligonucleotides containing an NF-kappaB binding motif and increased osteoclastogenesis. Our present results indicate a novel role for PPCA in osteoclastogenesis via down-regulation of NF-kappaB activity and suggest a new function for PPCA as an NF-kappaB-degrading enzyme in addition to its known multifunctional properties.

  7. Multifocal visual evoked responses to dichoptic stimulation using virtual reality goggles: Multifocal VER to dichoptic stimulation.

    Science.gov (United States)

    Arvind, Hemamalini; Klistorner, Alexander; Graham, Stuart L; Grigg, John R

    2006-05-01

    Multifocal visual evoked potentials (mfVEPs) have demonstrated good diagnostic capabilities in glaucoma and optic neuritis. This study aimed at evaluating the possibility of simultaneously recording mfVEP for both eyes with dichoptic stimulation using virtual reality goggles and also to determine the stimulus characteristics that yield maximum amplitude. ten healthy volunteers were recruited and temporally sparse pattern pulse stimuli were presented dichoptically using virtual reality goggles. Experiment 1 involved recording responses to dichoptically presented checkerboard stimuli and also confirming true topographic representation by switching off specific segments. Experiment 2 involved monocular stimulation and comparison of amplitude with Experiment 1. In Experiment 3, orthogonally oriented gratings were dichoptically presented. Experiment 4 involved dichoptic presentation of checkerboard stimuli at different levels of sparseness (5.0 times/s, 2.5 times/s, 1.66 times/s and 1.25 times/s), where stimulation of corresponding segments of two eyes were separated by 16.7, 66.7,116.7 & 166.7 ms respectively. Experiment 1 demonstrated good traces in all regions and confirmed topographic representation. However, there was suppression of amplitude of responses to dichoptic stimulation by 17.9+/-5.4% compared to monocular stimulation. Experiment 3 demonstrated similar suppression between orthogonal and checkerboard stimuli (p = 0.08). Experiment 4 demonstrated maximum amplitude and least suppression (4.8%) with stimulation at 1.25 times/s with 166.7 ms separation between eyes. It is possible to record mfVEP for both eyes during dichoptic stimulation using virtual reality goggles, which present binocular simultaneous patterns driven by independent sequences. Interocular suppression can be almost eliminated by using a temporally sparse stimulus of 1.25 times/s with a separation of 166.7 ms between stimulation of corresponding segments of the two eyes.

  8. Neurologic Complications of Psychomotor Stimulant Abuse.

    Science.gov (United States)

    Sanchez-Ramos, Juan

    2015-01-01

    Psychomotor stimulants are drugs that act on the central nervous system (CNS) to increase alertness, elevate mood, and produce a sense of well-being. These drugs also decrease appetite and the need for sleep. Stimulants can enhance stamina and improve performance in tasks that have been impaired by fatigue or boredom. Approved therapeutic applications of stimulants include attention deficit hyperactivity disorder (ADHD), narcolepsy, and obesity. These agents also possess potent reinforcing properties that can result in excessive self-administration and abuse. Chronic use is associated with adverse effects including psychosis, seizures, and cerebrovascular accidents, though these complications usually occur in individuals with preexisting risk factors. This chapter reviews the adverse neurologic consequences of chronic psychomotor stimulant use and abuse, with a focus on two prototypical stimulants methamphetamine and cocaine. © 2015 Elsevier Inc. All rights reserved.

  9. Optical stimulation of peripheral nerves in vivo

    Science.gov (United States)

    Wells, Jonathon D.

    This dissertation documents the emergence and validation of a new clinical tool that bridges the fields of biomedical optics and neuroscience. The research herein describes an innovative method for direct neurostimulation with pulsed infrared laser light. Safety and effectiveness of this technique are first demonstrated through functional stimulation of the rat sciatic nerve in vivo. The Holmium:YAG laser (lambda = 2.12 mum) is shown to operate at an optimal wavelength for peripheral nerve stimulation with advantages over standard electrical neural stimulation; including contact-free stimulation, high spatial selectivity, and lack of a stimulation artifact. The underlying biophysical mechanism responsible for transient optical nerve stimulation appears to be a small, absorption driven thermal gradient sustained at the axonal layer of nerve. Results explicitly prove that low frequency optical stimulation can reliably stimulate without resulting in tissue thermal damage. Based on the positive results from animal studies, these optimal laser parameters were utilized to move this research into the clinic with a combined safety and efficacy study in human subjects undergoing selective dorsal rhizotomy. The clinical Holmium:YAG laser was used to effectively stimulate human dorsal spinal roots and elicit functional muscle responses recorded during surgery without evidence of nerve damage. Overall these results predict that this technology can be a valuable clinical tool in various neurosurgical applications.

  10. Brain stimulation in migraine.

    Science.gov (United States)

    Brighina, Filippo; Cosentino, Giuseppe; Fierro, Brigida

    2013-01-01

    Migraine is a very prevalent disease with great individual disability and socioeconomic burden. Despite intensive research effort in recent years, the etiopathogenesis of the disease remains to be elucidated. Recently, much importance has been given to mechanisms underlying the cortical excitability that has been suggested to be dysfunctional in migraine. In recent years, noninvasive brain stimulation techniques based on magnetic fields (transcranial magnetic stimulation, TMS) and on direct electrical currents (transcranial direct current stimulation, tDCS) have been shown to be safe and effective tools to explore the issue of cortical excitability, activation, and plasticity in migraine. Moreover, TMS, repetitive TMS (rTMS), and tDCS, thanks to their ability to interfere with and/or modulate cortical activity inducing plastic, persistent effects, have been also explored as potential therapeutic approaches, opening an interesting perspective for noninvasive neurostimulation for both symptomatic and preventive treatment of migraine and other types of headache. In this chapter we critically review evidence regarding the role of noninvasive brain stimulation in the pathophysiology and treatment of migraine, delineating the advantages and limits of these techniques together with potential development and future application. © 2013 Elsevier B.V. All rights reserved.

  11. Effects of PTH and Ca2+ on renal adenyl cyclase

    International Nuclear Information System (INIS)

    Nielsen, S.T.; Neuman, W.F.

    1978-01-01

    The effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal-lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity. Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH- or Gpp(NH)p-stimulated activity was given by approximately 50 μM Ca 2+ . Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half-maximal stimulation of cAMP formation (1.8 x 10 -8 M) was unchanged by the presence of calcium. These data suggest that calcium acts at some point other than the initial hormone-receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex

  12. Comparing the force ripple during asynchronous and conventional stimulation.

    Science.gov (United States)

    Downey, Ryan J; Tate, Mark; Kawai, Hiroyuki; Dixon, Warren E

    2014-10-01

    Asynchronous stimulation has been shown to reduce fatigue during electrical stimulation; however, it may also exhibit a force ripple. We quantified the ripple during asynchronous and conventional single-channel transcutaneous stimulation across a range of stimulation frequencies. The ripple was measured during 5 asynchronous stimulation protocols, 2 conventional stimulation protocols, and 3 volitional contractions in 12 healthy individuals. Conventional 40 Hz and asynchronous 16 Hz stimulation were found to induce contractions that were as smooth as volitional contractions. Asynchronous 8, 10, and 12 Hz stimulation induced contractions with significant ripple. Lower stimulation frequencies can reduce fatigue; however, they may also lead to increased ripple. Future efforts should study the relationship between force ripple and the smoothness of the evoked movements in addition to the relationship between stimulation frequency and NMES-induced fatigue to elucidate an optimal stimulation frequency for asynchronous stimulation. © 2014 Wiley Periodicals, Inc.

  13. Modulating Hippocampal Plasticity with In Vivo Brain Stimulation

    Science.gov (United States)

    2016-11-17

    wires were left unhooked from stimulation device. Following stimulation , the animals were returned to their homecage until time of euthanasia and...current stimulation (tDCS) to enhance cognitive training: effect of timing of stimulation . Exp Brain Res 232:3345-3351. 15 DISTRIBUTION...AFRL-RH-WP-TR-2016-0082 MODULATING HIPPOCAMPAL PLASTICITY WITH IN-VIVO BRAIN STIMULATION Joyce G. Rohan Oakridge Institute

  14. Addictive drugs and brain stimulation reward.

    Science.gov (United States)

    Wise, R A

    1996-01-01

    Direct electrical or chemical stimulation of specific brain regions can establish response habits similar to those established by natural rewards such as food or sexual contact. Cocaine, mu and delta opiates, nicotine, phencyclidine, and cannabis each have actions that summate with rewarding electrical stimulation of the medial forebrain bundle (MFB). The reward-potentiating effects of amphetamine and opiates are associated with central sites of action where these drugs also have their direct rewarding effects, suggesting common mechanisms for drug reward per se and for drug potentiation of brain stimulation reward. The central sites at which these and perhaps other drugs of abuse potentiate brain stimulation reward and are rewarding in their own right are consistent with the hypothesis that the laboratory reward of brain stimulation and the pharmacological rewards of addictive drugs are habit forming because they act in the brain circuits that subserve more natural and biologically significant rewards.

  15. Neurotransmitter system of immune regulation as a marker of immunological disorders in pupils in the conditions of increased entry of strontium with drinking water

    Directory of Open Access Journals (Sweden)

    О.V. Dolgikh

    2015-09-01

    Full Text Available The evaluation of immunological markers in schoolchildren exposed to strontium is performed. It is shown that under the conditions of increased administration of strontium with drinking water the indication of spontaneous and induced levels of neurotransmitters in vitro allows to detect early functional disorders of the immune system. It was found that the following markers of specific hypersensitivity and mediators of intercellular immune regulation (IgG specific to strontium, cytokines IL-6, IL-10, IL-12, IL-17, α-TNF, GM-CSF, spontaneous and specifically stimulated, RANKL, OPG( may be proposed for the identification of health risk as early markers of immune disorders in school children living in areas of strontium geochemical provinces.

  16. A wireless wearable surface functional electrical stimulator

    Science.gov (United States)

    Wang, Hai-Peng; Guo, Ai-Wen; Zhou, Yu-Xuan; Xia, Yang; Huang, Jia; Xu, Chong-Yao; Huang, Zong-Hao; Lü, Xiao-Ying; Wang, Zhi-Gong

    2017-09-01

    In this paper, a wireless wearable functional electrical stimulator controlled by Android phone with real-time-varying stimulation parameters for multichannel surface functional electrical stimulation application has been developed. It can help post-stroke patients using more conveniently. This study focuses on the prototype design, including the specific wristband concept, circuits and stimulation pulse-generation algorithm. A novel stimulator circuit with a driving stage using a complementary current source technique is proposed to achieve a high-voltage compliance, a large output impedance and an accurate linear voltage-to-current conversion. The size of the prototype has been significantly decreased to 17 × 7.5 × 1 cm3. The performance of the prototype has been tested with a loaded resistor and wrist extension/flexion movement of three hemiplegic patients. According to the experiments, the stimulator can generate four-channel charge-balanced biphasic stimulation with a voltage amplitude up to 60 V, and the pulse frequency and width can be adjusted in real time with a range of 100-600 μs and 20-80 Hz, respectively.

  17. High frequency oscillations evoked by peripheral magnetic stimulation.

    Science.gov (United States)

    Biller, S; Simon, L; Fiedler, P; Strohmeier, D; Haueisen, J

    2011-01-01

    The analysis of somatosensory evoked potentials (SEP) and / or fields (SEF) is a well-established and important tool for investigating the functioning of the peripheral and central human nervous system. A standard technique to evoke SEPs / SEFs is the stimulation of the median nerve by using a bipolar electrical stimulus. We aim at an alternative stimulation technique enabling stimulation of deep nerve structures while reducing patient stress and error susceptibility. In the current study, we apply a commercial transcranial magnetic stimulation system for peripheral magnetic stimulation of the median nerve. We compare the results of simultaneously recorded EEG signals to prove applicability of our technique to evoke SEPs including low frequency components (LFC) as well as high frequency oscillations (HFO). Therefore, we compare amplitude, latency and time-frequency characteristics of the SEP of 14 healthy volunteers after electric and magnetic stimulation. Both low frequency components and high frequency oscillations were detected. The HFOs were superimposed onto the primary cortical response N20. Statistical analysis revealed significantly lower amplitudes and increased latencies for LFC and HFO components after magnetic stimulation. The differences indicate the inability of magnetic stimulation to elicit supramaximal responses. A psycho-perceptual evaluation showed that magnetic stimulation was less unpleasant for 12 out of the 14 volunteers. In conclusion, we showed that LFC and HFO components related to median nerve stimulation can be evoked by peripheral magnetic stimulation.

  18. Consensus paper: combining transcranial stimulation with neuroimaging

    DEFF Research Database (Denmark)

    Siebner, Hartwig R; Bergmann, Til O; Bestmann, Sven

    2009-01-01

    neuroimaging (online approach), TMS can be used to test how focal cortex stimulation acutely modifies the activity and connectivity in the stimulated neuronal circuits. TMS and neuroimaging can also be separated in time (offline approach). A conditioning session of repetitive TMS (rTMS) may be used to induce...... information obtained by neuroimaging can be used to define the optimal site and time point of stimulation in a subsequent experiment in which TMS is used to probe the functional contribution of the stimulated area to a specific task. In this review, we first address some general methodologic issues that need......In the last decade, combined transcranial magnetic stimulation (TMS)-neuroimaging studies have greatly stimulated research in the field of TMS and neuroimaging. Here, we review how TMS can be combined with various neuroimaging techniques to investigate human brain function. When applied during...

  19. Stuttering in Parkinson's disease after deep brain stimulation: A note on dystonia and low-frequency stimulation.

    Science.gov (United States)

    Mendonça, Marcelo D; Barbosa, Raquel; Seromenho-Santos, Alexandra; Reizinho, Carla; Bugalho, Paulo

    2018-04-01

    Stuttering, a speech fluency disorder, is a rare complication of Deep Brain Stimulation (DBS) in Parkinson's Disease (PD). We report a 61 years-old patient with PD, afflicted by severe On and Off dystonia, treated with Subthalamic Nucleus DBS that developed post-DBS stuttering while on 130 Hz stimulation. Stuttering reduction was noted when frequency was changed to 80 Hz, but the previously observed dystonia improvement was lost. There are no reports in literature on patients developing stuttering with low-frequency stimulation. We question if low-frequency stimulation could have a role for managing PD's post-DBS stuttering, and notice that stuttering improvement was associated with dystonia worsening suggesting that they are distinct phenomena. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Stimulation of hair cells with ultraviolet light

    Science.gov (United States)

    Azimzadeh, Julien B.; Fabella, Brian A.; Hudspeth, A. J.

    2018-05-01

    Hair bundles are specialized organelles that transduce mechanical inputs into electrical outputs. To activate hair cells, physiologists have resorted to mechanical methods of hair-bundle stimulation. Here we describe a new method of hair-bundle stimulation, irradiation with ultraviolet light. A hair bundle illuminated by ultraviolet light rapidly moves towards its tall edge, a motion typically associated with excitatory stimulation. The motion disappears upon tip-link rupture and is associated with the opening of mechanotransduction channels. Hair bundles can be induced to move sinusoidally with oscillatory modulation of the stimulation power. We discuss the implications of ultraviolet stimulation as a novel hair-bundle stimulus.

  1. Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Ju, E-mail: biohjk@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Hye-Jin [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Kyung-Ae [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Gwon, Mi-Ri; Jin Seong, Sook [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, Shin-Yoon [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Young-Ran, E-mail: yry@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    2015-06-10

    Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells. - Highlights: • LCN2 expression is regulated during osteoclast development. • LCN2 suppresses M-CSF-mediated osteoclast precursor proliferation. • LCN2 inhibits RANKL-induced osteoclast differentiation.

  2. Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

    Directory of Open Access Journals (Sweden)

    Shamik Das

    Full Text Available The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation in response to tumor cells. We have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of the ligand, IHH and transcription factor GLI1. Breast cancer cells interact with osteoblasts and cause an enhanced differentiation of pre-osteoblasts to osteoblasts that express increased levels of the osteoclastogenesis factors, RANKL and PTHrP. There is sustained expression of osteoclast-promoting factors, RANKL and PTHrP, even after the osteoblast differentiation ceases and apoptosis sets in. Moreover, tumor cells that are deficient in Hh signaling are compromised in their ability to induce osteoblast differentiation and consequently are inefficient in causing osteolysis. The stimulation of osteoblast differentiation sets the stage for osteoclast differentiation and overall promotes osteolysis. Thus, in the process of developing newer therapeutic strategies against breast cancer metastasis to bone it would worthwhile to keep in mind the role of the Hh pathway in osteoblast differentiation in an otherwise predominant osteolytic phenomenon.

  3. Can the human lumbar posterior columns be stimulated by transcutaneous spinal cord stimulation? A modeling study

    OpenAIRE

    Danner, Simon M.; Hofstoetter, Ursula S.; Ladenbauer, Josef; Rattay, Frank; Minassian, Karen

    2011-01-01

    Stimulation of different spinal cord segments in humans is a widely developed clinical practice for modification of pain, altered sensation and movement. The human lumbar cord has become a target for modification of motor control by epidural and more recently by transcutaneous spinal cord stimulation. Posterior columns of the lumbar spinal cord represent a vertical system of axons and when activated can add other inputs to the motor control of the spinal cord than stimulated posterior roots. ...

  4. Vertex Stimulation as a Control Site for Transcranial Magnetic Stimulation: A Concurrent TMS/fMRI Study

    OpenAIRE

    Jung, JeYoung; Bungert, Andreas; Bowtell, Richard; Jackson, Stephen R.

    2016-01-01

    Background A common control condition for transcranial magnetic stimulation (TMS) studies is to apply stimulation at the vertex. An assumption of vertex stimulation is that it has relatively little influence over on-going brain processes involved in most experimental tasks, however there has been little attempt to measure neural changes linked to vertex TMS. Here we directly test this assumption by using a concurrent TMS/fMRI paradigm in which we investigate fMRI blood-oxygenation-level-depen...

  5. Computational modeling of epidural cortical stimulation

    Science.gov (United States)

    Wongsarnpigoon, Amorn; Grill, Warren M.

    2008-12-01

    Epidural cortical stimulation (ECS) is a developing therapy to treat neurological disorders. However, it is not clear how the cortical anatomy or the polarity and position of the electrode affects current flow and neural activation in the cortex. We developed a 3D computational model simulating ECS over the precentral gyrus. With the electrode placed directly above the gyrus, about half of the stimulus current flowed through the crown of the gyrus while current density was low along the banks deep in the sulci. Beneath the electrode, neurons oriented perpendicular to the cortical surface were depolarized by anodic stimulation, and neurons oriented parallel to the boundary were depolarized by cathodic stimulation. Activation was localized to the crown of the gyrus, and neurons on the banks deep in the sulci were not polarized. During regulated voltage stimulation, the magnitude of the activating function was inversely proportional to the thickness of the CSF and dura. During regulated current stimulation, the activating function was not sensitive to the thickness of the dura but was slightly more sensitive than during regulated voltage stimulation to the thickness of the CSF. Varying the width of the gyrus and the position of the electrode altered the distribution of the activating function due to changes in the orientation of the neurons beneath the electrode. Bipolar stimulation, although often used in clinical practice, reduced spatial selectivity as well as selectivity for neuron orientation.

  6. An Implantable Mixed Analog/Digital Neural Stimulator Circuit

    DEFF Research Database (Denmark)

    Gudnason, Gunnar; Bruun, Erik; Haugland, Morten

    1999-01-01

    This paper describes a chip for a multichannel neural stimulator for functional electrical stimulation. The chip performs all the signal processing required in an implanted neural stimulator. The power and signal transmission to the stimulator is carried out via an inductive link. From the signals...... electrical stimulation is to restore various bodily functions (e.g. motor functions) in patients who have lost them due to injury or disease....

  7. Gamma-radiation-induced chromosal aberration in human lymphocytes: dose-rate effects in stimulated and non-stimulated cells

    International Nuclear Information System (INIS)

    Liniecki, J.; Bajerska, A.; Wyszynska, K.; Cisowska, B.

    1977-01-01

    Stimulated and non-stimulated human peripheral blood lymphocytes were irradiated acutely and chronically, over 24 h. Dose-effect relationships for dicentric chromosomes were established and various models were fitted to the data. At prolonged irradiations, the yield decreased in basic agreement with the linear-quadratic model of aberration induction. Dose-protraction experiments on PHA + and PHA - lymphocytes, irradiated under various conditions of oxygenation and suspension (culture medium, whole blood) showed that the rejoining time increased from about 3 h in non-stimulated cells to about 10 h after PHA stimulation, and that this retarded rejoining was most likely due to blastic transformation itself and not to other conditions of irradiation

  8. Can the human lumbar posterior columns be stimulated by transcutaneous spinal cord stimulation? A modeling study.

    Science.gov (United States)

    Danner, Simon M; Hofstoetter, Ursula S; Ladenbauer, Josef; Rattay, Frank; Minassian, Karen

    2011-03-01

    Stimulation of different spinal cord segments in humans is a widely developed clinical practice for modification of pain, altered sensation, and movement. The human lumbar cord has become a target for modification of motor control by epidural and, more recently, by transcutaneous spinal cord stimulation. Posterior columns of the lumbar spinal cord represent a vertical system of axons and when activated can add other inputs to the motor control of the spinal cord than stimulated posterior roots. We used a detailed three-dimensional volume conductor model of the torso and the McIntyre-Richard-Grill axon model to calculate the thresholds of axons within the posterior columns in response to transcutaneous lumbar spinal cord stimulation. Superficially located large-diameter posterior column fibers with multiple collaterals have a threshold of 45.4 V, three times higher than posterior root fibers (14.1 V). With the stimulation strength needed to activate posterior column axons, posterior root fibers of large and small diameters as well as anterior root fibers are coactivated. The reported results inform on these threshold differences, when stimulation is applied to the posterior structures of the lumbar cord at intensities above the threshold of large-diameter posterior root fibers. © 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  9. Transcranial Alternating Current Stimulation Attenuates Neuronal Adaptation.

    Science.gov (United States)

    Kar, Kohitij; Duijnhouwer, Jacob; Krekelberg, Bart

    2017-03-01

    We previously showed that brief application of 2 mA (peak-to-peak) transcranial currents alternating at 10 Hz significantly reduces motion adaptation in humans. This is but one of many behavioral studies showing that weak currents applied to the scalp modulate neural processing. Transcranial stimulation has been shown to improve perception, learning, and a range of clinical symptoms. Few studies, however, have measured the neural consequences of transcranial current stimulation. We capitalized on the strong link between motion perception and neural activity in the middle temporal (MT) area of the macaque monkey to study the neural mechanisms that underlie the behavioral consequences of transcranial alternating current stimulation. First, we observed that 2 mA currents generated substantial intracranial fields, which were much stronger in the stimulated hemisphere (0.12 V/m) than on the opposite side of the brain (0.03 V/m). Second, we found that brief application of transcranial alternating current stimulation at 10 Hz reduced spike-frequency adaptation of MT neurons and led to a broadband increase in the power spectrum of local field potentials. Together, these findings provide a direct demonstration that weak electric fields applied to the scalp significantly affect neural processing in the primate brain and that this includes a hitherto unknown mechanism that attenuates sensory adaptation. SIGNIFICANCE STATEMENT Transcranial stimulation has been claimed to improve perception, learning, and a range of clinical symptoms. Little is known, however, how transcranial current stimulation generates such effects, and the search for better stimulation protocols proceeds largely by trial and error. We investigated, for the first time, the neural consequences of stimulation in the monkey brain. We found that even brief application of alternating current stimulation reduced the effects of adaptation on single-neuron firing rates and local field potentials; this mechanistic

  10. Modern management of epilepsy: Vagus nerve stimulation.

    Science.gov (United States)

    Ben-Menachem, E

    1996-12-01

    Vagus nerve stimulation (VNS) was first tried as a treatment for seizure patients in 1988. The idea to stimulate the vagus nerve and disrupt or prevent seizures was proposed by Jacob Zabarra. He observed a consistent finding among several animal studies which indicated that stimulation of the vagus nerve could alter the brain wave patterns of the animals under study. His hypothesis formed the basis for the development of the vagus nerve stimulator, an implantable device similar to a pacemaker, which is implanted in the left chest and attached to the left vagus nerve via a stimulating lead. Once implanted, the stimulator is programmed by a physician to deliver regular stimulation 24 hours a day regardless of seizure activity. Patients can also activate extra 'on-demand' stimulation with a handheld magnet. Clinical studies have demonstrated VNS therapy to be a safe and effective mode of treatment when added to the existing regimen of severe, refractory patients with epilepsy. Efficacy ranges from seizure free to no response with the majority of patients (> 50%) reporting at least a 50% improvement in number of seizures after 1.5 years of treatment. The side-effect profile is unique and mostly includes stimulation-related sensations in the neck and throat. The mechanism of action for VNS is not clearly understood although two theories have emerged. First, the direct connection theory hypothesizes that the anticonvulsant action of VNS is caused by a threshold raising effect of the connections to the nucleus of the solitary tract and on to other structures. The second is the concept that chronic stimulation of the vagus nerve increases the amount of inhibitory neurotransmitters and decreases the amount of excitatory neurotransmitters. Additional research into the optimal use of VNS is ongoing. Animal and clinical research have produced some interesting new data suggesting there are numerous ways to improve the clinical performance of vagus nerve stimulation as a

  11. Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

    Science.gov (United States)

    Lin, Jia-Hui; Yang, Ya-Chun; Shih, Ya-Chen; Hung, Szu-Ying; Lu, Chi-Yu; Tseng, Wei-Lung

    2016-03-15

    Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

    International Nuclear Information System (INIS)

    Dreslor, S.L.; Frattini, M.G.

    1987-01-01

    Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N 2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

  13. [A prospective study of adenosine triphosphate-tumor chemosensitivity assay directed chemotherapy in patients with recurrent ovarian cancer].

    Science.gov (United States)

    Gao, Yu-tao; Wu, Ling-ying; Zhang, Wei; Zhao, Dan; Li, Ning; Tian, Hai-mei; Wang, Xiao-bing; Li, Mo; Sun, Yang-chun; Li, Nan; Li, Xiao-guang

    2013-05-01

    To investigate the efficacy of adenosine triphosphate (ATP)-tumor chemosensitivity assay (TCA) directed chemotherapy in patients with recurrent epithelial ovarian cancer. From August 2010 to June 2012, recurrent epithelial ovarian cancer patients were prospectively enrollmented in Cancer Hospital, Peking Union Medical College,Chinese Academy of Medical Sciences.The entry criteria are as follows: (1) Histologically proven to be epithelial ovarian cancer. (2) Patients of recurrent ovarian cancer with bidimensionally measurable tumor, or ascitic or pleural fluid for testing. (3) Karnofsky performance status > 60. (4) A life expectancy of at least more than 6 months.According to patients desires, they were assigned into two groups: assay-directed therapy group and physician's-choice therapy group, patients' clinical and pathological characteristics, response rate to chemotherapy and progression-free survival (PFS) were compared between two groups. A total of 113 patients with recurrent epithelial ovarian cancer were prospectively enrollmented to assay-directed chemotherapy (n = 56) or physician's-choice chemotherapy (n = 57).There was no difference in median age,types of recurrence, surgical-pathological stage, pathological type, tumor grade, times of recurrence, residual disease at secondary cytoreductive surgery between assay-directed group and physician's-choice group. The overall response rate (ORR) and median PFS in the ATP-TCA group was 66% (37/56) and 7 months, while the ORR in the control group was 46% (26/57, P = 0.037), the median PFS was 4 months (P = 0.040). For platinum-resistant patients, the ORR between ATP-TCA directed chemotherapy 59% (16/27) and control group 25% (7/28) were significantly different (P = 0.010), and the median PFS between two groups were also significantly different (5 months and 2 months, respectively, P = 0.003). ATP-TCA directed chemotherapy could improve ORR and PFS in patients with recurrent epithelial ovarian cancer, especially

  14. Comparison of myocardial blood flow induced by adenosine triphosphate and dipyridamole in patients with coronary artery disease

    International Nuclear Information System (INIS)

    Mamede, M.; Tadamura, Eiji; Hosokawa, Ryohei

    2005-01-01

    Myocardial perfusion imaging with adenosine triphosphate (ATP) has been used increasingly to diagnose coronary artery disease (CAD) and assess risk for this disease. This study compared absolute myocardial blood flow (MBF) and myocardial flow reserve index (MFR) with ATP and dipyridamole (DIP) in patients with CAD. MBF was quantified by 15 O-H 2 O PET in 21 patients with CAD (17 male, 4 female), aged 55 to 81 years. MBF was measured at rest, during intravenous injection of ATP (0.16 mg/kg/min), and again after DIP infusion (0.56 mg/kg). Regions of interest were drawn in nonischemic and ischemic segments based on findings from thallium-201 ( 201 Tl) scintigraphy and coronary angiography (CAG). Absolute MBF values and indexes of MFR were calculated in nonischemic and ischemic segments. Intravenous injection of ATP and DIP significantly increased MBF in nonischemic (2.4±0.9 and 2.1±0.8 ml/g/min, respectively; p<0.01, for both) and in ischemic segments (1.3±0.4 and 1.5±0.4 ml/g/min, respectively; p<0.01, for both). There was a significant difference in MBF values between ATP and DIP in nonischemic segments (p<0.05), which was not observed in ischemic segments. In nonischemic segments, ATP produced higher MFR than DIP (2.1±0.8 and 1.8±0.7, respectively; p<0.05), while no significant difference was observed in ischemic segments (1.5±0.6 and 1.7±0.3, respectively). ATP produced a greater hyperemia than DIP between the ischemic and nonischemic myocardium in patients with CAD. ATP is as effective as DIP for the diagnosis of CAD. (author)

  15. Transcranial electrical stimulation accelerates human sleep homeostasis.

    Directory of Open Access Journals (Sweden)

    Davide Reato

    Full Text Available The sleeping brain exhibits characteristic slow-wave activity which decays over the course of the night. This decay is thought to result from homeostatic synaptic downscaling. Transcranial electrical stimulation can entrain slow-wave oscillations (SWO in the human electro-encephalogram (EEG. A computational model of the underlying mechanism predicts that firing rates are predominantly increased during stimulation. Assuming that synaptic homeostasis is driven by average firing rates, we expected an acceleration of synaptic downscaling during stimulation, which is compensated by a reduced drive after stimulation. We show that 25 minutes of transcranial electrical stimulation, as predicted, reduced the decay of SWO in the remainder of the night. Anatomically accurate simulations of the field intensities on human cortex precisely matched the effect size in different EEG electrodes. Together these results suggest a mechanistic link between electrical stimulation and accelerated synaptic homeostasis in human sleep.

  16. Effect of Low-Level Laser Stimulation on EEG

    Directory of Open Access Journals (Sweden)

    Jih-Huah Wu

    2012-01-01

    Full Text Available Conventional laser stimulation at the acupoint can induce significant brain activation, and the activation is theoretically conveyed by the sensory afferents. Whether the insensible low-level Laser stimulation outside the acupoint could also evoke electroencephalographic (EEG changes is not known. We designed a low-level laser array stimulator (6 pcs laser diode, wavelength 830 nm, output power 7 mW, and operation frequency 10 Hz to deliver insensible laser stimulations to the palm. EEG activities before, during, and after the laser stimulation were collected. The amplitude powers of each EEG frequency band were analyzed. We found that the low-level laser stimulation was able to increase the power of alpha rhythms and theta waves, mainly in the posterior head regions. These effects lasted at least 15 minutes after cessation of the laser stimulation. The amplitude power of beta activities in the anterior head regions decreased after laser stimulation. We thought these EEG changes comparable to those in meditation.

  17. Effect of low-level laser stimulation on EEG.

    Science.gov (United States)

    Wu, Jih-Huah; Chang, Wen-Dien; Hsieh, Chang-Wei; Jiang, Joe-Air; Fang, Wei; Shan, Yi-Chia; Chang, Yang-Chyuan

    2012-01-01

    Conventional laser stimulation at the acupoint can induce significant brain activation, and the activation is theoretically conveyed by the sensory afferents. Whether the insensible low-level Laser stimulation outside the acupoint could also evoke electroencephalographic (EEG) changes is not known. We designed a low-level laser array stimulator (6 pcs laser diode, wavelength 830 nm, output power 7 mW, and operation frequency 10 Hz) to deliver insensible laser stimulations to the palm. EEG activities before, during, and after the laser stimulation were collected. The amplitude powers of each EEG frequency band were analyzed. We found that the low-level laser stimulation was able to increase the power of alpha rhythms and theta waves, mainly in the posterior head regions. These effects lasted at least 15 minutes after cessation of the laser stimulation. The amplitude power of beta activities in the anterior head regions decreased after laser stimulation. We thought these EEG changes comparable to those in meditation.

  18. Electrical stimulation and motor recovery.

    Science.gov (United States)

    Young, Wise

    2015-01-01

    In recent years, several investigators have successfully regenerated axons in animal spinal cords without locomotor recovery. One explanation is that the animals were not trained to use the regenerated connections. Intensive locomotor training improves walking recovery after spinal cord injury (SCI) in people, and >90% of people with incomplete SCI recover walking with training. Although the optimal timing, duration, intensity, and type of locomotor training are still controversial, many investigators have reported beneficial effects of training on locomotor function. The mechanisms by which training improves recovery are not clear, but an attractive theory is available. In 1949, Donald Hebb proposed a famous rule that has been paraphrased as "neurons that fire together, wire together." This rule provided a theoretical basis for a widely accepted theory that homosynaptic and heterosynaptic activity facilitate synaptic formation and consolidation. In addition, the lumbar spinal cord has a locomotor center, called the central pattern generator (CPG), which can be activated nonspecifically with electrical stimulation or neurotransmitters to produce walking. The CPG is an obvious target to reconnect after SCI. Stimulating motor cortex, spinal cord, or peripheral nerves can modulate lumbar spinal cord excitability. Motor cortex stimulation causes long-term changes in spinal reflexes and synapses, increases sprouting of the corticospinal tract, and restores skilled forelimb function in rats. Long used to treat chronic pain, motor cortex stimuli modify lumbar spinal network excitability and improve lower extremity motor scores in humans. Similarly, epidural spinal cord stimulation has long been used to treat pain and spasticity. Subthreshold epidural stimulation reduces the threshold for locomotor activity. In 2011, Harkema et al. reported lumbosacral epidural stimulation restores motor control in chronic motor complete patients. Peripheral nerve or functional electrical

  19. Shared Neural Mechanisms for the Evaluation of Intense Sensory Stimulation and Economic Reward, Dependent on Stimulation-Seeking Behavior.

    Science.gov (United States)

    Norbury, Agnes; Valton, Vincent; Rees, Geraint; Roiser, Jonathan P; Husain, Masud

    2016-09-28

    Why are some people strongly motivated by intense sensory experiences? Here we investigated how people encode the value of an intense sensory experience compared with economic reward, and how this varies according to stimulation-seeking preference. Specifically, we used a novel behavioral task in combination with computational modeling to derive the value individuals assigned to the opportunity to experience an intense tactile stimulus (mild electric shock). We then examined functional imaging data recorded during task performance to see how the opportunity to experience the sensory stimulus was encoded in stimulation-seekers versus stimulation-avoiders. We found that for individuals who positively sought out this kind of sensory stimulation, there was common encoding of anticipated economic and sensory rewards in the ventromedial prefrontal cortex. Conversely, there was robust encoding of the modeled probability of receiving such stimulation in the insula only in stimulation-avoidant individuals. Finally, we found preliminary evidence that sensory prediction error signals may be positively signed for stimulation-seekers, but negatively signed for stimulation-avoiders, in the posterior cingulate cortex. These findings may help explain why high intensity sensory experiences are appetitive for some individuals, but not for others, and may have relevance for the increased vulnerability for some psychopathologies, but perhaps increased resilience for others, in high sensation-seeking individuals. People vary in their preference for intense sensory experiences. Here, we investigated how different individuals evaluate the prospect of an unusual sensory experience (electric shock), compared with the opportunity to gain a more traditional reward (money). We found that in a subset of individuals who sought out such unusual sensory stimulation, anticipation of the sensory outcome was encoded in the same way as that of monetary gain, in the ventromedial prefrontal cortex

  20. Effect of transcutaneous electrical nerve stimulation induced parotid stimulation on salivary flow

    Directory of Open Access Journals (Sweden)

    Sreenivasulu Pattipati

    2013-01-01

    Full Text Available Aims and Objectives: The main objective of this study was to evaluate the duration of stimulation over the parotid salivary flow following the use of transcutaneous electric nerve stimulation (TENS in different age groups. Materials and Methods: The study was carried out in three different age groups. Under group A individuals from 21 to 35 years of age, group B 36-50 years and group C above 51 years were considered. In each group 30 subjects were taken of whom 15 were males and 15 were females. The placement of pads was approximated bilaterally over the parotid glands. The working parameters of TENS unit were fixed at 50 Hz and the unit was in normal mode. Results: Subjects belonging to group B were showing statistically significant increases in the duration of stimulated parotid salivary flow following the use of TENS. Conclusion: TENS can be considered as a non-pharmacological alternative to improve salivation for longer period in xerostomia patients.