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Sample records for human gd3 synthase

  1. Isolation and functional analysis of the human glioblastoma-specific promoter region of the human GD3 synthase (hST8Sia I) gene

    Institute of Scientific and Technical Information of China (English)

    Hyun-Mi Dae; Haw-Young Kwon; Nam-Young Kang; Na-Ree Song; Kyoung-Sook Kim; Cheorl-Ho Kim; Jai-Heon Lee; Young-Choon Lee

    2009-01-01

    We identified the promoter region of the human GD3 synthase (hST8Sia I) gene to elucidate the mechanism underlying the regulation of hST8Sia I expression in human glioblastoma cells. The 5-rapid amplification of cDNA end using mRNA prepared from U-87MG cells revealed the presence of transcription start site of hST8Sia I gene, and the 5'-terminal analysis of its product showed that transcription started from 648 nucleotides upstream of the translational initiation site. Functional analysis of the 5'-flanking region of the hST8Sia I gene by transient expression method revealed that the region from 638 to 498 is important for transcriptional activity of the hST8Sia I gene in U-87MG and T98G cells. This region lacks apparent TATA and CAAT boxes, but contains putative binding sites for transcription factors AREB6 and Elk-1. Site-directed mutagenesis and transient transfection assays demonstrated that both AREB6 and Elk-1 elements in this region were required for the promoter activity in U-87MG and T98G cells. These results indicated that both AREB6 and Elk-1 might play an essential role in the transcriptional activity of hST8Sia I gene essential for GD3 synthesis in human glioblastoma cells.

  2. GD3 synthase overexpression sensitizes hepatocarcinoma cells to hypoxia and reduces tumor growth by suppressing the cSrc/NF-kappaB survival pathway.

    Directory of Open Access Journals (Sweden)

    Josep M Lluis

    Full Text Available BACKGROUND: Hypoxia-mediated HIF-1alpha stabilization and NF-kappaB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth. METHODOLOGY/PRINCIPAL FINDINGS: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3, which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold, no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O(2 enhanced reactive oxygen species (ROS generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kappaB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts. CONCLUSION: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kappaB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

  3. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6.

    Science.gov (United States)

    Miyata, Maiko; Ichihara, Masatoshi; Tajima, Orie; Sobue, Sayaka; Kambe, Mariko; Sugiura, Kazumitsu; Furukawa, Koichi; Furukawa, Keiko

    2014-03-07

    Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.

  4. Increased a-series gangliosides positively regulate leptin/Ob receptor-mediated signals in hypothalamus of GD3 synthase-deficient mice.

    Science.gov (United States)

    Ji, Shuting; Tokizane, Kyohei; Ohkawa, Yuki; Ohmi, Yuhsuke; Banno, Ryoichi; Okajima, Tetsuya; Kiyama, Hiroshi; Furukawa, Koichi; Furukawa, Keiko

    2016-10-21

    Gangliosides are widely involved in the regulation of cells and organs. However, little is known about their roles in adipose tissues and hypothalamus. In GD3 synthase-knockout (GD3S KO) mice, deletion of b-series gangliosides resulted in the reduction of serum leptin due to disturbed secretion from adipocytes. To examine whether leptin signals altered, leptin/leptin receptor (ObR)-mediated signaling in hypothalamus was analyzed. Hypothalamus of GD3S KO mouse showed increased expression of GM1 and GD1a, and increased activation of ObR-mediated signals such as pSTAT3 and c-Fos. Leptin stimulation of hypothalamus-derived N-41 cells and their transfectants with GD3S cDNA showed that a-series gangliosides positively regulate leptin/ObR-mediated signals. Co-precipitation analysis revealed that ObR interacts with a-series gangliosides with increased association by leptin stimulation. In brown adipose tissues (BAT) of GD3S KO mice, their weights and adipocyte numbers were increased, and BAT markers such as PGC1α and UCP-1 were also up-regulated. These results suggested that leptin/ObRb-mediated signals were enhanced in hypothalamus of GD3S KO mice due to increased a-series gangliosides, leading to the apparently similar features of energy expenditure between the KO and wild type mice. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3.

    Science.gov (United States)

    Hamamura, Kazunori; Tsuji, Momoko; Hotta, Hiroshi; Ohkawa, Yuki; Takahashi, Masataka; Shibuya, Hidenobu; Nakashima, Hideyuki; Yamauchi, Yoshio; Hashimoto, Noboru; Hattori, Hisashi; Ueda, Minoru; Furukawa, Keiko; Furukawa, Koichi

    2011-05-27

    The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.

  6. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

    Energy Technology Data Exchange (ETDEWEB)

    Miyata, Maiko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Ichihara, Masatoshi; Tajima, Orie; Sobue, Sayaka; Kambe, Mariko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Sugiura, Kazumitsu [Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Furukawa, Koichi, E-mail: koichi@med.nagoya-u.ac.jp [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Furukawa, Keiko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan)

    2014-03-07

    Highlights: • Melanocytes showed low ST8SIA1 and high B3GALT4 levels in contrast with melanomas. • Direct UVB irradiation of melanocytes did not induce ganglioside synthase genes. • Culture supernatants of UVB-irradiated keratinocytes induced ST8SIA1 in melanocytes. • TNFα and IL-6 secreted from keratinocytes enhanced ST8SIA1 expression in melanocytes. • Inflammatory cytokines induced melanoma-related ST8SIA1 in melanocytes. - Abstract: Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.

  7. GM3 alpha2,8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells.

    Science.gov (United States)

    Daniotti, J L; Martina, J A; Giraudo, C G; Zurita, A R; Maccioni, H J

    2000-04-01

    GD3 synthase (Sial-T2) is a key enzyme of ganglioside synthesis that, in concert with GM2 synthase (GalNAc-T), regulates the ratio of a- and b-pathway gangliosides. In this work, we study the sub-Golgi location of an epitope-tagged version of chicken Sial-T2 transfected to CHO-K1 cells. The expressed protein was enzymatically active both in vitro and in vivo and showed a molecular mass of approximately 47 or approximately 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of, respectively, beta-mercaptoethanol. The 95-kDa form of Sial-T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impaired glycosylation, indicating that it was formed in the ER. Confocal immunofluorescence microscopy showed Sial-T2 localized to the Golgi complex and, within the organelle, partially co-localizing with the mannose-6-phosphate receptor, a marker of the trans-Golgi network (TGN). In cells treated with brefeldin A, a major fraction of Sial-T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overloading. In experiments of incorporation of sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP-NeuAc were converted to GD2 upon incubation with UDP-GalNAc. These results indicate that Sial-T2 localizes mainly to the proximal Golgi, although a fraction is located in the TGN functionally coupled to GalNAc-T. Consistent with this, most of the enzyme was in an endoglycosidase H (Endo-H)-sensitive, neuraminidase (NANase)-insensitive form. A minor secreted form lacking approximately 40 amino acids was Endo-H-resistant and NANase-sensitive, indicating that the cells were able to process N-glycans to Endo-H-resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proximal Golgi and that a functional fraction

  8. Accumulation of unusual gangliosides G(Q3) and G(P3) in breast cancer cells expressing the G(D3) synthase.

    Science.gov (United States)

    Steenackers, Agata; Vanbeselaere, Jorick; Cazet, Aurélie; Bobowski, Marie; Rombouts, Yoann; Colomb, Florent; Le Bourhis, Xuefen; Guérardel, Yann; Delannoy, Philippe

    2012-08-10

    Glycosphingolipids from the ganglio-series are usually classified in four series according to the presence of 0 to 3 sialic acid residues linked to lactosylceramide. The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases that show high specificity toward glycolipid substrates. ST8Sia I (EC 2.4.99.8, SAT-II, SIAT 8a) is the key enzyme controlling the biosynthesis of b- and c-series gangliosides. ST8Sia I is expressed at early developmental stages whereas in adult human tissues, ST8Sia I transcripts are essentially detected in brain. ST8Sia I together with b- and c-series gangliosides are also over-expressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, neuroblastoma and in estrogen receptor (ER) negative breast cancer, where they play a role in cell proliferation, migration, adhesion and angiogenesis. We have stably expressed ST8Sia I in MCF-7 breast cancer cells and analyzed the glycosphingolipid composition of wild type (WT) and GD3S+ clones. As shown by mass spectrometry, MCF-7 expressed a complex pattern of neutral and sialylated glycosphingolipids from globo- and ganglio-series. WT MCF-7 cells exhibited classical monosialylated gangliosides including G(M3), G(M2), and G(M1a). In parallel, the expression of ST8Sia I in MCF-7 GD3S+ clones resulted in a dramatic change in ganglioside composition, with the expression of b- and c-series gangliosides as well as unusual tetra- and pentasialylated lactosylceramide derivatives G(Q3) (II(3)Neu5Ac(4)-Gg(2)Cer) and G(P3) (II(3)Neu5Ac(5)-Gg(2)Cer). This indicates that ST8Sia I is able to act as an oligosialyltransferase in a cellular context.

  9. An epitope common to gangliosides O-acetyl-GD3 and GD3 recognized by antibodies in melanoma patients after active specific immunotherapy.

    Science.gov (United States)

    Ravindranath, M H; Morton, D L; Irie, R F

    1989-07-15

    GD3 is a major ganglioside of human melanoma and was shown to be an effective target for passive immunotherapy with murine monoclonal antibodies. It was noted earlier that GD3 neither purified nor in melanoma cell vaccine (MCV), could elicit an antibody response in melanoma patients. In this study, we demonstrate that melanoma patients who received MCV had autoantibodies against a derivative of GD3, O-acetylated GD3 (O-AcGD3), a minor ganglioside expressed on human melanoma cells, and that the antibodies cross-reacted with GD3. Thin layer chromatographic immunostaining revealed that all of the sera containing antibodies against O-AcGD3 also reacted to GD3. None of the other sera responded only to GD3, although the MCV contained 7- to 12-fold higher GD3 than O-AcGD3. Furthermore, the antibody activity was completely abolished by absorption with animal erythrocytes expressing either O-acetyl disialogangliosides or GD3, indicating that the antibodies recognize an epitope commonly shared by GD3 and O-AcGD3. The antibodies bound only to the sialyloligosaccharide moiety but not to the ceramide portion of GD3 after endoglycosylceramidase treatment. The antibodies failed to bind to GD3 after neuraminidase treatment. These results indicate that the sialyloligosaccharides of the gangliosides are important components of the epitope. Periodate oxidation abolished reactivity of the antibodies to GD3 but not that to O-AcGD3, revealing that the glycerol side chain of the sialic acids in both GD3s was an important structure of the epitope. The binding of the antibodies to melanoma cell surface gangliosides was confirmed by an absorption with a GD3- and O-AcGD3-positive melanoma cell line. These results in the light of previous reports on the inability of GD3 to elicit immune response in humans suggest that anti-GD3 antibodies found in the melanoma patients were induced by immunization with O-AcGD3 and O-AcGD3 present in the MCV would serve as an antigen source for GD3-targeted

  10. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...... endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle....

  11. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...... in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than...... in the longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather...

  12. Effect of Gd3+ on the colloidal stability of liposomes.

    Science.gov (United States)

    Sabín, Juan; Prieto, Gerardo; Sennato, Simona; Ruso, Juan M; Angelini, Roberta; Bordi, Federico; Sarmiento, Félix

    2006-09-01

    Lanthanide ions such as La3+ and Gd3+ are well known to have large effects on the structure of phospholipid membranes. Unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) were prepared by sonication method and confirmed by transmission electron microscopy. The effects of concentration of gadolinium ions Gd3+ on DPPC unilamellar vesicles in aqueous media were studied by different techniques. As physical techniques, photon correlation spectroscopy, electrophoretic mobility, and differential scanning calorimetry were used. The theoretical predictions of the colloidal stability of liposomes were followed using the Derjaguin-Landau-Verwey-Overbeek theory. Changes in the size of liposomes and high polydispersities values were observed as Gd3+ concentration increases, suggesting that this cation induces the aggregation of vesicles. Electrophoretic mobility measurements on unilamellar vesicles as a function of Gd3+ ion concentration show that the vesicles adsorb Gd3+ ions. Above Gd3+ concentrations of 0.1 mol dm-3, the zeta potential and light scattering measurements indicate the beginning of aggregation process. For comparison with similar phospholipids, the zeta potential of phosphatidylcholine interacting with Gd3+ was measured, showing an analogous behavior. Differential scanning calorimetry has been used to determine the effect of Gd3+ on the transition temperature (Tc) and on the enthalpy (DeltaHc) associated with the process.

  13. Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor α and Yes Kinase*

    Science.gov (United States)

    Ohkawa, Yuki; Momota, Hiroyuki; Kato, Akira; Hashimoto, Noboru; Tsuda, Yusuke; Kotani, Norihiro; Honke, Koichi; Suzumura, Akio; Furukawa, Keiko; Ohmi, Yuhsuke; Natsume, Atsushi; Wakabayashi, Toshihiko; Furukawa, Koichi

    2015-01-01

    There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas. PMID:25940087

  14. Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor α and Yes Kinase.

    Science.gov (United States)

    Ohkawa, Yuki; Momota, Hiroyuki; Kato, Akira; Hashimoto, Noboru; Tsuda, Yusuke; Kotani, Norihiro; Honke, Koichi; Suzumura, Akio; Furukawa, Keiko; Ohmi, Yuhsuke; Natsume, Atsushi; Wakabayashi, Toshihiko; Furukawa, Koichi

    2015-06-26

    There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.

  15. Overcoming artificial broadening in Gd(3+)-Gd(3+) distance distributions arising from dipolar pseudo-secular terms in DEER experiments.

    Science.gov (United States)

    Cohen, Marie Ramirez; Frydman, Veronica; Milko, Petr; Iron, Mark A; Abdelkader, Elwy H; Lee, Michael D; Swarbrick, James D; Raitsimring, Arnold; Otting, Gottfried; Graham, Bim; Feintuch, Akiva; Goldfarb, Daniella

    2016-05-14

    By providing accurate distance measurements between spin labels site-specifically attached to bio-macromolecules, double electron-electron resonance (DEER) spectroscopy provides a unique tool to probe the structural and conformational changes in these molecules. Gd(3+)-tags present an important family of spin-labels for such purposes, as they feature high chemical stability and high sensitivity in high-field DEER measurements. The high sensitivity of the Gd(3+) ion is associated with its high spin (S = 7/2) and small zero field splitting (ZFS), resulting in a narrow spectral width of its central transition at high fields. However, under the conditions of short distances and exceptionally small ZFS, the weak coupling approximation, which is essential for straightforward DEER data analysis, becomes invalid and the pseudo-secular terms of the dipolar Hamiltonian can no longer be ignored. This work further explores the effects of pseudo-secular terms on Gd(3+)-Gd(3+) DEER measurements using a specifically designed ruler molecule; a rigid bis-Gd(3+)-DOTA model compound with an expected Gd(3+)-Gd(3+) distance of 2.35 nm and a very narrow central transition at the W-band (95 GHz). We show that the DEER dipolar modulations are damped under the standard W-band DEER measurement conditions with a frequency separation, Δν, of 100 MHz between the pump and observe pulses. Consequently, the DEER spectrum deviates considerably from the expected Pake pattern. We show that the Pake pattern and the associated dipolar modulations can be restored with the aid of a dual mode cavity by increasing Δν from 100 MHz to 1.09 GHz, allowing for a straightforward measurement of a Gd(3+)-Gd(3+) distance of 2.35 nm. The increase in Δν increases the contribution of the |-5/2〉→|-3/2〉 and |-7/2〉→|-5/2〉 transitions to the signal at the expense of the |-3/2 〉→|-1/2〉 transition, thus minimizing the effect of dipolar pseudo-secular terms and restoring the validity of the weak

  16. From bacterial to human dihydrouridine synthase: automated structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Fiona, E-mail: fiona.whelan@york.ac.uk; Jenkins, Huw T., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Griffiths, Samuel C. [University of Oxford, Headington, Oxford OX3 7BN (United Kingdom); Byrne, Robert T. [Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich (Germany); Dodson, Eleanor J.; Antson, Alfred A., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom)

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  17. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  18. The crystal structure of human GDP-L-fucose synthase.

    Science.gov (United States)

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  19. Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.

    Directory of Open Access Journals (Sweden)

    Roberta d'Emmanuele di Villa Bianca

    Full Text Available Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.

  20. Gd3Al2-xGax系合金磁热效应的研究%Study on Magnetocaloric Effect of Gd3Al2-xGax

    Institute of Scientific and Technical Information of China (English)

    生利英; 曹朝霞; 黄焦宏; 齐勇田; 宋博宇

    2016-01-01

    The organization structure and the magnetocaloric effect of Gd3 Al2-x Gax (0.1 x =0,0.2,0.3) series alloy prepared by smelting in a a high-frequency floating furnace and annealing treatment at 900 ℃ for 30 h were investigated.The results show that Gd3Al2-xGax series alloy keeps simple tetragonal crystal structure of Gd3Al2.The content of base phase Gd3Al2 increases after heat treatment.The Curie temperature of Gd3 Al2-xGaxseries alloy changes between 1 K ~ 2 K along with the increase of the content of Ga.The half peak width of △Sm-T curve is almost the same.The maximum magnetic entropy changes of Gd3Al1.8Ga0.2 alloy is the largest under magnetic field changes of 1.5 T,which exceeds 30% of Gd3Al2 alloy.A small amount of Ga to replace Al can improve the magnetocaloric effect of Gd3Al2 alloy.%对经真空高频磁悬浮炉熔炼、900℃30 h热处理的Gd3 Al2-XGax(x=0,0.1,0.2,0.3)系列合金的组织结构和磁热效应进行了研究.研究发现,Gd3Al2-XGax系列合金保持了Gd3Al2的简单正方晶体结构,热处理后基相Gd3Al2的含量增加,随着Ga含量的增加,Gd3Al2-XGax合金的居里温度变化在1K~2K之间,在1.5T的外加磁场下,△Sm-T曲线半峰宽几乎不变,Gd3Al1.8Ga0.2合金的最大磁熵变最大,超过Gd3Al2的最大磁熵变30%.少量Ga替代Al可以提高Gd3Al2合金的磁热效应.

  1. Gd3Fe5O12 near the compensation temperature

    DEFF Research Database (Denmark)

    Hansen, Britt Rosendahl; Kuhn, Luise Theil; Bahl, Christian Robert Haffenden;

    2010-01-01

    Applying a magnetic eld to a magnetic material will usually cause a change in the magnetic entropy of the material. If done adiabatically, the temperature of the material will change - this is the magnetocaloric eect. Gadolinium Iron Garnet (GdIG, Gd3Fe5O12) is a complex magnetic system with three...... magnetic sublattices, one Gd and two Fe. The Fe sites are strongly coupled antiferromagnetically, i.e. the magnetic moments are anti-parallel, while the Gd magnetic moment is weakly coupled antiferromagnetically to the resulting Fe moment. This results in a compensation temperature, where the total...

  2. Energy transfer between Gd3+ and Tb3+ in phosphate glass

    Institute of Scientific and Technical Information of China (English)

    HE Dongbing; YU Chunlei; CHENG Jimeng; LI Shunguang; HU Lili

    2011-01-01

    The phosphate glass doped with Gd3+,Tb3+ and Gd3+/Tb3+ were prepared by high temperature melting. The photo-luminescence behavior of Gd3+ and Tb3+ in phosphate glass were investigated by absorption, excitation, and emission spectroscopy. Energy transfer between Gd3+ and Tb3+ in phosphate glass was studied, and it was found that there were two energy transfer mechanisms between Gd3+ and Tb3+ in phosphate glass: one was from 4f7 level of Gd3+ to the 4f8 level of Tb3+, and the other was from 5d level of Tb3+ to 4f7 level of Gd3+. The new findings would be beneficial for the study of Tb3+-doped scintillating phosphate glass.

  3. Facile combustion synthesis of novel CaZrO3:Eu3+, Gd3+ red phosphor and remarkably enhanced photoluminescence by Gd3+ doping

    Indian Academy of Sciences (India)

    Qingqing Du; Guangjun Zhou; Shuo Zhang; Xiao Jia; Haifeng Zhou; Zhongsen Yang

    2015-02-01

    A facile sol–gel combustion route was reported for the direct preparation of CaZrO3:Eu3+ and CaZrO3:Eu3+, Gd3+. The obtained deposits were characterized by XRD, TGA-DSC, SEM, EDS, PL measurements and microscope fluorescence. When the Gd3+ ions were introduced in this compound, the emissions of CaZrO3:Eu3+ were remarkably enhanced. The emission spectrum of CaZrO3:Eu3+, Gd3+ nanocrystals exhibited a red shift compared with CaZrO3:Eu3+ samples. A new mechanism of the improved photoluminescent properties of Eu3+ by Gd3+ was investigated. The optimized phosphor CaZrO3:5%Eu3+, 2%Gd3+ could be considered an efficient red-emitting component for white lighting devices excited in the near-ultraviolet region.

  4. Human leucocytes in asthenozoospermic patients: endothelial nitric oxide synthase expression.

    Science.gov (United States)

    Buldreghini, E; Hamada, A; Macrì, M L; Amoroso, S; Boscaro, M; Lenzi, A; Agarwal, A; Balercia, G

    2014-12-01

    In a basic study at the Andrology Unit, Department of Clinical and Molecular Sciences, Polytechnic University of Marche, Ancona, Italy, we evaluated the pattern of mRNA endothelial nitric oxide synthase (eNOS) expression in human blood leucocytes isolated from normozoospermic fertile and asthenozoospermic infertile men to elucidate any pathogenic involvement in sperm cell motility. Forty infertile men with idiopathic asthenozoospermia and 45 normozoospermic fertile donors, age-matched, were included. Semen parameters were evaluated, and expression analysis of mRNA was performed in human leucocytes using reverse transcription polymerase chain reaction. Sperm volume, count, motility and morphology were determined, and eNOS expression and Western blotting analyses were performed. A positive correlation was observed between the concentrations of NO and the percentage of immotile spermatozoa. The mRNA of eNOS was more expressed in peripheral blood leucocytes isolated from asthenozoospermic infertile men versus those of fertile normozoospermic men (7.46 ± 0.38 versus 7.06 ± 0.56, P = 0.0355). A significant up-regulation of eNOS gene in peripheral blood leucocytes was 1.52-fold higher than that of fertile donors. It is concluded that eNOS expression and activity are enhanced in blood leucocytes in men with idiopathic asthenozoospermia.

  5. Transcriptional regulation of human thromboxane synthase gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K.D.; Baek, S.J.; Fleischer, T [Univ. of Maryland Medical School, Baltimore, MD (United States)] [and others

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  6. Gd$^{3+}$ - Gd$^{3+}$ distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance

    CERN Document Server

    Clayton, Jessica A; Godt, Adelheid; Goldfarb, Daniella; Han, Songi; Sherwin, Mark S

    2016-01-01

    Electron paramagnetic resonance spectroscopy in combination with site-directed spin-labeling is a very powerful tool for elucidating the structure and organization of biomolecules. Gd$^{3+}$ complexes have recently emerged as a new class of spin labels for distance determination by pulsed EPR spectroscopy at Q- and W-band. We present CW EPR measurements at 240 GHz (8.6 Tesla) on a series of Gd-rulers of the type Gd-PyMTA---spacer---Gd-PyMTA, with Gd-Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these Gd-rulers show that significant dipolar broadening of the central $|-1/2\\rangle\\rightarrow|1/2\\rangle$ transition occurs at 30 K for Gd-Gd distances up to $\\sim$ 3.4 nm with Gd-PyMTA as the spin label. This represents a significant extension for distances accessible by CW EPR, as nitroxide-based spin labels at X-band frequencies can typically only access distances up to $\\sim$ 2 nm. We show that this broadening persists at biologically relevant temperatures above 200 K, and that this method i...

  7. Gd(3+)-Gd(3+) distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance.

    Science.gov (United States)

    Clayton, Jessica A; Qi, Mian; Godt, Adelheid; Goldfarb, Daniella; Han, Songi; Sherwin, Mark S

    2017-02-15

    Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling is a very powerful tool for elucidating the structure and organization of biomolecules. Gd(3+) complexes have recently emerged as a new class of spin labels for distance determination by pulsed EPR spectroscopy at Q- and W-band. We present CW EPR measurements at 240 GHz (8.6 Tesla) on a series of Gd-rulers of the type Gd-PyMTA-spacer-Gd-PyMTA, with Gd-Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these Gd-rulers show that significant dipolar broadening of the central |-1/2〉 → |1/2〉 transition occurs at 30 K for Gd-Gd distances up to ∼3.4 nm with Gd-PyMTA as the spin label. This represents a significant extension for distances accessible by CW EPR, as nitroxide-based spin labels at X-band frequencies can typically only access distances up to ∼2 nm. We show that this broadening persists at biologically relevant temperatures above 200 K, and that this method is further extendable up to room temperature by immobilizing the sample in glassy trehalose. We show that the peak-to-peak broadening of the central transition follows the expected 1/r(3) dependence for the electron-electron dipolar interaction, from cryogenic temperatures up to room temperature. A simple procedure for simulating the dependence of the lineshape on interspin distance is presented, in which the broadening of the central transition is modeled as an S = 1/2 spin whose CW EPR lineshape is broadened through electron-electron dipolar interactions with a neighboring S = 7/2 spin.

  8. Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine

    Energy Technology Data Exchange (ETDEWEB)

    Še; #269; kut; #279; , Jolita; McCloskey, Diane E.; Thomas, H. Jeanette; Secrist III, John A.; Pegg, Anthony E.; Ealick, Steven E. (Cornell); (Southern Research); (UPENN-MED)

    2011-11-17

    Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose-dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X-ray crystallography at 2.0 {angstrom} resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K{sub d} of 1.1 {+-} 0.3 {mu}M in the absence of putrescine and 3.2 {+-} 0.1 {mu}M in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.

  9. Constitutive expression of inducible nitric oxide synthase in the normal human colonic epithelium

    DEFF Research Database (Denmark)

    Perner, A; Andresen, L; Normark, M

    2002-01-01

    Inducible nitric oxide synthase (iNOS) in the human colon is considered expressed only in inflammatory states such as ulcerative or collagenous colitis. As subtle iNOS labelling was previously observed in some colonic mucosal biopsies from a heterogeneous group of controls with non-inflamed bowel...

  10. Constitutive expression of inducible nitric oxide synthase in the normal human colonic epithelium

    DEFF Research Database (Denmark)

    Perner, A; Andresen, Lars; Normark, M;

    2002-01-01

    Inducible nitric oxide synthase (iNOS) in the human colon is considered expressed only in inflammatory states such as ulcerative or collagenous colitis. As subtle iNOS labelling was previously observed in some colonic mucosal biopsies from a heterogeneous group of controls with non-inflamed bowel...

  11. Structural organization of the human neuronal nitric oxide synthase gene (NOS1).

    Science.gov (United States)

    Hall, A V; Antoniou, H; Wang, Y; Cheung, A H; Arbus, A M; Olson, S L; Lu, W C; Kau, C L; Marsden, P A

    1994-12-30

    Neuronal nitric oxide (NO) synthase, localized to human chromosome 12, uniquely participates in diverse biologic processes; neurotransmission, the regulation of body fluid homeostasis, neuroendocrine physiology, control of smooth muscle motility, sexual function, and myocyte/myoblast biology, among others. Restriction enzyme mapping, subcloning, and DNA sequence analysis of bacteriophage- and yeast artificial chromosome-derived human genomic DNA indicated that the mRNA for neuronal NO synthase is dispersed over a minimum of 160 kilobases of human genomic DNA. Analysis of intron-exon splice junctions predicted that the open reading frame is encoded by 28 exons, with translation initiation and termination in exon 2 and exon 29, respectively. Determination of transcription initiation sites in brain poly(A) RNA with primer extension analysis and RNase protection revealed a major start site 28 nucleotides downstream from a TATA box. Sequence inspection of 5'-flanking regions revealed potential cis-acting DNA elements: AP-2, TEF-1/MCBF, CREB/ATF/c-Fos, NRF-1, Ets, NF-1, and NF-kappa B-like sequences. Diversity appears to represent a major theme apparent upon analysis of human neuronal NO synthase mRNA transcripts. A microsatellite of the dinucleotide variety was detected within the 3'-untranslated region of exon 29. Multiple alleles were evident in normal individuals indicating the existence of allelic mRNA sequence variation. Characterization of variant human neuronal NO synthase cDNAs indicated the existence of casette exon 9/10 and exon 10 deletions as examples of structural mRNA diversity due to alternative splicing. The latter deletion of a 175-nucleotide exon introduces a frame-shift and premature stop codon indicating the potential existence of a novel NH2 terminus protein. In summary, analysis of the human neuronal NO synthase locus reveals a complex genomic organization and mRNA diversity that is both allelic and structural.

  12. Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression.

    Science.gov (United States)

    Sciannamblo, Mariateresa; Chigorno, Vanna; Passi, Alberto; Valaperta, Rea; Zucchi, Ileana; Sonnino, Sandro

    2002-03-01

    In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density. GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation. Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase.

  13. Preparation and Characterization of Gd3+-doped Monodisperse TiO2 Hollow Microsphere

    Institute of Scientific and Technical Information of China (English)

    JI; Feng; SHANG; Pengbo; ZHENG; Yuying

    2015-01-01

    Gd3+-doped monodisperse TiO2 hollow microspheres with various molar ratios of Gd3+/TBOT were synthesized via a novel process, which involved the preparation of SiO2 templates, deposition of Gd3+-doped TiO2 by sol-gel, SiO2 coating, heat treatment to induce crystallization of TiO2, and finally etching away the inner SiO2 templates and outer SiO2 layers. The synthesized samples were characterized by transmission electron microscopy(TEM), scanning electron microscopy(SEM), X-ray diffraction(XRD), diffuse reflection spectroscopy(DRS), specific surface area measurement(BET) and X-ray photoelectron spectroscopy(XPS), respectively. The photocatalytic activity of Gd3+-doped samples was evaluated via photocatalytic degradation of Methyl orange under UV irradiation. The results show that the SiO2 layers prevent aggregation of TiO2 hollow microspheresand improve the thermal stability of the synthesized samples. Also, the photocatalytic activity of monodisperse TiO2 hollow microspheres can be enhanced at the optimal molar ration of Gd3+/TBOT of 0.7%.

  14. Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase%Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase

    Institute of Scientific and Technical Information of China (English)

    张亚; 林赋; 邓晓东; 王任小; 叶德泳

    2011-01-01

    Sphingomyelin synthase (SMS) produces sphingomyelin and diacylglycerol from ceramide and phosphatidyl- choline. It plays an important role in cell survival and apoptosis, inflammation, and lipid homeostasis, and therefore has been noticed in recent years as a novel potential drug target. In this study, we combined homology modeling, molecular docking, molecular dynamics simulation, and normal mode analysis to derive a three-dimensional struc- ture of human sphingomyelin synthase (hSMS 1) in complex with sphingomyelin. Our model provides a reasonable explanation on the catalytic mechanism of hSMS 1. It can also explain the high selectivity of hSMS 1 towards phos- phocholine and sphingomyelin as well as some other known experimental results about hSMS1. Moreover, we also derived a complex model of D609, the only known small-molecule inhibitor of hSMS 1 so far. Our hSMS 1 model may serve as a reasonable structural basis for the discovery of more effective small-molecule inhibitors of hSMS 1.

  15. Human uroporphyrinogen-III synthase: genomic organization, alternative promoters, and erythroid-specific expression.

    Science.gov (United States)

    Aizencang, G; Solis, C; Bishop, D F; Warner, C; Desnick, R J

    2000-12-01

    Uroporphyrinogen-III (URO) synthase is the heme biosynthetic enzyme defective in congenital erythropoietic porphyria. The approximately 34-kb human URO-synthase gene (UROS) was isolated, and its organization and tissue-specific expression were determined. The gene had two promoters that generated housekeeping and erythroid-specific transcripts with unique 5'-untranslated sequences (exons 1 and 2A) followed by nine common coding exons (2B to 10). Expression arrays revealed that the housekeeping transcript was present in all tissues, while the erythroid transcript was only in erythropoietic tissues. The housekeeping promoter lacked TATA and SP1 sites, consistent with the observed low level expression in most cells, whereas the erythroid promoter contained GATA1 and NF-E2 sites for erythroid specificity. Luciferase reporter assays demonstrated that the housekeeping promoter was active in both erythroid K562 and HeLa cells, while the erythroid promoter was active only in erythroid cells and its activity was increased during hemin-induced erythroid differentiation. Thus, human URO-synthase expression is regulated during erythropoiesis by an erythroid-specific alternative promoter.

  16. Crystallization and preliminary X-ray crystallographic analysis of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    CHEN Yushu; LI Xiaofeng; LOU Zhiyong; WANG Weina; PANG Hai; RAO Zihe

    2006-01-01

    Human geranylgeranyl pyrophosphate synthase (GGPS) is an enzyme that catalyzes the synthesis of geranylgeranyl py rophosphate (GGPP) from farnesyldiphosphate and isopentenyldiphosphate. Recombinant human GGPS was crystallized by the hanging-drop vapor diffusion method. Crystals were grown at 18℃ using PEG 4000 as precipitant. Diffraction data were obtained to a resolution of 2.8 (A) from a single frozen crystal belonging to space group PI, with unit-cell parameters: α = 68.9 (A), b = 107.7 (A), c = 137.4 (A),α=99.6°, β=97.6°, γ=97.8°.

  17. Luminescent Properties of Gd3PO7:Eu in UV/VUV Region

    Institute of Scientific and Technical Information of China (English)

    ZENG Xiao-Qing; KWON I1-Eok; HONG Guang-Yan; YOU Hong-Peng; WU Xue-Yan; KIM Chang-Hong; PYUN Chong-Hong; YU Byung-Yong; BAE Hyun-Sook; PARK Cheal-Hee

    2001-01-01

    The luminescent properties of Gd3PO7:Eu were investigated in UV and VUV regions. This phosphor has such strong emissions around 618.5 nm under UV excitation, especially around 209nm, that it has a better colour saturation than that of Y2O3:Eu. It could be a new potential red phosphor for lamp and other applications with UV-excitation source or even for displaying devices. In the VUV excitation spectrum of Eu3+ emissions, energy absorptions by Gd3+, Gd-O and PO34- have been observed, on which it can be inferred that there are three kinds of energy transferring processes to Eu3+ ions. The energy absorptions of Gd3+, Gd-O and PO34- are induced by 4 f → 4 f transitions, a bandgap transition of Gd3PO7 and intramolecular (2t2 → 2a, 3t2) transition, respectively.The energy transfer efficiency of the PO34- intramolecular transition is lower compared with that of the bandgap transition (Gd-O).

  18. Inter-atomic bonding and dielectric polarization in Gd3+ incorporated Co-Zn ferrite nanoparticles

    Science.gov (United States)

    Pawar, R. A.; Desai, S. S.; Patange, S. M.; Jadhav, S. S.; Jadhav, K. M.

    2017-04-01

    A series of ferrite with a chemical composition Co0.7Zn0.3GdxFe2-xO4 (where x=0.0 to x=0.1) were prepared by sol-gel auto-combustion method. X-ray diffraction pattern were used to determine the crystal structure and phase formation of the prepared samples. Scanning electron microscopy is used to study the surface morphology of the prepared samples. Elastic properties were determined from the infrared spectroscopy. Debye temperature, wave velocities, elastic constants found to increase with the increase in Gd3+ substitution. Dielectric properties such as dielectric constant and dielectric loss were studied as a function of Gd3+ substitution and frequency. Dielectric constant decreased with the increase in frequency and Gd3+ substitution. Behavior of dielectric properties was explained on the basis of Maxwell-Wagner interfacial polarization which in accordance with Koops phenomenological theory. Real and imaginary part of impedance was studied as a function of resistance and Gd3+ substitution. The behavior of impedance is systematically discussed on the basis of resistance-capacitance circuit.

  19. Regulation by GD3 of the proinflammatory response of microglia mediated by interleukin-15.

    Science.gov (United States)

    Gómez-Nicola, Diego; Doncel-Pérez, Ernesto; Nieto-Sampedro, Manuel

    2006-04-01

    The interleukin (IL)-15-dependent immune responses of murine microglia were strongly affected by low concentrations of the ganglioside GD3. The ganglioside binding to IL-15 inhibited the proinflammatory effects of the cytokine, reducing IL-15-dependent T-cell proliferation as well as mRNA expression for IL-15Ralpha, p65, and NFATc2 in the N13 murine microglial cell line. Treatment of primary murine microglial cultures with GD3 abolished IL-15 production, without affecting cellular viability, but decreased the production of nitric oxide, a direct sensor of inflammation and nuclear factor-kappaB activity. We conclude that low doses of GD3 could inhibit specific proinflammatory mechanisms and modulate the inflammatory environment, leading to a less reactive scene. Microglial cells are one of the main actors in the inflammatory events that follow CNS trauma or an autoimmune disease episode, modulating the internal production of cytokines, growth factors, and other homeostatic molecules that may determine the evolution and outcome of tissue damage. Proinflammatory cytokines have a relevant role in the initial events, and modulation of their activity by gangliosides could cut down their harmful effects and interfere with invasion of the CNS by peripheral immune cells. The antiinflammatory properties of GD3 could be significant in the treatment of pain subsequent to CNS damage.

  20. Nanostructured Gd3+-TiO2 surfaces for self-cleaning application

    Science.gov (United States)

    Saif, M.; El-Molla, S. A.; Aboul-Fotouh, S. M. K.; Ibrahim, M. M.; Ismail, L. F. M.; Dahn, Douglas C.

    2014-06-01

    Preparation of self-cleaning surfaces based on lanthanide modified titanium dioxide nanoparticles has rarely been reported. In the present work, gadolinium doped titanium dioxide thin films (x mol Gd3+-TiO2 where x = 0.000, 0.005, 0.008, 0.010, 0.020 and 0.030 mol) were synthesized by sol-gel method and deposited using doctor-blade method. These films were characterized by studying their structural, optical and electrical properties. Doping with gadolinium decreases the band gap energy and increase conductivity of thin films. The photo self-cleaning activity in term of quantitative determination of the active oxidative species (rad OH) produced on the thin film surfaces was evaluated using fluorescent probe method. The results show that, the highly active thin film is the 0.020 Gd3+-TiO2. The structural, morphology, optical, electrical and photoactivity properties of Gd3+-TiO2 thin films make it promising surfaces for self-cleaning application. Mineralization of commercial textile dye (Remazol Red RB-133, RR) and durability using 0.020Gd3+-TiO2 film surface was studied.

  1. Milk-derived GM3 and GD3 differentially inhibit dendritic cell maturation and effector functionalities

    DEFF Research Database (Denmark)

    Brønnum, H.; Seested, T.; Hellgren, Lars;

    2005-01-01

    value of gangliosides in breast milk has yet to be elucidated but when milk is ingested, dietary gangliosides might conceptually affect immune cells, such as dendritic cells (DCs). In this study, we address the in vitro effect of GD(3) and GM(3) on DC effector functionalities. Treatment of bone marrow...

  2. Phase formation and UV luminescence of Gd3+ doped perovskite-type YScO3

    Science.gov (United States)

    Shimizu, Yuhei; Ueda, Kazushige

    2016-10-01

    Synthesis of pure and Gd3+doped perovskite-type YScO3 was attempted by a polymerized complex (PC) method and solid state reaction (SSR) method. Crystalline phases and UV luminescence of samples were examined with varying heating temperatures. The perovskite-type single phase was not simply formed in the SSR method, as reported in some literatures, and two cubic C-type phases of starting oxide materials remained forming slightly mixed solid solutions. UV luminescence of Gd3+ doped samples increased with an increase in heating temperatures and volume of the perovskite-type phase. In contrast, a non-crystalline precursor was crystallized to a single C-type phase at 800 °C in the PC method forming a completely mixed solid solution. Then, the phase of perovskite-type YScO3 formed at 1200 °C and its single phase was obtained at 1400 °C. It was revealed that high homogeneousness of cations was essential to generate the single perovskite-phase of YScO3. Because Gd3+ ions were also dissolved into the single C-type phase in Gd3+ doped samples, intense UV luminescence was observed above 800 °C in both C-type phase and perovskite-type phase.

  3. Influence of Rare Earth (Gd3+) on Structural, Gigahertz Dielectric and Magnetic Studies of Cobalt ferrite

    Science.gov (United States)

    Pervaiz, Erum; Gul, I. H.

    2013-06-01

    A series of Gd3+ doped nanocrystalline Co-ferrites CoGdxFe2-xO4 (x = 0.0 to 0.1) has been prepared by sol-gel auto combustion technique. Structural and morphology studies were performed using X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM). Indexed XRD patterns confirm the formation of pure cubic spinel phase. Average crystallite sizes ranges from 16 nm to 25 nm ±2, were calculated from Sherrer's formula and Williamson Hall plots. Crystal strain increases with increase in doping amount of trivalent Gd ion. Lattice constant (a) and crystallite size D (311) increases with increase in Gd3+ concentration due to large ionic radii (0.94nm) of Gd3+ replacing Fe3+ (0.64nm). SEM images show the spherical morphology and uniform size distribution. Room temperature DC electrical resistivity decreases (~106) for x=0.025 then increases up to x=0.1 ~ (4.5×107). Dielectric properties have been studied using RF Impedance/material analyzer in the frequency range of 1 MHz to 1GHz. All the studied samples show a semi-conducting behavior as Permittivity and tangent loss (tanδ) decreases with the substitution of Gd3+ in parent crystal structure and have values of 4.92 and 0.016 at 1 GHz respectively. Complex impedance and Complex electric modulus plots were further studied for complete contribution of grains and grain boundary resistances to conduction and resonance frequencies respectively. Magnetic studies by Vibrating Sample Magnetometer (VSM) shows that magnetization (Ms) decreases with increase in Gd3+ concentration from 63 emu/gm to 27.26 emu/gm. Coercivity (Hc) first decreases for x=0.025, after which it increases to 2308 Oe for x=0.1.

  4. Gold nanoparticles functionalised with fast water exchanging Gd3+ chelates: linker effects on the relaxivity.

    Science.gov (United States)

    Ferreira, Miguel F; Gonçalves, Janaina; Mousavi, Bibimaryam; Prata, Maria I M; Rodrigues, Sérgio P J; Calle, Daniel; López-Larrubia, Pilar; Cerdan, Sebastian; Rodrigues, Tiago B; Ferreira, Paula M; Helm, Lothar; Martins, José A; Geraldes, Carlos F G C

    2015-03-07

    The relaxivity displayed by Gd(3+) chelates immobilized onto gold nanoparticles is the result of the complex interplay between the nanoparticle size, the water exchange rate and the chelate structure. In this work we study the effect of the length of ω-thioalkyl linkers, anchoring fast water exchanging Gd(3+) chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd(3+) chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(α-amino)propionate chelator were prepared and studied as potential CA for MRI. High relaxivities per chelate, of the order of magnitude 28-38 mM(-1) s(-1) (30 MHz, 25 °C), were attained thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. Fast local rotational motions of the immobilized chelates around connecting linkers (internal flexibility) still limit the attainable relaxivity. The degree of internal flexibility of the immobilized chelates seems not to be correlated with the length of the connecting linkers. Biodistribution and MRI studies in mice suggest that the in vivo behavior of the gold nanoparticles was determined mainly by size. Small nanoparticles (HD = 3.9 nm) undergo fast renal clearance and avoidance of the RES organs while larger nanoparticles (HD = 4.8 nm) undergo predominantly hepatobiliary excretion. High relaxivities, allied to chelate and nanoparticle stability and fast renal clearance in vivo suggest that functionalized gold nanoparticles hold great potential for further investigation as MRI contrast agents. This study contributes to a better understanding of the effect of linker length on the relaxivity of gold nanoparticles functionalized with Gd(3+) complexes. It is a relevant contribution towards "design rules" for nanostructures functionalized with Gd(3+) chelates as Contrast Agents for MRI and multimodal imaging.

  5. Characterization of the Phytochelatin Synthase from the Human Parasitic Nematode Ancylostoma ceylanicum

    Science.gov (United States)

    Rigouin, Coraline; Vermeire, Jon J.; Nylin, Elyse; Williams, David L.

    2013-01-01

    Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention. PMID:23916800

  6. Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum.

    Science.gov (United States)

    Rigouin, Coraline; Vermeire, Jon J; Nylin, Elyse; Williams, David L

    2013-09-01

    Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependent and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention.

  7. Human gene encoding prostacyclin synthase (PTGIS): Genomic organization, chromosomal localization, and promoter activity

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Chieko; Yabuki, Tomoko; Inoue, Hiroyasu [National Cardiovascular Center Research Institute, Osaka (Japan)] [and others

    1996-09-01

    The prostacyclin synthase gene isolated from human genomic libraries (PTGIS) consists of 10 exons spanning approximately 60 kb. All the splice donor and acceptor sites conform to the GT/AG rule. Genomic Southern blot and fluorescence in situ hybridization analyses revealed that the human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q13.11-q13.13. The 1.5-kb sequence of the 5{prime} of the translational initiation site contained both GC-rich and pyrimidine-rich regions and consensus sequences of the transcription factor recognition sites such as Sp1, AP-2, the interferon-{gamma} response element, GATA, NF-{kappa}B, the CACCC box, and the glucocorticoid response element. The core binding sequence (GAGACC) of the shear stress responsive element was also found in the 5{prime}-flanking region of the gene. The major product of the primer extension analysis suggested that the transcription of the gene started from the positions around 49 bp upstream of the translational initiation codon. Transient transfection experiments using human aortic and bovine arterial endothelial cells demonstrated that the GC-rich region (positions -145 to -10) possessed a significant promoter activity. The 6-kb downstream sequence of the translational termination codon contained multiple polyadenylation signals, Alu repeat sequences, and the consensus sequence of the primate-repetitive DNA element, MER1. Two sizes of the prostacyclin synthase mRNAs (approximately 6 and 3.3 kb) were detected with the human aorta and lung. RNA blot hybridization analysis using the 3{prime}-untranslated region as probe indicated that the sizes of the 3{prime}-flanking regions were different in the major 6-kb and minor 3.3-kb mRNAs. 54 refs., 7 figs.

  8. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe Brandt; Von Wettstein-Knowles, Penny

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

  9. Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA

    Institute of Scientific and Technical Information of China (English)

    Yong-Zhong Wang; You-Qing Cao; Jian-Nong Wu; Miao Chen; Xiao-Ying Cha

    2005-01-01

    AIM: To investigate the expression of NOS in gastric carcinoma, and to explore the relationship between the expression of nitric oxide synthases (NOS) and p53, PCNA,pathological features and clinical staging of gastric cancer.METHODS: The activity of NOS protein was investigated in 85 samples of human gastric carcinoma and 25 samples of normal gastric mucosal tissue by biochemical assay. We then examined the expression of NOS, p53, PCNA in 85 samples of human gastric cancer was examined by immunohistochemistry, and NOS mRNA expression in 85 gastric cancer tissue specimens by in situ hybridization.RESULTS: Biochemical assay showed that the activity of NOS was significantly higher in gastric carcinoma than in normal gastric mucosal tissues (t = 0.4161, P<0.01).Immunohistochemistry revealed that endothelial nitric oxide synthase (eNOS) expressed in all samples of normal gastric mucosa, but only 6 cases of 85 gastric cancer specimens showed weak positive immunohistochemical reactions to eNOS (20%). Inducible nitric oxide synthase (iNOS) was expressed strongly in human gastric carcinoma (81.2%). In situ hybridization analysis showed that iNOS mRNA expression was significantly stronger than eNOS mRNA expression in gastric cancer tissue (x2 = 10.23, P<0.01). The expression of iNOS in gastric cancer was associated with differentiation, clinical stages or lymph node metastases (r= 0.3426, P<0.05). However,iNOS expression did not correlate with histological classifications and morphological types. The expression of iNOS was significantly correlated with p53 or PCNA expression (r = 0.3612, P<0.05). The expression of neuronal nitric oxide synthase (nNOS) was not examined by immunohistochemistry and in situ hybridization in gastric cancer specimens and normal gastric mucosa.CONCLUSION: In human gastric cancer, there is an enhanced expression of iNOS, but not of eNOS. NOS promotes the proliferation of tumor cells and plays an important role in gastric cancer spread

  10. Gd3Al2-xSix系合金磁热效应的研究%Study on Magnetocaloric Effects of Gd3Al2-xSix Alloys

    Institute of Scientific and Technical Information of China (English)

    生利英

    2013-01-01

    采用真空高频磁悬浮炉制备出一系列Gd3Al2-xSix (x=0,0.1,0.2,0.5,0.8)合金.通过Si对Al的部分替代,研究了Si元素微量替代对Gd3Al2合金磁热效应的影响.结果表明,Gd3Al2-xSix系合金的结构与Gd3Al2相同;随着Si含量的增加,Gd3Al2-xSix系合金的居里温度和磁热效应发生了变化,这说明Si对Al的部分替代改变了GdAl2合金的磁热效应.%A series of Gd3Al2-χSiχ, (χ=0,0.1, 0.2, 0.5, 0.8) alloys were prepared in a high-frequency floating furnace. The effects of minor substitution of Al by Si on the magnetocaloric effects of Gd3Al2 alloy were investigated. The results show that the microstructure of Gd3Al2, alloy is the same with Gd2Al2 alloy. The Cure temperature and magnetocaloric effects of Gd3Al2-χSiχ alloys change with Si content increasing. The minor addition of Si can change the magnetocaloric effects of Gd3Al2-χSiχ alloy.

  11. Dermatan sulfate domains defined by the novel antibody GD3A12, in normal tissues and ovarian adenocarcinomas.

    NARCIS (Netherlands)

    Dam, G.B. ten; Yamada, S.; Kobayashi, F.; Purushothaman, A.; Westerlo, E.M.A. van de; Bulten, J.; Malmstrom, A.; Sugahara, K.; Massuger, L.F.A.G.; Kuppevelt, A.H.M.S.M. van

    2009-01-01

    Dermatan sulfate (DS) expression in normal tissue and ovarian cancer was investigated using the novel, phage display-derived antibody GD3A12 that was selected against embryonic glycosaminoglycans (GAGs). Antibody GD3A12 was especially reactive with DS rich in IdoA-GalNAc4S disaccharide units. IdoA

  12. Highly biocompatible TiO2:Gd3+ nano-contrast agent with enhanced longitudinal relaxivity for targeted cancer imaging

    Science.gov (United States)

    Chandran, Parwathy; Sasidharan, Abhilash; Ashokan, Anusha; Menon, Deepthy; Nair, Shantikumar; Koyakutty, Manzoor

    2011-10-01

    We report the development of a novel magnetic nano-contrast agent (nano-CA) based on Gd3+ doped amorphous TiO2 of size ~25 nm, exhibiting enhanced longitudinal relaxivity (r1) and magnetic resonance (MR) contrasting together with excellent biocompatibility. Quantitative T1 mapping of phantom samples using a 1.5 T clinical MR imaging system revealed that the amorphous phase of doped titania has the highest r1 relaxivity which is ~2.5 fold higher than the commercially used CA Magnevist™. The crystalline (anatase) samples formed by air annealing at 250 °C and 500 °C showed significant reduction in r1 values and MR contrast, which is attributed to the loss of proton-exchange contribution from the adsorbed water and atomic re-arrangement of Gd3+ ions in the crystalline host lattice. Nanotoxicity studies including cell viability, plasma membrane integrity, reactive oxygen stress and expression of pro-inflammatory cytokines, performed on human primary endothelial cells (HUVEC), human blood derived peripheral blood mononuclear cells (PBMC) and nasopharyngeal epidermoid carcinoma (KB) cell line showed excellent biocompatibility up to relatively higher doses of 200 μg ml-1. The potential of this nano-CA to cause hemolysis, platelet aggregation and plasma coagulation were studied using human peripheral blood samples and found no adverse effects, illustrating the possibility of the safe intravenous administration of these agents for human applications. Furthermore, the ability of these agents to specifically detect cancer cells by targeting molecular receptors on the cell membrane was demonstrated on folate receptor (FR) positive oral carcinoma (KB) cells, where the folic acid conjugated nano-CA showed receptor specific accumulation on cell membrane while leaving the normal fibroblast cells (L929) unstained. This study reveals that the Gd3+ doped amorphous TiO2 nanoparticles having enhanced magnetic resonance contrast and high biocompatibility is a promising candidate for

  13. 5-Fluorouracil incorporation into RNA and DNA in relation to thymidylate synthase inhibition of human colorectal cancers.

    NARCIS (Netherlands)

    Noordhuis, P.; Holwerda, U.; Wilt, van der C.L.; Groeningen, van C.J.; Smid, K.; Meijer, S.; Pinedo, H.M.; Peters, G.J.

    2004-01-01

    BACKGROUND: The mechanism of action of 5-fluorouracil (5-FU) has been associated with inhibition of thymidylate synthase (TS) and incorporation of 5-FU into RNA and DNA, but limited data are available in human tumor tissue for the latter. We therefore measured incorporation in human tumor biopsy spe

  14. Persistent luminescence in powdered and ceramic polycrystalline Gd3Al2Ga3O12:Ce

    Science.gov (United States)

    Dosovitskiy, G.; Fedorov, A.; Mechinsky, V.; Borisevich, A.; Dosovitskiy, A.; Tret'jak, E.; Korjik, M.

    2017-02-01

    This paper studies powders of Gd3Ga3Al2O12:Ce, a promising scintillator composition, as a possible object for express pre-characterization of scintillation kinetics and level of persistent luminescence. Garnet phase powders with uniform microstructure, consisting of 1-2 μm grains, were obtained by co-precipitation approach. It was shown, that both scintillation decay time and presence of persistent luminescence are influenced by both powder thermal treatment temperature and strong Ga deficit.

  15. Enhanced relaxivity of Gd3+-based contrast agents geometrically confined within porous nanoconstructs

    OpenAIRE

    Sethi, Richa; Ananta, Jeyarama S.; Karmonik, Christof; Zhong, Meng; Steve H. Fung; Liu, Xuewu; Li, King; Ferrari, Mauro; Wilson, Lon J.; Decuzzi, Paolo

    2012-01-01

    Gadolinium chelates, which are currently approved for clinical MRI use, provide relaxivities well below their theoretical limit, and they also lack tissue specificity. Recently, the geometrical confinement of Gd3+-based contrast agents (CAs) within porous structures has been proposed as a novel, alternative strategy to improve relaxivity without chemical modification of the CA. Here, we have characterized and optimized the performance of MRI nanoconstructs obtained by loading [Gd(DTPA)(H2O)]2...

  16. Electron paramagnetic resonance parameters and local structure for Gd3+ in KY3F10

    Indian Academy of Sciences (India)

    Shao-Yi Wu; Hua-Ming Zhang; Guang-Duo Lu; Zhi-Hong Zhang

    2007-09-01

    The electron paramagnetic resonance parameters, zero-field splittings (ZFSs) b$_{2}^{0}$, b$_{4}^{0}$, b$_{4}^{4}$, b$_{6}^{0}$, b$_{6}^{4}$ and the factors for Gd3+ on the tetragonal Y3+ site in KY3F10 are theoretically studied from the superposition model for the ZFSs and the approximation formula for the factor containing the admixture of the ground 8S7/2 and the excited 6L7/2 (L=P, D, F, G) states via the spin–orbit coupling interactions, respectively. By analysing the above ZFSs, the local structure information for the impurity Gd3+ is obtained, i.e., the impurity–ligand bonding angles related to the four-fold (C4) axis for the impurity Gd3+ center are found to be about 0.6° larger than those for the host Y3+ site in KY3F10. The calculated ZFSs based on the above angular distortion as well as the factors are in reasonable agreement with the observed values. The present studies on the ZFSs and the local structure would be helpful to understand the optical and magnetic properties of this material with Gd dopants.

  17. Inhibitor of fatty acid synthase induced apoptosis in human colonic cancer cells

    Institute of Scientific and Technical Information of China (English)

    Pei Lin Huang; Zhen Sheng Dai; Yue Lin Jin; Shi Neng Zhu; Shi Lun Lu

    2000-01-01

    @@INTRODUCTION The treatment of human epithelial malignancies is limited by drug resistance and toxic and side effects,which results in the failure in the treatment of majority of advanced cancer victims. To seek for a new, and specific antineoplastic therapy will provide hope for tumor treatment. Although disordered intermediary metabolism in cancer cells has been known for many years, much of the work focused on abnormal glucose catabolism. At the same time, little attention has been paid to fatty acid synthasis in tumor tissues, dispite of the significance of fatty acid synthase (FAS) in some clinical human ovarian[1], breast[2], colorectal[3],and prostatic cancers[4,5]. Tumor cells which express high levels of fatty acid synthesizing enzymes use endogeneously synthesized fatty acids for membrance biosynthesis and appear to export large amounts of lipid. In contrast, normal cells preferentially utilize diary lipid.

  18. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  19. The basal kinetic parameters of glycogen synthase in human myotube cultures are not affected by chronic high insulin exposure

    DEFF Research Database (Denmark)

    Gaster, M; Schrøder, H D; Handberg, A

    2001-01-01

    There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic...... parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV(0.1)) of GS, increased...

  20. Tuning the selectivity of Gd3N cluster endohedral metallofullerene reactions with Lewis acids.

    Science.gov (United States)

    Stevenson, Steven; Rottinger, Khristina A; Fahim, Muska; Field, Jessica S; Martin, Benjamin R; Arvola, Kristine D

    2014-12-15

    We demonstrate the manipulation of the Lewis acid strength to selectively fractionate different types of Gd3N metallofullerenes that are present in complex mixtures. Carbon disulfide is used for all Lewis acid studies. CaCl2 exhibits the lowest reactivity but the highest selectivity by precipitating only those gadolinium metallofullerenes with the lowest first oxidation potentials. ZnCl2 selectively complexes Gd3N@C88 during the first 4 h of reaction. Reaction with ZnCl2 for an additional 7 days permits a selective precipitation of Gd3N@C84 as the dominant endohedral isolated. A third fraction is the filtrate, which possesses Gd3N@C86 and Gd3N@C80 as the two dominant metallofullerenes. The order of increasing reactivity and decreasing selectivity (left to right) is as follows: CaCl2 Lewis acids and have the highest selectivity because of their very low precipitation onsets, which are below +0.19 V (i.e., endohedrals with first oxidation potentials below +0.19 V are precipitated). For CaCl2, the precipitation threshold is estimated at a remarkably low value of +0.06 V. Because most endohedrals possess first oxidation potentials significantly higher than +0.06 V, CaCl2 is especially useful in its ability to precipitate only a select group of gadolinium metallofullerenes. The Lewis acids of intermediate reactivity (i.e., precipitation onsets estimated between +0.19 and +0.4 V) are MgCl2, MnCl2, CuCl2, and WCl4. The strongest Lewis acids (WCl6, ZrCl4, AlCl3, and FeCl3) are the least selective and tend to precipitate the entire family of gadolinium metallofullerenes. Tuning the Lewis acid for a specific type of endohedral should be useful in a nonchromatographic purification method. The ability to control which metallofullerenes are permitted to precipitate and which endohedrals would remain in solution is a key outcome of this work.

  1. Modification of human U4 RNA requires U6 RNA and multiple pseudouridine synthases.

    Science.gov (United States)

    Zerby, D B; Patton, J R

    1997-12-01

    Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore unmodified, U4 RNA. The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, since the activities in the extracts were micrococcal nuclease (MN) sensitive. Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glycerol gradient analysis we determined that exogenously added U4 RNA that is associated with U6 RNA (sedimentation velocity 16 S) was significantly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containing (5-FU) wild-type and mutant U4 RNAs, were used to investigate formation of Psi in U4 RNA. Deletions and point mutations in these 5-FU-containing U4 RNAs affected their ability to inhibit Psi synthase in vitro. With the aid of these potent inhibitors it was determined that at least two separate activities modify the uridines at these positions.

  2. Insights into the phosphatase and the synthase activities of human bisphosphoglycerate mutase: a quantum mechanics/molecular mechanics simulation.

    Science.gov (United States)

    Chu, Wen-Ting; Zheng, Qing-Chuan; Zhang, Hong-Xing

    2014-03-07

    Bisphosphoglycerate mutase (BPGM) is a multi-activity enzyme. Its main function is to synthesize the 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. This enzyme can also catalyze the 2,3-bisphosphoglycerate to the 3-phosphoglycerate. In this study, the reaction mechanisms of both the phosphatase and the synthase activities of human bisphosphoglycerate mutase were theoretically calculated by using the quantum mechanics/molecular mechanics method based on the metadynamics and umbrella sampling simulations. The simulation results not only show the free energy curve of the phosphatase and the synthase reactions, but also reveal the important role of some residues in the active site. Additionally, the energy barriers of the two reactions indicate that the activity of the synthase in human bisphosphoglycerate mutase is much higher than that of the phosphatase. The estimated reaction barriers are consistent with the experimental data. Therefore, our work can give important information to understand the catalytic mechanism of the bisphosphoglycerate mutase family.

  3. Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes.

    Science.gov (United States)

    Johannesen, J; Pie, A; Pociot, F; Kristiansen, O P; Karlsen, A E; Nerup, J

    2001-06-01

    Exposure of human pancreatic islets to a mixture of cytokines induces expression of the inducible nitric oxide synthase (iNOS), impairs beta-cell function, and induces apoptosis. We performed a mutational scanning of all 27 exons of the human NOS2 gene and linkage transmission disequilibrium testing of identified NOS2 polymorphisms in a Danish nationwide type 1 diabetes mellitus (IDDM) family collection. Mutational screening was performed using PCR-amplified exons, followed by single stranded conformation polymorphism and verification of potential polymorphisms by sequencing. The transmission disequilibrium test was performed in an IDDM family material comprising 257 Danish families; 154 families were affected sibling pair families, and 103 families were simplex families. In total, 10 polymorphisms were identified in 8 exons, of which 4 were tested in the family material. A C/T single nucleotide polymorphism in exon 16 resulting in an amino acid substitution, Ser(608)Leu, showed linkage to IDDM in human leukocyte antigen DR3/4-positive affected offspring (P = 0.008; corrected P = 0.024). No other distorted transmission patterns were found for any other tested single nucleotide polymorphism or constructed haplotypes with the exception of those including data from exon 16. In conclusion, linkage of the human NOS2 gene to IDDM in a subset of patients supports a pathogenic role of nitric oxide in human IDDM.

  4. Inhibition of human lanosterol synthase by the constituents of Colocasia esculenta (taro).

    Science.gov (United States)

    Sakano, Yuichi; Mutsuga, Motoh; Tanaka, Rie; Suganuma, Hiroyuki; Inakuma, Takahiro; Toyoda, Masatake; Goda, Yukihiro; Shibuya, Masaaki; Ebizuka, Yutaka

    2005-02-01

    Ethanol extracts of lyophilized vegetables were tested for inhibition of human lanosterol synthase (hOSC) in order to find the compounds to suppress cholesterol biosynthesis. Of 130 samples tested, twelve samples showed significant inhibition. Among them, Colocasia esculenta (taro) showed the highest inhibition (55% inhibition at 300 microg/ml). Examination of activity variation among eight taro cultivars indicated that "Aichi-wase" and "Yatsugashira" had the most potent activity for hOSC inhibition. In order to identify the active constituent of taro, ethanol extracts of "Aichi-wase" were partitioned with hexane and aqueous methanol, and fractionated by silica gel column chromatography. Inhibitory activity was concentrated in two major active fractions. Further purification of these fractions by preparative HPLC gave three monogalactosyldiacylglycerols and five digalactosyldiacylglycerols as active compounds that showed 28 to 67% inhibitory activities at the concentration 300 microg/ml.

  5. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas

  6. Role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas.

    Directory of Open Access Journals (Sweden)

    Maurizio Sorice

    Full Text Available We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM. Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution.

  7. The Regulation of Nitric Oxide Synthase Isoform Expression in Mouse and Human Fallopian Tubes: Potential Insights for Ectopic Pregnancy

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    Junting Hu

    2014-12-01

    Full Text Available Nitric oxide (NO is highly unstable and has a half-life of seconds in buffer solutions. It is synthesized by NO-synthase (NOS, which has been found to exist in the following three isoforms: neuro nitric oxide synthase (nNOS, inducible nitric oxide synthase (iNOS, and endothelial nitric oxide synthase (eNOS. NOS activity is localized in the reproductive tracts of many species, although direct evidence for NOS isoforms in the Fallopian tubes of mice is still lacking. In the present study, we investigated the expression and regulation of NOS isoforms in the mouse and human Fallopian tubes during the estrous and menstrual cycles, respectively. We also measured isoform expression in humans with ectopic pregnancy and in mice treated with lipopolysaccharide (LPS. Our results confirmed the presence of different NOS isoforms in the mouse and human Fallopian tubes during different stages of the estrous and menstrual cycles and showed that iNOS expression increased in the Fallopian tubes of women with ectopic pregnancy and in LPS-treated mice. Elevated iNOS activity might influence ovulation, cilia beats, contractility, and embryo transportation in such a manner as to increase the risk of ectopic pregnancy. This study has provided morphological and molecular evidence that NOS isoforms are present and active in the human and mouse Fallopian tubes and suggests that iNOS might play an important role in both the reproductive cycle and infection-induced ectopic pregnancies.

  8. GD3+ cells in the adult rat optic nerve are ramified microglia rather than O-2Aadult progenitor cells.

    Science.gov (United States)

    Wolswijk, G

    1994-04-01

    The adult central nervous system (CNS) contains a population of adult oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells (O-2Aadult progenitor cells). These cells may provide a source of the new oligodendrocytes that are needed to repair demyelinated lesions. In order to examine the role of O-2Aadult progenitor cells in the regeneration of the oligodendrocyte population following demyelinating damage, it is essential to be able to identify such cells unambiguously in sections of adult CNS tissue. The present study examined whether antibodies to the ganglioside GD3 specifically label O-2Aadult progenitor cells in cultures and sections of adult optic nerve, since previous studies on the developing CNS had suggested that O-2Aperinatal progenitor cells were GD3+ in vitro and in vivo. Evidence is presented indicating that, although O-2Aadult progenitor cells in vitro were labelled with the R24 mAb (an anti-GD3 mAb), all GD3+ cells in sections of adult optic nerve bound the OX-42 mAb and the B4 isolectin derived from Griffonia Simplicifolia, and thus were not O-2Aadult progenitor cells, but ramified microglia. The data suggest that O-2Aadult progenitor cells become GD3+ when placed in culture and that ramified microglia lose GD3-expression in vitro.

  9. Nerve Growth Factor Inhibits Gd3+-sensitive Calcium Influx and Reduces Chemical Anoxic Neuronal Death

    Institute of Scientific and Technical Information of China (English)

    Hui JIANG; Shunlian TIAN; Yan ZENG; Jing SHI

    2008-01-01

    To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca2+ concentration ([Ca2+]i) were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min,[Ca2+]i was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died.When combination of 20 μmol/L MK-801 (glutamate receptor antagonist), 40 μmol/L CNQX (AMPA receptor antagonist) and 5 μmol/L nimodipine (voltage-gated calcium channel antagonist) (hereafter denoted as MCN) were administrated to hippocampal cultures, levels of [Ca2+]i and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd3+ (10μmol/L) almost completely blocked KCN-mediated [Ca2+]i elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN,inhibited KCN-induced [Ca2+]i increase by 77.4% and reduced cell death by 87.1%. Only PLC inhibitor U73122 (10 μmol/L) abolished NGF effects. It is concluded that Gd3+-sensitive calcium influx,which is NMDA (glutamate receptor) and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd3+-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.

  10. Expression of nitric oxide synthase and guanylate cyclase in the human ciliary body and trabecular meshwork

    Institute of Scientific and Technical Information of China (English)

    WU Ren-yi; MA Ning

    2012-01-01

    Background The role played by the nitric oxide (NO) signaling pathway in the aqueous humor dynamics is still unclear.This study was designed to investigate the expression and distribution of NO synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body,trabecular meshwork and the Schlemm's canal.Methods Twelve eyes after corneal transplantation were used.Expression of three NOS isoforms (i.e.neuronal NOS (nNOS),inducible NOS (iNOS) and endothelial NOS (eNOS)) and GC were assessed in 10 eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC.Ciliary bodies were dissected free and the total proteins were extracted.Western blotting was performed to confirm the protein expression of 3 NOS isoforms and GC.Results Expression of 3 NOS isoforms and GC were observed in the ciliary epithelium,ciliary muscle,trabecular meshwork and the endothelium of the Schlemm's canal.Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells.Protein expressions of 3 NOS isoforms and GC were confirmed in isolated human ciliary body by Western blotting.Conclusions The expression of NOS isoforms and GC in human ciliary body suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP,cGMP) signaling pathway in the ciliary body,and may play a role in both processes of aqueous humor formation and drainage.

  11. Potential Opponent for 18FDG: Gd3+-DTPA-DG: A New Synthetic MRI Contrast Agent

    Directory of Open Access Journals (Sweden)

    Mehdi Shafiee Ardestani

    2010-05-01

    Full Text Available Glucose mediated imaging tools have recently be-come an area of research interest in the field of nuclear medicine, especially Positron Emission Tomography (PET imaging. The difficulties in the use, preparation and cost of radioactively-labeled glycosylated compounds led to the research and development in this present study of a new gadolinium-labeled glucose that does not have a radioactive half-life or difficulties in its synthesis and utilization. Based on the structure of fluorodeoxyglucose (18FDG, a new compound consisting of a D-glucose conjugated to a well-known chelator DTPA was synthesized, labeled with Gd3+ and examined in vitro and in vivo. The results showed a good anti-cancer potency for the prepared complex. Gd3+ -DTPA-DG did not produce any significant alteration in the blood glucose level and was also phosphorylated by hexokinase enzyme. In vitro relaxation times T1 and T2 were determined and compared with a standard compound Magnevist®. Finally, imaging studies were performed in tumor-bearing mice and the tumor areas were shown successfully. This compound may have great potential in the future in radio-oncology.

  12. Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase.

    Science.gov (United States)

    Fako, Valerie E; Zhang, Jian-Ting; Liu, Jing-Yuan

    2014-10-03

    Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser(2308)) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His(2481) of TE that in turn leads to an increased hydrogen bonding between water molecules and His(2481) and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser(2308). Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance.

  13. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  14. Inducible nitric oxide synthase (NOS II) is constitutive in human neutrophils.

    Science.gov (United States)

    Cedergren, Jan; Follin, Per; Forslund, Tony; Lindmark, Maria; Sundqvist, Tommy; Skogh, Thomas

    2003-10-01

    The objective was to study the expression of inducible nitric oxide synthase (NOS II) in and NO production by human blood neutrophils and in in vivo exudated neutrophils. Cellular expression of NOS II was evaluated by flow cytometry in whole blood, in isolated blood neutrophils, and in neutrophils obtained by exudation in vivo into skin chambers. Neutrophil NOS II was also demonstrated by Western blotting. Uptake of 3H-labelled L-arginine was studied in vitro and NOS activity measured in a whole cell assay by the conversion of 3H-arginine to 3H-citrulline. In contrast to unseparated blood cells, NOS II was demonstrable both in isolated blood neutrophils and exudated cells. The failure to detect NOS II by flow cytometry in whole blood cells thus proved to be due to the quenching effect of hemoglobin. Western blotting revealed a 130 kD band corresponding to NOS II in isolated blood neutrophils, but detection was dependent on diisopropylfluorophosphate for proteinase inhibition. L-arginine was taken up by neutrophils, but enzymatic activity could not be demonstrated. We conclude that human neutrophils constitutively express NOS II, but that its demonstration by FITC-labelling is inhibited by hemoglobin-mediated quenching in whole blood samples.

  15. Bioenergetic relevance of hydrogen sulfide and the interplay between gasotransmitters at human cystathionine β-synthase.

    Science.gov (United States)

    Vicente, João B; Malagrinò, Francesca; Arese, Marzia; Forte, Elena; Sarti, Paolo; Giuffrè, Alessandro

    2016-08-01

    Merely considered as a toxic gas in the past, hydrogen sulfide (H2S) is currently viewed as the third 'gasotransmitter' in addition to nitric oxide (NO) and carbon monoxide (CO), playing a key signalling role in human (patho)physiology. H2S can either act as a substrate or, similarly to CO and NO, an inhibitor of mitochondrial respiration, in the latter case by targeting cytochrome c oxidase (CcOX). The impact of H(2)S on mitochondrial energy metabolism crucially depends on the bioavailability of this gaseous molecule and its interplay with the other two gasotransmitters. The H(2)S-producing human enzyme cystathionine β-synthase (CBS), sustaining cellular bioenergetics in colorectal cancer cells, plays a role in the interplay between gasotransmitters. The enzyme was indeed recently shown to be negatively modulated by physiological concentrations of CO and NO, particularly in the presence of its allosteric activator S-adenosyl-l-methionine (AdoMet). These newly discovered regulatory mechanisms are herein reviewed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

  16. Influence of Gd3+ concentration on luminescence properties of Eu3+ ions in sol-gel materials

    Science.gov (United States)

    Szpikowska-Sroka, Barbara; Pawlik, Natalia; Pisarski, Wojciech A.

    2016-12-01

    The sol-gel powders doubly-doped with Gd3+/Eu3+ ions with different concentration of Gd3+ have been successfully obtained. The spectroscopic characterization of prepared samples was conducted based on excitation and emission spectra as well as luminescence decay analysis. Upon direct excitation of Eu3+ active ions, the characteristic 5D0 → 7F1 (orange) and 5D0 → 7F2 (red) emission bands were observed. The energy transfer from Gd3+ to Eu3+ ions was registered upon λexc = 273 nm excitation. An efficient conversion of ultraviolet radiation (UV) into visible luminescence was successfully observed. The energy transfer process from Gd3+ to Eu3+ led to longer luminescence decay from the 5D0 state in comparison to that obtained under direct excitation of Eu3+ ions (λexc = 393 nm). Generally, obtained results clearly indicated the beneficial influence of increasing concentration of Gd3+ ions on luminescence properties of Eu3+ in studied silica sol-gel phosphors.

  17. Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families

    Energy Technology Data Exchange (ETDEWEB)

    Suchi, Mariko; Mizuno, Haruo; Tsuboi, Takashi [Nagoya City Univ. Medical School (Japan)] [and others

    1997-03-01

    Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5{prime}-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a {lambda}EMBL-3 human genomic library and report a single-copy gene spanning {approximately}15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5{prime} flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A- to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A ({nu} = .26) and 440 Gpoly ({nu} = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function. 76 refs., 5 figs., 7 tabs.

  18. Molecular docking analysis of selected Clinacanthus nutans constituents as xanthine oxidase, nitric oxide synthase, human neutrophil elastase, matrix metalloproteinase 2, matrix metalloproteinase 9 and squalene synthase inhibitors

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Narayanaswamy

    2016-01-01

    Full Text Available Background: Clinacanthus nutans (Burm. f. Lindau has gained popularity among Malaysians as a traditional plant for anti-inflammatory activity. Objective: This prompted us to carry out the present study on a selected 11 constituents of C. nutans which are clinacoside A–C, cycloclinacoside A1, shaftoside, vitexin, orientin, isovitexin, isoorientin, lupeol and β-sitosterol. Materials and Methods: Selected 11 constituents of C. nutans were evaluated on the docking behavior of xanthine oxidase (XO, nitric oxide synthase (NOS, human neutrophil elastase (HNE, matrix metalloproteinase (MMP 2 and 9, and squalene synthase (SQS using Discovery Studio Version 3.1. Also, molecular physicochemical, bioactivity, absorption, distribution, metabolism, excretion, and toxicity (ADMET, and toxicity prediction by computer assisted technology analyzes were also carried out. Results: The molecular physicochemical analysis revealed that four ligands, namely clinacoside A–C and cycloclinacoside A1 showed nil violations and complied with Lipinski's rule of five. As for the analysis of bioactivity, all the 11 selected constituents of C. nutans exhibited active score (>0 toward enzyme inhibitors descriptor. ADMET analysis showed that the ligands except orientin and isoorientin were predicted to have Cytochrome P4502D6 inhibition effect. Docking studies and binding free energy calculations revealed that clinacoside B exhibited the least binding energy for the target enzymes except for XO and SQS. Isovitexin and isoorientin showed the potentials in the docking and binding with all of the six targeted enzymes, whereas vitexin and orientin docked and bound with only NOS and HNE. Conclusion: This present study has paved a new insight in understanding these 11 C. nutans ligands as potential inhibitors against XO, NOS, HNE, MMP 2, MMP 9, and SQS.

  19. Influence of functional variant of neuronal nitric oxide synthase on impulsive behaviors in humans.

    Science.gov (United States)

    Reif, Andreas; Jacob, Christian P; Rujescu, Dan; Herterich, Sabine; Lang, Sebastian; Gutknecht, Lise; Baehne, Christina G; Strobel, Alexander; Freitag, Christine M; Giegling, Ina; Romanos, Marcel; Hartmann, Annette; Rösler, Michael; Renner, Tobias J; Fallgatter, Andreas J; Retz, Wolfgang; Ehlis, Ann-Christine; Lesch, Klaus-Peter

    2009-01-01

    Human personality is characterized by substantial heritability but few functional gene variants have been identified. Although rodent data suggest that the neuronal isoform of nitric oxide synthase (NOS-I) modifies diverse behaviors including aggression, this has not been translated to human studies. To investigate the functionality of an NOS1 promoter repeat length variation (NOS1 Ex1f variable number tandem repeat [VNTR]) and to test whether it is associated with phenotypes relevant to impulsivity. Molecular biological studies assessed the cellular consequences of NOS1 Ex1f VNTR; association studies were conducted to investigate the impact of this genetic variant on impulsivity; imaging genetics was applied to determine whether the polymorphism is functional on a neurobiological level. Three psychiatric university clinics in Germany. More than 3200 subjects were included in the association study: 1954 controls, 403 patients with personality disorder, 383 patients with adult attention-deficit/hyperactivity disorder (ADHD), 151 with familial ADHD, 189 suicide attempters, and 182 criminal offenders. For the association studies, the major outcome criteria were phenotypes relevant to impulsivity, namely, the dimensional phenotype conscientiousness and the categorical phenotypes adult ADHD, aggression, and cluster B personality disorder. A novel functional promoter polymorphism in NOS1 was associated with traits related to impulsivity, including hyperactive and aggressive behaviors. Specifically, the short repeat variant was more frequent in adult ADHD, cluster B personality disorder, and autoaggressive and heteroaggressive behavior. This short variant came along with decreased transcriptional activity of the NOS1 exon 1f promoter and alterations in the neuronal transcriptome including RGS4 and GRIN1. On a systems level, it was associated with hypoactivation of the anterior cingulate cortex, which is involved in the processing of emotion and reward in behavioral

  20. Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase.

    Science.gov (United States)

    Cheng, Tsung-Lin; Liao, Ching-Chun; Tsai, Wen-Hui; Lin, Chin-Chih; Yeh, Chin-Wei; Teng, Chiao-Fang; Chang, Wen-Tsan

    2009-08-01

    Citrate synthase (CS), the first and rate-limiting enzyme of the tricarboxylic acid (TCA) cycle, plays a decisive role in regulating energy generation of mitochondrial respiration. Most mitochondrial proteins are synthesized in the cytoplasm as preproteins with an amino (N)-terminal mitochondrial targeting sequence (MTS) that directs mitochondria-specific sorting of the preprotein. However, the MTS and targeting mechanism of the human CS protein are not fully characterized. The human CS gene is a single nuclear gene which transcribes into two mRNA variants, isoform a (CSa) and b (CSb), by alternative splicing of exon 2. CSa encodes 466 amino acids, including a putative N-terminal MTS, while CSb expresses 400 residues with a shorter N terminus, lacking the MTS. Our results indicated that CSa is localized in the mitochondria and the N-terminal 27 amino acids, including a well-conserved RXY downward arrow (S/A) motif (the RHAS sequence), can efficiently target the enhanced green fluorescent protein (EGFP) into the mitochondria. Furthermore, site-directed mutagenesis analysis of the conserved basic amino acids and serine/threonine residues revealed that the R9 residue is essential but all serine/threonine residues are dispensable in the mitochondrial targeting function. Moreover, RNA interference (RNAi)-mediated gene silencing of the preprotein import receptors, including TOM20, TOM22, and TOM70, showed that all three preprotein import receptors are required for transporting CSa into the mitochondria. In conclusion, we have experimentally identified the mitochondrial targeting sequence of human CSa and elucidated its targeting mechanism. These results provide an important basis for the study of mitochondrial dysfunction due to aberrant CSa trafficking.

  1. Expression of differential nitric oxide synthase isoforms in human gastric mucosa infected with Helicobacter pylori

    Institute of Scientific and Technical Information of China (English)

    屠振兴; 龚燕芳; 丁华; 许国铭; 李兆申; 满晓华

    2003-01-01

    Objective: To study the relationship between nitric oxide synthase (NOS) expression in human gastric mucosa and Helicobacter pylori (H.pylori) infection. Methods: Gastric mucosa samples were obtained from antrum of 33 patients received gastroendoscopy. H.pylori infection was confirmed by Giems staining and bacteria culture under microaerophilic conditions. Expression of iNOS, eNOS and nitrotyrosine were detected by immunohistochemistry. Results: (1) The positive rate of H. pylori infection was 66.7%(22/33). (2) iNOS positive staining in inflammatory cells was detected in 77.3%(17/22) of samples with H.pylori and 27.3%(3/11) without H.pylori infection (P0.05). (5) Moderate and severe infiltrations of inflammatory cells were found in 86.4%(19/22) of gastric biopsies with H. pylori and 9.1%(1/11) of samples without H. pylori infection (P<0.01). Conclusion: H.pylori infection might promote infiltration of mononuclear cells and macrophages in gastric mucosa and induce iNOS expression in these cells. The accumulated nitric oxide in local area may result in gastric mucosa damage.

  2. Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer.

    Science.gov (United States)

    Uotila, P; Valve, E; Martikainen, P; Nevalainen, M; Nurmi, M; Härkönen, P

    2001-02-01

    Cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase-2 (NOS-2) each have an important role in angiogenesis. The expression of these genes was investigated in human prostate cancer by immunohistochemistry, the expression of COX-1 and COX-2 being confirmed by mRNA analysis. Prostate cancer specimens from 12 patients were compared to control prostates from 13 patients operated on for bladder carcinoma. The intensity of COX-2 and NOS-2 immunostaining was significantly stronger in prostate cancer cells than in the non-malignant glandular epithelium of the control prostates. COX-2 and NOS-2 were clearly also expressed in the lesions of prostatic intraepithelial neoplasia (PIN) in control prostates. COX-2 was detected in the muscle fibres of the hyperplastic stroma of some control prostates. No significant difference was detected in COX-1 expression between control and cancer prostates. These results indicate that the expression of COX-2 and NOS-2 is elevated in prostatic adenocarcinoma and in PIN.

  3. Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Kentaro Wakasa

    Full Text Available Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

  4. Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation.

    Science.gov (United States)

    Sawada, N; Nagahara, N; Arisaka, F; Mitsuoka, K; Minami, M

    2011-06-01

    The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

  5. The physical properties of Gd3Ru: A real candidate for a practical cryogenic refrigerator

    Science.gov (United States)

    Monteiro, J. C. B.; dos Reis, R. D.; Gandra, F. G.

    2015-05-01

    The magnetization, the specific heat, and the magnetocaloric effect (MCE) for Gd3Ru are presented as function of temperature at different magnetic fields. The results show a maximum entropy change - Δ S = 30 J/kg K @ 5 T, which is the highest value for the R3M compounds. With a non-hysteretic transition of first order type at TC = 54 K, it presents a temperature change ΔTmax = 5.7 K around 59 K with a refrigerating cooling power of 700 J/kg and these results are comparable to values found for giant MCE materials. This compound is stable and able to operate at temperatures between 90 K and 40 K with a minimum - Δ S = 5 J/kg K. These figures were obtained by sweeping the magnetic field without using sample preparation routines. This methodology is appropriate to evaluate the MCE for the cycling process of a cryogenic magnetic refrigerator.

  6. Mapping of second-nearest-neighbor fluoride ions of orthorhombic Gd 3+-Ag + complexes in CaF 2

    Science.gov (United States)

    Nakata, R.; Den Hartog, H. W.

    The ENDOR technique is applied to determine the positions of 24 second-nearest-neighbor F - ions around an orthorhombic Gd 3+-Ag + complex in CaF 2 crystals. Experimental ENDOR data of the second-nearest-neighbor F - ions are analyzed by using the usual spin Hamiltonian and a least-squares fitting method. The best fits of the experimental results give superhyperfine (shf) constants and the F - directions ( K, L, M) with respect to the Gd 3+ ion, from which the distance between the second-nearest-neighbor F - ion and the Gd 3+ ion is determined by assuming that the hyperfine interaction is due to the classical dipole-dipole interaction. The displacements of the F - ions are estimated and compared with the theoretical values calculated by Bijvank and den Hartog on the basis of a polarizable point charge model.

  7. Prostaglandin-endoperoxide H synthase-2 expression and activity increases with term labor in human chorion

    National Research Council Canada - National Science Library

    Mijovic, J E; Zakar, T; Nairn, T K; Olson, D M

    1997-01-01

    We investigated the changes in prostaglandin-endoperoxide H synthase (PGHS) specific activity and the levels and distribution of PGHS-1 and PGHS-2 mRNA in chorion collected at term before the onset of labor (CS...

  8. Effect of Gd3+ substitution on structural, magnetic, dielectric and optical properties of nanocrystalline CoFe2O4

    Science.gov (United States)

    Joshi, Seema; Kumar, Manoj; Chhoker, Sandeep; Kumar, Arun; Singh, Mahavir

    2017-03-01

    Nanoparticles of CoGdxFe2-xO4 with x=0.0, 0.03, 0.05, 0.07, 0.10 and 0.15 were synthesized by co-precipitation method. Gd3+ substitution effect on different properties of nanocrystalline CoFe2O4 has been studied. X-ray diffraction and Raman spectroscopy confirmed the formation of single phase cubic mixed spinel structure. Cation distribution has been proposed from Rietveld refined data. Mössbauer spectra at room temperature showed two ferrimagnetic Zeeman sextets with one superparamagnetic doublet. Mössbauer parameters suggested that Gd3+ ions occupy the octahedral site in CoFe2O4. Room temperature magnetic measurements exhibited that the saturation magnetization decreased from 91 emu/gm to 54 emu/gm for x=0.0 to 0.15 samples. The coercivity decreased from 1120 Oe to 340 Oe for x=0.0 to 0.07 samples and increased from 400 Oe to 590 Oe for x=0.10 and 0.15 samples, respectively. Raman analysis showed that the degree of inversion with Gd3+ substitution supporting the variation of coercivity. Electron spin resonance spectra revealed the dominancy of superexchange interactions in these samples. Optical band gap measurement suggested that all samples are indirect band gap materials and band gap has been decreased with Gd3+ substitution. Both dielectric constant and dielectric loss is found to decrease because of the decrease in hopping rate with the Gd3+ substitution for Fe3+ at the octahedral sites. Low dielectric loss suggested the applicability of Gd3+ doped CoFe2O4 nanoparticles for high frequency microwave device applications.

  9. Synthesis and luminescence properties of nanocrystalline Gd_3Ga_5O_(12):Eu~(3+) by a homogeneous precipitation method

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    Nanocrystalline Gd3Ga5O12:Eu3+ with cubic phase was prepared by a urea homogeneous precipitation method. X-ray diffraction (XRD), field emission scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), thermo-gravimetric and differential thermal analysis (TG-DTA) and photoluminescence spectra were used to characterize the samples. The effects of the initial solution pH value and urea content on the structure of the sample were studied. The XRD results show that pure phase Gd3Ga5O...

  10. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

  11. Vascular endothelial growth factor and nitric oxide synthase expression in human tooth germ development.

    Science.gov (United States)

    Mastrangelo, F; Sberna, M T; Tettamanti, L; Cantatore, G; Tagliabue, A; Gherlone, E

    2016-01-01

    Vascular Endothelia Growth Factor (VEGF) and Nitric Oxide Synthase (NOS) expression, were evaluated in human tooth germs at two different stages of embryogenesis, to clarify the role of angiogenesis during tooth tissue differentiation and growth. Seventy-two third molar germ specimens were selected during oral surgery. Thirty-six were in the early stage and 36 in the later stage of tooth development. The samples were evaluated with Semi-quantitative Reverse Transcription-Polymerase chain Reaction analyses (RT-PcR), Western blot analysis (WB) and immunohistochemical analysis. Western blot and immunohistochemical analysis showed a VEGF and NOS 1-2-3 positive reaction in all samples analysed. VEGF high positive decrease reaction was observed in stellate reticulum cells, ameloblast and odontoblast clusters in early stage compared to later stage of tooth germ development. Comparable VEGF expression was observed in endothelial cells of early and advanced stage growth. NOS1 and NOS3 expressions showed a high increased value in stellate reticulum cells, and ameloblast and odontoblast clusters in advanced stage compared to early stage of development. The absence or only moderate positive reaction of NOS2 was detected in all the different tissues. Positive NOS2 expression showed in advanced stage of tissue development compared to early stage. The action of VEGF and NOS molecules are important mediators of angiogenesis during dental tissue development. VEGF high positive expression in stellate reticulum cells in the early stage of tooth development compared to the later stage and the other cell types, suggests a critical role of the stellate reticulum during dental embryo-morphogenesis.

  12. Significance of redox-active cysteines in human FAD synthase isoform 2.

    Science.gov (United States)

    Miccolis, Angelica; Galluccio, Michele; Nitride, Chiara; Giancaspero, Teresa Anna; Ferranti, Pasquale; Iametti, Stefania; Indiveri, Cesare; Bonomi, Francesco; Barile, Maria

    2014-12-01

    FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

  13. Luminescence and Luminescence Quenching in Gd3(Ga,Al)5O12 Scintillators Doped with Ce3+

    NARCIS (Netherlands)

    Ogiegło, J.M.; Katelnikovas, A.; Zych, A.K.|info:eu-repo/dai/nl/31407564X; Jüstel, T.; Meijerink, A.|info:eu-repo/dai/nl/075044986; Ronda, R.C.

    2013-01-01

    The optical properties of gadolinium gallium aluminum garnet, Gd3(Ga,Al)5O12, doped with Ce3+ are investigated as a function of the Ga/Al ratio, aimed at an improved understanding of the energy flow and luminescence quenching in these materials. A decrease of both the crystal field strength and band

  14. Nanometer Scale Distance Measurements for Biological Systems using Gd^3+-based Spin Probes at High Magnetic Fields

    Science.gov (United States)

    Edwards, Devin; Goldfarb, Daniella; Han, Songi; Sherwin, Mark

    2012-02-01

    Determination of nanometer-scale distances is critical for understanding structure and dynamics of proteins. Electron Paramagnetic Resonance (EPR), primarily below 1 T, is used to complement other structural techniques by quantifying sparse distances up to 8 nm in biomolecules labeled with nitroxide-based radicals. EPR becomes more powerful with increasing magnetic fields and frequencies. At 95 GHz (3.5 T), Gd^3+ ions have shown clear advantages over nitroxide probes (Potapov, JACS 2010). We show that these advantages are even more dramatic at 240 GHz (8.5 T). The width of Gd^3+'s central EPR transition narrows with increasing average distance between Gd^3+ ions out to distances as long as 5 nm. This doubles the distances accessible with nitroxides in continuous wave measurements, which can be carried out above the 200K protein-glass transition and with broad distance distributions. Temperature-dependent measurements of the phase memory times at 8.5 T and low temperatures show distance dependence out to 10 nm. Measurements of Gd^3+ labeled Proteorhodopsin confirm that phase memory times remain long enough to observe distance dependence in a spin-labeled protein. This work is supported by the National Science Foundation and the Binational Science Foundation.

  15. Luminescence and Luminescence Quenching in Gd3(Ga,Al)5O12 Scintillators Doped with Ce3+

    NARCIS (Netherlands)

    Ogiegło, J.M.; Katelnikovas, A.; Zych, A.K.; Jüstel, T.; Meijerink, A.; Ronda, R.C.

    2013-01-01

    The optical properties of gadolinium gallium aluminum garnet, Gd3(Ga,Al)5O12, doped with Ce3+ are investigated as a function of the Ga/Al ratio, aimed at an improved understanding of the energy flow and luminescence quenching in these materials. A decrease of both the crystal field strength and band

  16. Synthesis and photoluminescence study of narrow-band UVB-emitting LiSr$_4$(BO$_3$)$_3$:Gd$^{3+}$, Pr$^{3+}$ phosphor

    Indian Academy of Sciences (India)

    A O CHAUHAN; N S BAJAJ; S K OMANWAR

    2017-02-01

    A series of Pr$^{3+}$, Gd$^{3+}$ and Pr$^{3+}$–Gd$^{3+}$-doped inorganic borate phosphors LiSr$_4$(BO$_3$)$_3$ were successfully synthesized by a modified solid-state diffusion method. The crystal structures and the phase purities of samples were characterized by powder X-ray diffraction. Surface morphology of the sample was studied by scanning electronic microscopy (SEM). The optimal concentrations of dopant Gd$^{3+}$ ions in compound LiSr$_4$(BO$_3$)$_3$ were determined through the measurements of photoluminescence (PL) spectra of phosphors. Gd$^{3+}$-doped phosphorsLiSr$_4$(BO$_3$)$_3$ show strong band absorption in UV spectral region and narrow-band UVB emission under the excitation of 276 nm was only due to ${}^{6}$P$_J$ $\\to$ ${}^{8}$S$_{7/2}$ transition of Gd$^{3+}$ ions. The effect of Pr$^{3+}$ ion on excitation of LiSr$_4$(BO$_3$)$_3$:Gd$^{3+}$ was also studied. The excitation of LiSr$_4$(BO$_3$)$_3$:Gd$^{3+}$, Pr$^{3+}$ gives a broad-band spectra, which show very good overlap with the Hg 253.7 nm line. The photoluminescence spectra of LiSr$_4$(BO$_3$)$_3$ with differentdoping concentrations Pr$^{3+}$ and keeping the concentration of Gd$^{3+}$ constant at 0.03 mol have also been studied. The emission intensity of LiSr$_4$(BO$_3$)$_3$:Pr$^{3+}$–Gd$^{3+}$ phosphors increases with increasing Pr$^{3+}$ doping concentration and reaches a maximum at 0.01 mol. From the photoluminescence study of LiSr$_4$(BO$_3$)$_3$:Gd$^{3+}$, Pr$^{3+}$ we conclude that there was efficient energy transfer from Pr$^{3+} $\\to$ Gd$^{3+}$ ions in LiSr$_{4−x−y}$Pr$_x$Gd$_y$(BO$_3$)$_3$ phosphors.

  17. Green-Emitting Gd3Ga5O12: Tb3+ Nanoparticles Phosphor: Synthesis, Structure, and Luminescence

    Science.gov (United States)

    Luchechko, A.; Kostyk, L.; Varvarenko, S.; Tsvetkova, O.; Kravets, O.

    2017-04-01

    Nano- and microceramics of Gd3Ga5O12 garnet doped with 1 mol % Tb3+ ions were synthesized via co-precipitation and high-temperature solid-state reaction methods. X-ray diffraction measurements confirmed the formation of the garnet structure with Ia3d space group in all investigated samples. Atomic force microscopy surface images and grain-size distribution diagrams of Gd3Ga5O12: 1 mol % Tb3+ nanoceramics with 300 and 400 g/mol of polyethylene glycol (PEG) were obtained. The relationship between the content of polyethylene glycol and the particle size of Gd3Ga5O12: Tb3+ phosphors was revealed. An intense broad band ( λ m = 266 nm) related to spin-allowed 4 f 8-4 f 75 d 1 transitions of Tb3+ ions was found in photoluminescence excitation spectra of Gd3Ga5O12: Tb3+ nanocrystalline ceramics with PEG-300 and PEG-400 at 300 K. The broad excitation band caused by spin-forbidden ( λ m = 295 nm) 4 f-5 d transitions in Tb3+ ions was additionally observed in the photoluminescence excitation spectra of Gd3Ga5O12: Tb3+ microceramics. Emission of Tb3+ ions under X-ray and UV excitations is presented by two groups of sharp lines which correspond to 5D3 and 5D4 → 7Fj transitions of Tb3+ ions with the most intense line at 546 nm (5D4 → 7F5). It was established that the increasing of PEG content leads to the decreasing of the X-ray and photoluminescence emission intensities.

  18. Surfactant-free Gd3+-ion-containing carbon nanotube MRI contrast agents for stem cell labeling

    Science.gov (United States)

    Gizzatov, Ayrat; Hernández-Rivera, Mayra; Keshishian, Vazrik; Mackeyev, Yuri; Law, Justin J.; Guven, Adem; Sethi, Richa; Qu, Feifei; Muthupillai, Raja; Cabreira-Hansen, Maria Da Graça; Willerson, James T.; Perin, Emerson C.; Ma, Qing; Bryant, Robert G.; Wilson, Lon J.

    2015-07-01

    There is an ever increasing interest in developing new stem cell therapies. However, imaging and tracking stem cells in vivo after transplantation remains a serious challenge. In this work, we report new, functionalized and high-performance Gd3+-ion-containing ultra-short carbon nanotube (US-tube) MRI contrast agent (CA) materials which are highly-water-dispersible (ca. 35 mg ml-1) without the need of a surfactant. The new materials have extremely high T1-weighted relaxivities of 90 (mM s)-1 per Gd3+ ion at 1.5 T at room temperature and have been used to safely label porcine bone-marrow-derived mesenchymal stem cells for MR imaging. The labeled cells display excellent image contrast in phantom imaging experiments, and TEM images of the labeled cells, in general, reveal small clusters of the CA material located within the cytoplasm with 109 Gd3+ ions per cell.There is an ever increasing interest in developing new stem cell therapies. However, imaging and tracking stem cells in vivo after transplantation remains a serious challenge. In this work, we report new, functionalized and high-performance Gd3+-ion-containing ultra-short carbon nanotube (US-tube) MRI contrast agent (CA) materials which are highly-water-dispersible (ca. 35 mg ml-1) without the need of a surfactant. The new materials have extremely high T1-weighted relaxivities of 90 (mM s)-1 per Gd3+ ion at 1.5 T at room temperature and have been used to safely label porcine bone-marrow-derived mesenchymal stem cells for MR imaging. The labeled cells display excellent image contrast in phantom imaging experiments, and TEM images of the labeled cells, in general, reveal small clusters of the CA material located within the cytoplasm with 109 Gd3+ ions per cell. Electronic supplementary information (ESI) available: NMRD profiles, the Fourier transforms of the EXAFS data, EXAFS curve fitting data, cell viability data. See DOI: 10.1039/c5nr02078f

  19. Influence of Li+ charge compensator ion on the energy transfer from Pr3 + to Gd3 + ions in Ca9Mg(PO4)6F2:Gd3 +, Pr3 +, Li+ phosphor

    Science.gov (United States)

    Tamboli, Sumedha; Dhoble, S. J.

    2017-09-01

    Phototherapy is a renowned treatment for curing skin diseases since ancient times. Phototherapeutic treatment for psoriasis and many other diseases require narrow band ultra violet-B (NB-UVB) light with peak intensity at 313 nm to be exposed to the affected part of body. In this paper, we report combustion synthesis of NB-UVB - 313 nm emitting Ca9Mg(PO4)6F2 phosphors doped with Gd3 +, Pr3 + and Li+ ions. The phase formation was confirmed by obtaining X-ray diffraction (XRD) pattern and morphology was studied with the Scanning electron microscopy (SEM) images. Photoluminescence (PL) emission spectra show intense narrow band emission at 313 nm under 274 nm excitation wavelengths. Emission intensity was enhanced when Ca9Mg(PO4)6F2 compound is co-doped with Pr3 + ions. Excitation spectra of Ca9Mg(PO4)6F2:Gd3 +, Pr3 + doped samples shows broad excitation in ultra violet C (UVC) region. Diffuse reflectance spectra (DRS), obtained by UV-visible spectrophotometer, measures the absorption properties of the material. By applying Kubelka Munk function on the diffuse reflectance spectra, band gap of the material is determined. PL decay curves were examined which indicates efficient energy transfer between Pr3 + and Gd3 + ions. Charge compensation effect was also studied by co-doping Li+ ion in host. Emission intensity was found to increase with the addition of charge compensator. The prepared phosphor has potential to convert UVC light into NB-UVB. The luminescence intensity of Gd3 + shows remarkable increase when it is sensitized with Pr3 +, and an addition of charge compensator in the form of Li+, show even better results. This phosphor surely has the potential to be used as phototherapy lamp phosphor.

  20. A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site.

    Science.gov (United States)

    Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

    2014-09-01

    Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

  1. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

    Directory of Open Access Journals (Sweden)

    Yi-Hsuan Wu

    Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  2. Recent progress in advanced optical materials based on gadolinium aluminate garnet (Gd3Al5O12).

    Science.gov (United States)

    Li, Ji-Guang; Sakka, Yoshio

    2015-02-01

    This review article summarizes the recent achievements in stabilization of the metastable lattice of gadolinium aluminate garnet (Gd3Al5O12, GAG) and the related developments of advanced optical materials, including down-conversion phosphors, up-conversion phosphors, transparent ceramics, and single crystals. Whenever possible, the materials are compared with their better known YAG and LuAG counterparts to demonstrate the merits of the GAG host. It is shown that novel emission features and significantly improved luminescence can be attained for a number of phosphor systems with the more covalent GAG lattice and the efficient energy transfer from Gd(3+) to the activator. Ce(3+) doped GAG-based single crystals and transparent ceramics are also shown to simultaneously possess the advantages of high theoretical density, fast scintillation decay, and high light yields, and hold great potential as scintillators for a wide range of applications. The unresolved issues are also pointed out.

  3. Thermodynamic properties of the gadolinium gallium garnet, Gd3Ga5O12, between 0.05 and 25 K

    Science.gov (United States)

    Daudin, B.; Lagnier, R.; Salce, B.

    1982-06-01

    The specific heat, the entropy, the thermal conductivity and the diffusivity of Gd 3Ga 5O 12 have been measured in the presence of an applied magnetic field. Calculations have been performed in order to explain simultaneously all these results within the framework of a simple molecular field theory. The agreement is good above 5 K and at low temperatures the deviations may be explained by the limitations of the simple model.

  4. Decreased cyclooxygenase inhibition by aspirin in polymorphic variants of human prostaglandin H synthase-1.

    Science.gov (United States)

    Liu, Wen; Poole, Elizabeth M; Ulrich, Cornelia M; Kulmacz, Richard J

    2012-07-01

    Aspirin (ASA), a major antiplatelet and cancer-preventing drug, irreversibly blocks the cyclooxygenase (COX) activity of prostaglandin H synthase-1 (PGHS-1). Considerable differences in ASA effectiveness are observed between individuals, and some of this variability may be due to PGHS-1 protein variants. Our overall aim is to determine which, if any, of the known variants in the mature PGHS-1 protein lead to functional alterations in COX catalysis or inhibition by ASA. The present study targeted four PGHS-1 variants: R53H, R108Q, L237M, and V481I. Wild-type human PGHS-1 and the four polymorphic variants were expressed as histidine-tagged, homodimeric proteins in insect cells using baculovirus vectors, solubilized with a detergent, and purified by affinity chromatography. The purified proteins were characterized in vitro to evaluate COX and peroxidase (POX) catalytic parameters and the kinetics of COX inhibition by ASA and NS-398. Compared with the wild type, several variants showed a higher COX/POX ratio (up to 1.5-fold, for R108Q), an elevated arachidonate Km (up to 1.9-fold, for R108Q), and/or a lower ASA reactivity (up to 60% less, for R108Q). The decreased ASA reactivity in R108Q reflected both a 70% increase in the Ki for ASA and a 30% decrease in the rate constant for acetyl group transfer to the protein. Computational modeling of the brief ASA pulses experienced by PGHS-1 in circulating platelets during daily ASA dosing predicted that the 60% lower ASA reactivity in R108Q yields a 15-fold increase in surviving COX activity; smaller, approximately two-fold increases in surviving COX activity were predicted for L237M and V481I. NS-398 competitively inhibited COX catalysis of the wild type (Ki=6 µmol/l) and inhibited COX inactivation by 1.0 mmol/l ASA in both the wild type (IC50=0.8 µmol/l) and R108Q (IC50=2.1 µmol/l). Of the four PGHS-1 variants examined, R108Q exerts the largest functional effects, with evidence for impaired interactions with a COX

  5. Li3Gd3Te2O12:Eu3+- an intense red phosphor for solid state lighting applications

    Science.gov (United States)

    Tiwari, Hansnath; Asiri Naidu, S.; Varadaraju, U. V.

    2017-02-01

    Li3Gd3-3xEu3xTe2O12 (x=0.05-1.0) phases with garnet structure were synthesized by high temperature solid state reaction and the photoluminescence properties were investigated. The appearance of bands due to intra 4 f transitions of Gd3+ in the excitation spectra recorded by monitoring the 612 nm emission line of the activator indicates Gd3+→Eu3+ energy transfer in this host lattice. Under 395 nm excitation, the electric dipole transition is predominant in the emission spectrum of Eu3+ and is in agreement with the C2 point group (noncentrosymmetric) of the EuO8 polyhedron. The critical concentration of the Eu3+ activator in this series was found to be 0.6 (x=0.2) above which, concentration quenching occurs. The emission intensity of the phosphor composition, Li3Gd2.4Eu0.6Te2O12 is 4 times that of the commercial sample of Y2O3:Eu3+ phosphor.

  6. The effects of Gd3+ doping on the physical structure and photocatalytic performance of Bi2MoO6 nanoplate crystals

    Science.gov (United States)

    Yu, Changlin; Wu, Zhen; Liu, Renyue; He, Hongbo; Fan, Wenhong; Xue, Shuangshuang

    2016-06-01

    Gd3+ doped Bi2MoO6 nanoplate crystals were fabricated by solvothermal combined calcination method. The effects of Gd3+ doping with different concentrations on the texture, crystal and optical properties of Bi2MoO6 were investigated by N2 physical adsorption, X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR) and ultraviolet-visible diffuse reflection spectrum (UV-vis DRS), photoluminescence (PL) spectroscopy, and X-ray photoelectron spectroscopy (XPS). Under simulated solar light irradiation, the influences of Gd3+doping on photocatalytic activity of Bi2MoO6 were evaluated by photocatalytic degradation of Rhodamine B. The characterization results showed that with Gd3+ doping, a contraction of lattice and a decrease in crystallite size occurred. Meanwhile, an increase in surface area over Gd3+ doped Bi2MoO6 was observed. Moreover, Gd3+ doping could obviously enhance the visible light harvesting of Bi2MoO6 and promoted the separation of photogenerated electrons and holes. With optimum Gd3+(6 wt%) doping, Gd/Bi2MoO6 exhibited the best activity and stability in degradation of Rhodamine B.

  7. Human immunodeficiency virus-1 negative factor protein promotes human herpesvirus-8 viral interleukin-6-induced angiogenesis: role of glycogen synthase kinase-3β/ β-catenin signaling pathway

    Institute of Scientific and Technical Information of China (English)

    姚水洪

    2014-01-01

    Objective To explore the role of glycogen synthase kinase(GSK)-3β/β-catenin signaling pathway on human immunodeficiency virus-1(HIV-1)negative factor(Nef)protein promoting of human herpesvirus-8(HHV-8)viral interleukin-6(v IL-6)induced angiogenesis.Methods GSK-3βmutant plasmid GSK-3β-S9A,dominant-negative(DN)form GSK-3β-DN and the control vector pc DNA3.1+were transfected into endothelial cells which

  8. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  9. Lentivirus-mediated gene transfer of uroporphyrinogen III synthase fully corrects the porphyric phenotype in human cells.

    Science.gov (United States)

    Géronimi, F; Richard, E; Lamrissi-Garcia, I; Lalanne, M; Ged, C; Redonnet-Vernhet, I; Moreau-Gaudry, F; de Verneuil, H

    2003-05-01

    Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the uroporphyrinogen III synthase, the fourth enzyme of the heme biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor, with death often occurring early in adult life. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, the autografting of genetically modified primitive/stem cells may be the only alternative. In vitro gene transfer experiments have documented the feasibility of gene therapy via hematopoietic cells to treat this disease. In the present study lentiviral transduction of porphyric cell lines and primary CD34(+) cells with the therapeutic human uroporphyrinogen III synthase (UROS) cDNA resulted in both enzymatic and metabolic correction, as demonstrated by the increase in UROS activity and the suppression of porphyrin accumulation in transduced cells. Very high gene transfer efficiency (up to 90%) was achieved in both cell lines and CD34(+) cells without any selection. Expression of the transgene remained stable over long-term liquid culture. Furthermore, gene expression was maintained during in vitro erythroid differentiation of CD34(+) cells. Therefore the use of lentiviral vectors is promising for the future treatment of CEP patients by gene therapy.

  10. Trimeric form of intracellular ATP synthase subunit β of Aggregatibacter actinomycetemcomitans binds human interleukin-1β.

    Directory of Open Access Journals (Sweden)

    Annamari Paino

    Full Text Available Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.

  11. Accurate and Sensitive Detection of Plasmodium Species in Humans by Use of the Dihydrofolate Reductase-Thymidylate Synthase Linker Region▿ †

    Science.gov (United States)

    Tanomsing, Naowarat; Imwong, Mallika; Theppabutr, Sasikrit; Pukrittayakamee, Sasithon; Day, Nicholas P. J.; White, Nicholas J.; Snounou, Georges

    2010-01-01

    A nested-PCR protocol based on the linker region of the Plasmodium dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) was developed. This provides highly sensitive specific detection and identification of the five parasite species that infect humans. PMID:20702666

  12. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    Energy Technology Data Exchange (ETDEWEB)

    Manceur, Aziza P. [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Tseng, Michael [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Holowacz, Tamara [Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Witterick, Ian [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Department of Otolaryngology, Head and Neck Surgery, University of Toronto, ON (Canada); Weksberg, Rosanna [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); McCurdy, Richard D. [The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); Warsh, Jerry J. [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Audet, Julie, E-mail: julie.audet@utoronto.ca [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada)

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  13. Expression of Prostacyclin-Synthase in Human Breast Cancer: Negative Prognostic Factor and Protection against Cell Death In Vitro

    Directory of Open Access Journals (Sweden)

    Thomas Klein

    2015-01-01

    Full Text Available Endogenously formed prostacyclin (PGI2 and synthetic PGI2 analogues have recently been shown to regulate cell survival in various cell lines. To elucidate the significance of PGI2 in human breast cancer, we performed immunohistochemistry to analyze expression of prostacyclin-synthase (PGIS in 248 human breast cancer specimens obtained from surgical pathology files. We examined patients’ 10-year survival retrospectively by sending a questionnaire to their general practitioners and performed univariate analysis to determine whether PGIS expression correlated with patient survival. Lastly, the effects of PGI2 and its analogues on cell death were examined in a human breast cancer cell line (MCF-7 and a human T-cell leukemia cell line (CCRF-CEM. PGIS expression was observed in tumor cells in 48.7% of samples and was associated with a statistically significant reduction in 10-year survival (P=0.038; n=193. Transient transfection of PGIS into MCF-7 cells exposed to sulindac increased cell viability by 50% and exposure to carbaprostacyclin protected against sulindac sulfone induced apoptosis in CCRF-CEM cells. Expression of PGIS is correlated with a reduced patient survival and protects against cell death in vitro, suggesting that PGIS is a potential therapeutic target in breast cancer.

  14. Magnetic and ferroelectric characteristics of Gd$^{3+}$ and Ti$^{4+}$ co-doped BiFeO$_3$ ceramics

    Indian Academy of Sciences (India)

    SHIVANAND MADOLAPPA; A V ANUPAMA; P W JASCHIN; K B R VARMA; B SAHOO

    2016-04-01

    Polycrystalline BiFeO3 and Bi$_{0.9}$Gd$_{0.1}$Fe$_{1−x}$Ti$_x$O$_3$ ($x = 0$, 0.01, 0.05 and 0.1) samples were synthesized by solid-state reaction route. Structural, magnetic and ferroelectric properties of these samples were investigated. X-ray powder diffraction (XRD) results confirmed the presence of a significant amount of Bi$_2$Fe$_4$O$_9$ impurity phase in the undoped BiFeO$_3$ sample. Mössbauer spectroscopy studies corroborated the XRD studies to confirm the presence of impurity phase.We have observed that gadolinium (Gd$^{3+}$) and titanium (Ti$^{4+}$) doping, respectively, on Bi$^{3+}$ and Fe$^{3+}$ sites facilitated a significant reduction in the impurity phase formation in BiFeO$_3$. Interestingly, Gd$^{3+}$-doping significantly reduced the impurity phase formation as compared to the undoped BiFeO$_3$ sample. This impurity phase formation was further overcome by doping higher ($x \\ge 0.05$) amounts of Ti in BiFeO$_3$. The crystallographicsite occupancies of Gd and Ti were confirmed by Rietveld refinement of XRD data,Mössbauer spectroscopy and magnetization measurements. An enhancement in ferromagnetic properties along with moderate ferroelectricproperties have been observed after co-doping. There was an increasing trend in remnant polarization (Pr) with the increase in Ti concentration besides an improvement in the characteristic saturation magnetization. Our resultsdemonstrate that Gd$^{3+}$ and Ti$^{4+}$ doping could be used to enhance multifunctional properties of BiFeO3 ceramics to enable them as potential material for various devices.

  15. Spectroscopic investigations of Gd3Sc2Ga3O12 garnet doped with Cr3+ and Nd3+ ions

    Institute of Scientific and Technical Information of China (English)

    K.Lema(n)ski; P.J.Dere(n); A.Gagor; W.Strek

    2009-01-01

    Absorption,excitation and emission spectra as well as decay time measurements at 10,77,and 300 K were performed for Ga3Gd3Sc2O12 garnet single-crystal doped with Cr3+ and Nd3+ ions.Strong reabsorption of Cr3+ emission by Nd3+ absorption lines was observed.The assignments of f-f and d-d transitions were proposed.The broad emission band of chromium ions is an indication that weak/intermediate crystal field strength is present at the Cr3+ site.

  16. [Luminescence properties of Pr(3+) and energy transfer characteristics of Pr(3+)-->Gd(3+) in CaSiO3].

    Science.gov (United States)

    Chu, Ben-li; Liu, Xing-ren; Wang, Xiao-jun; Zhang, Jia-hua; Jiang, Xue-yin

    2002-08-01

    The excitation spectrum and the emission spectrum of Pr3+ in CaSiO3 under the room temperature were studied. The emission spectrum was constituted of three emission bands, corresponding to the emissions of the lowest 4f5d states to the 3H4, 3H6, 1G4 of the 4f2 states. The emission of the 3P0 and 1D2 were not observed. The concentration quenching of Pr3+ was due to the radiative and nonradiative energy transfer. There was energy transfer from Pr3+ to Gd3+, with the transfer rate of 10% of the Pr3+ emission rate.

  17. Influence of heat treatment process on magnetocaloric effect in Gd3Al2-xGax%热处理对Gd3Al2-xGax系合金磁热效应的影响

    Institute of Scientific and Technical Information of China (English)

    生利英

    2015-01-01

    采用真空高频磁悬浮炉制备了Gd3Al2-Gax(x=0,0.1,0.2,0.3)4种样品.把样品分2份:1份不进行热处理,1份置于真空烧结炉中进行退火处理.首先用磁热效应直接测量仪测量热处理前后样品在1.5T磁场下的绝热温变;然后利用XRD测量样品的结构;最后选取典型样品采用SEM观察其组织形貌.结果表明,热处理前后Gd3Al2-xGax合金的结构相同;最大居里温度下降2K;主相增加,杂相减少;绝热温变提高.

  18. Cystathionine β-synthase-derived hydrogen sulfide is involved in human malignant hyperthermia.

    Science.gov (United States)

    Vellecco, Valentina; Mancini, Antonio; Ianaro, Angela; Calderone, Vincenzo; Attanasio, Chiara; Cantalupo, Anna; Andria, Barbara; Savoia, Gennaro; Panza, Elisabetta; Di Martino, Antonietta; Cirino, Giuseppe; Bucci, Mariarosaria

    2016-01-01

    Hydrogen sulfide is an endogenous gasotransmitter and its mechanism of action involves activation of ATP-sensitive K(+) channels and phosphodiesterase inhibition. As both mechanisms are potentially involved in malignant hyperthermia (MH), in the present study we addressed the involvement of the L-cysteine/hydrogen sulfide pathway in MH. Skeletal muscle biopsies obtained from 25 MH-susceptible (MHS) and 56 MH-negative (MHN) individuals have been used to perform the in vitro contracture test (IVCT). Quantitative real-time PCR (qPCR) and Western blotting studies have also been performed. Hydrogen sulfide levels are measured in both tissue samples and plasma. In MHS biopsies an increase in cystathionine β-synthase (CBS) occurs, as both mRNA and protein expression compared with MHN biopsies. Hydrogen sulfide biosynthesis is increased in MHS biopsies (0.128±0.12 compared with 0.943±0.13 nmol/mg of protein per min for MHN and MHS biopsies, respectively; Phydrogen sulfide pathway in MH, giving new insight into MH molecular mechanisms. This finding has potential implications for clinical care and could help to define less invasive diagnostic procedures.

  19. Mutations and polymorphisms in the human N-acetylglutamate synthase (NAGS) gene.

    Science.gov (United States)

    Caldovic, Ljubica; Morizono, Hiroki; Tuchman, Mendel

    2007-08-01

    N-acetylglutamate synthase (NAGS) deficiency, an autosomal recessive disorder, is the last urea cycle disorder for which molecular testing became available. This is the first comprehensive report of 21 mutations that cause NAGS deficiency and of commonly found polymorphisms in the NAGS gene. Five mutations are reported here for the first time. A total of 10 disease-causing mutations are associated with acute neonatal hyperammonemia; the remaining mutations were found in patients with late onset disease. Residual enzymatic activities are included in this report and the deleterious effects of eight mutations were confirmed by expression studies. Mutations in the NAGS gene are distributed throughout its reading frame. No mutations have been found in exon 1, which encodes for the putative mitochondrial targeting signal and variable segment of NAGS. Three polymorphisms have been found. Early, accurate, and specific diagnosis of NAGS deficiency is critical since this condition can be successfully treated with N-carbamylglutamate (NCG, Carbaglu; Orphan Europe). Treatment with NCG should be initiated as soon as a patient is suspected of having NAGS deficiency. Molecular testing represents the most reliable method of diagnosis.

  20. Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate.

    Science.gov (United States)

    Yokoyama, Takeshi; Ostermann, Andreas; Mizuguchi, Mineyuki; Niimura, Nobuo; Schrader, Tobias E; Tanaka, Ichiro

    2014-04-01

    Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS-risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5 mm (∼3.5 mm(3)) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4 Å resolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported.

  1. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    Science.gov (United States)

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro.

  2. Improving the MR Imaging Sensitivity of Upconversion Nanoparticles by an Internal and External Incorporation of the Gd(3+) Strategy for in Vivo Tumor-Targeted Imaging.

    Science.gov (United States)

    Du, Hongli; Yu, Jiani; Guo, Dongcai; Yang, Weitao; Wang, Jun; Zhang, Bingbo

    2016-02-01

    Gd(3+)-ion-doped upconversion nanoparticles (UCNPs), integrating the advantages of upconversion luminescence and magnetic resonance imaging (MRI) modalities, are capturing increasing attention because they are promising to improve the accuracy of diagnosis. The embedded Gd(3+) ions in UCNPs, however, have an indistinct MRI enhancement owing to the inefficient exchange of magnetic fields with the surrounding water protons. In this study, a novel approach is developed to improve the MR imaging sensitivity of Gd(3+)-ion-doped UCNPs. Bovine serum albumin (BSA) bundled with DTPA-Gd(3+) (DTPA(Gd)) is synthesized both as the MR imaging sensitivity synergist and phase-transfer ligand for the surface engineering of UCNPs. The external Gd(3+) ion attachment strategy is found to significant improve the MR imaging sensitivity of Gd(3+)-ion-doped UCNPs. The relaxivity analysis shows that UCNPs@BSA·DTPA(Gd) exhibit higher relaxivity values than do UCNPs@BSA without DTPA(Gd) moieties. Another relaxivity study discloses a striking message that the relaxivity value does not always reflect the realistic MRI enhancement capability. The high concentration of Gd(3+)-ion-containing UCNPs with further surface-engineered BSA·DTPA(Gd) (denoted as UCNPs-H@BSA·DTPA(Gd)) exhibits a more pronounced MRI enhancement capability compared to the other two counterparts [UCNPs-N@BSA·DTPA(Gd) and UCNPs-L@BSA·DTPA(Gd) (-N and -L are denoted as zero and low concentrations of Gd(3+) ion doping, respectively)], even though it holds the lowest r1 of 1.56 s(-1) per mmol L(-1) of Gd(3+). The physicochemical properties of UCNPs are essentially maintained after BSA·DTPA(Gd) surface decoration with good colloidal stability, in addition to improving the MR imaging sensitivity. In vivo T1-weighted MRI shows potent tumor-enhanced MRI with UCNPs-H@BSA·DTPA(Gd). An in vivo biodistribution study indicates that it is gradually excreted from the body via hepatobiliary and renal processing with no obvious

  3. Synthesis and Spectral Properties of Nd3+:Gd3Ga5O12 Nanopowder for Transparent Laser Ceramics

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Nd3+:Gd3Ga5O12 (Nd:GGG) nanopowder for transparent laser ceramics was synthesized using sol-gel method. XRD, SEM, and fluorescence spectrum were used to study the properties of Nd3+:Gd3Ga5O12 nanopowder. XRD patterns of samples show that it has a cubic structure. Meanwhile, pure Nd:GGG crystals were obtained at 1000 ℃ for 12 h. SEM photographs show that dispersed, uniform, ball-like Nd:GGG nanopowder is obtained. Both XRD and SEM results show that the crystallization degree and the grain size increase with the increase in calcining temperature. Analysis of fluorescence spectrum shows that fluorescence emission occurs at 1062.7 nm, which is the result of Nd3+(4F3/2→4I11/2) transition. Homogenous Nd:GGG nanopowder with a small grain size synthesized using the sol-gel method is favorable for sintering the transparent ceramic, which proves that the nanopowder obtained is suitable as a precursor for preparing GGG transparent ceramics.

  4. Luminescence Enhanced Eu(3+)/Gd(3+) Co-Doped Hydroxyapatite Nanocrystals as Imaging Agents In Vitro and In Vivo.

    Science.gov (United States)

    Xie, Yunfei; He, Wangmei; Li, Fang; Perera, Thalagalage Shalika Harshani; Gan, Lin; Han, Yingchao; Wang, Xinyu; Li, Shipu; Dai, Honglian

    2016-04-27

    Biocompatible, biodegradable, and luminescent nano material can be used as an alternative bioimaging agent for early cancer diagnosis, which is crucial to achieve successful treatment. Hydroxyapatite (HAP) nanocyrstals have good biocompatibility and biodegradability, and can be used as an excellent host for luminescent rare earth elements. In this study, based on the energy transfer from Gd(3+) to Eu(3+), the luminescence enhanced imaging agent of Eu/Gd codoping HAP (HAP:Eu/Gd) nanocrystals are obtained via coprecipitation with plate-like shape and no change in crystal phase composition. The luminescence can be much elevated (up to about 120%) with a nonlinear increase versus Gd doping content, which is due to the energy transfer ((6)PJ of Gd(3+) → (5)HJ of Eu(3+)) under 273 nm and the possible combination effect of the cooperative upconversion and the successive energy transfer under 394 nm, respectively. Results demonstrate that the biocompatible HAP:Eu/Gd nanocrystals can successfully perform cell labeling and in vivo imaging. The intracellular HAP:Eu/Gd nanocrystals display good biodegradability with a cumulative degradation of about 65% after 72 h. This biocompatible, biodegradable, and luminescence enhanced HAP:Eu/Gd nanocrystal has the potential to act as a fluorescent imaging agent in vitro and in vivo.

  5. Czochralski growth of Gd3(Al5-xGax)O12 (GAGG) single crystals and their scintillation properties

    Science.gov (United States)

    Kurosawa, Shunsuke; Shoji, Yasuhiro; Yokota, Yuui; Kamada, Kei; Chani, Valery I.; Yoshikawa, Akira

    2014-05-01

    Ce:Gd3(AlxGa1-x)5O12 (x=2.5/5 and 3/5, Ce:GAGG-2.5 and Ce:GAGG-3) crystals were grown by the Czochralski process in order to reduce cost of the starting materials as compared with conventional Ce:Gd3Al2Ga3O12 (Ce:GAGG-2) crystal which have high light output. Although perovskite phase was detected in Ce:GAGG-3, Ce:GAGG-2.5 had single-phase garnet structure. Solidification fraction for the Ce:GAGG-2.5 growth was 0.52. Optical properties including transmittance, emission, and excitation spectra of 30 samples cut from the Ce:GAGG-2.5 bulk ingot did not depend on their original position along the growth axis. These samples had light outputs of approximately 58,000±3000 photons/MeV. However, scintillation decay times varied from 140 to 200 ns and depended on the position clearly.

  6. Luminescence and luminescence quenching in Gd3(Ga,Al)5O12 scintillators doped with Ce3+.

    Science.gov (United States)

    Ogiegło, Joanna M; Katelnikovas, Arturas; Zych, Aleksander; Jüstel, Thomas; Meijerink, Andries; Ronda, Cees R

    2013-03-28

    The optical properties of gadolinium gallium aluminum garnet, Gd3(Ga,Al)5O12, doped with Ce(3+) are investigated as a function of the Ga/Al ratio, aimed at an improved understanding of the energy flow and luminescence quenching in these materials. A decrease of both the crystal field strength and band gap with increasing content of Ga(3+) is observed and explained by the geometrical influence of Ga(3+) on the crystal field splitting of the 5d level in line with theoretical work of Muñoz-García et al. ( uñoz-García, A. B.; Seijo, L. Phys. Rev. B 2010, 82, 184118 ). Thermal quenching results in shorter decay times as well as reduced emission intensities for all samples in the temperature range from 100 to 500 K. An activation energy for emission quenching is calculated from the data. The band gap of the host is measured upon Ga substitution and the decrease in band gap is related to Ga(3+) substitution into tetrahedral sites after all octahedral sites are occupied in the garnet material. Based on the change in band gap and crystal field splitting, band diagrams can be constructed explaining the low thermal quenching temperatures in the samples with high Ga content. The highest luminescence intensity is found for Gd3(Ga,Al)5O12 with 40% of Al(3+) replaced by Ga(3+).

  7. Nitric oxide synthase and heme oxygenase expressions in human liver cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Beatrice J Goh; Bee Tee Tan; Wei Min Hon; Kang Hoe Lee; Hoon Eng Khoo

    2006-01-01

    AIM: Portal hypertension is a common complication of liver cirrhosis. Intrahepatic pressure can be elevated in several ways. Abnormal architecture affecting the vasculature, an increase in vasoconstrictors and increased circulation from the splanchnic viscera into the portal system may all contribute. It follows that endogenous vasodilators may be able to alleviate the hypertension. We therefore aimed to investigate the levels of endogenous vasodilators, nitric oxide (NO) and carbon monoxide (CO) through the expression of nitric oxide synthase (NOS) and heme oxygenase (HO).METHOD: Cirrhotic (n= 20) and non-cirrhotic (n = 20) livers were obtained from patients who had undergone surgery. The mRNA and protein expressions of the various isoforms of NOS and HO were examined using competitive PCR, Western Blot and immunohistochemistry.RESULTS: There was no significant change in either inducible NOS (iNOS) or neuronal NOS (nNOS) expressions while endothelial NOS (eNOS) was upregulated in cirrhotic livers. Concomitantly, caveolin-1, an established down-regulator of eNOS, was up-regulated.Inducible HO-1 and constitutive HO-2 were found to show increased expression in cirrhotic livers albeit in different localizations.CONCLUSION: The differences of NOS expression might be due to their differing roles in maintaining liver homeostasis and/or involvement in the pathology of cirrhosis. Sheer stress within the hypertensive liver may induce increased expression of eNOS. In turn, caveolin-1 is also increased. Whether this serves as a defense mechanism against further cirrhosis or is a consequence of cirrhosis, is yet unknown. The elevated expression of HO-1 and HO-2 suggest that CO may compensate in its role as a vasodilator albeit weakly. It is possible that CO and NO have parallel or coordinated functions within the liver and may work antagonistically in the pathophysiology of portal hypertension.

  8. Triptolide Inhibits Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression in Human Colon Cancer and Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    Xiangmin TONG; Shui ZHENG; Jie JIN; Lifen ZHU; Yinjun LOU; Hangping YAO

    2007-01-01

    Triptolide (TP), a traditional Chinese medicine, has been reported to be effective in the treatment of autoimmune diseases and exerting antineoplastic activity in several human tumor cell lines. This study investigates the antitumor effect of TP in human colon cancer cells (SW114) and myelocytic leukemia (K562), and elucidates the possible molecular mechanism involved. SW114 and K562 cells were treated with different doses of TP (0, 5, 10, 20, or 50 ng/ml). The cell viability was assessed by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results demonstrated that TP inhibited the proliferation of both tumor cell lines in a dose-dependent manner. To further investigate its mechanisms, the products prostaglandin E2 (PGE2) and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay (ELISA). Our data showed that TP strongly inhibited the production of NO and PGE2. Consistent with these results, the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was up-regulated both at the mRNA level and the protein expression level, as shown by real-time RT-PCR and Western blotting. These results indicated that the inhibition of the inflammatory factor COX-2 and iNOS activity could be involved in the antitumor mechanisms of TP.

  9. Crystal structures of human HMG-CoA synthase isoforms provide insights into inherited ketogenesis disorders and inhibitor design.

    Science.gov (United States)

    Shafqat, Naeem; Turnbull, Andrew; Zschocke, Johannes; Oppermann, Udo; Yue, Wyatt W

    2010-05-14

    3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.

  10. Structural Injury after Lithium Treatment in Human and Rat Kidney involves Glycogen Synthase Kinase-3β Positive Epithelium

    DEFF Research Database (Denmark)

    Kjærsgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    2011-01-01

    Lithium is reabsorbed by distal nephron segments in sodium depleted states. It was hypothesized that lithium causes permanent injury to the developing kidney particularly in the sodium-retaining phase around weaning through entry into epithelial cells of the distal nephron and inhibition of glyco......Lithium is reabsorbed by distal nephron segments in sodium depleted states. It was hypothesized that lithium causes permanent injury to the developing kidney particularly in the sodium-retaining phase around weaning through entry into epithelial cells of the distal nephron and inhibition...... of glycogen synthase kinase-3β (GSK-3β). GSK-3β and pGSK-3β was investigated in a developing series of rat kidney cortex and medulla. Li+ was given to female wistar rats with litters through food pellets at postnatal (P) days 7-28. In human fetal and adult kidney the expression of GSK-3β was examined and also...... a kidney from a lithium treated patient was investigated. GSK-3β was associated with connecting tubule and collecting ducts in developing and adult human and rat kidney. Renal abundance of inactive, serine9 phosphorylated GSK-3β protein decreased significantly with postnatal development. At P28, plasma Li...

  11. Active Smoking Increases Microsomal PGE2-Synthase-1/PGE-Receptor-4 Axis in Human Abdominal Aortic Aneurysms

    Directory of Open Access Journals (Sweden)

    Jaime-Félix Dilmé

    2014-01-01

    Full Text Available Background. The cyclooxygenase- (COX- 2/microsomal PGE-synthase- (mPGES- 1/PGE-receptor- (EP- 4 axis could play a key role in the physiopathology of abdominal aortic aneurysm (AAA in humans. In this study, we investigated the influence of cardiovascular risk factors on the expression of the PGE2 pathway in human AAA. Methods. Aortic (n=89 and plasma (n=79 samples from patients who underwent AAA repair were collected. Patients were grouped according to risk factors. COX-isoenzymes, mPGES-1, EPs, α-actin, and CD45 and CD68 transcripts levels were quantified by QRT-PCR and plasma PGE2 metabolites by EIA. Results. Current smoking (CS patients compared to no-CS had significantly higher local levels of mPGES-1 (P=0.009, EP-4 (P=0.007, and PGE2 metabolites plasma levels (P=0.008. In the multiple linear regression analysis, these parameters remained significantly enhanced in CS after adding confounding factors. Results from association studies with cell type markers suggested that the increased mPGES-1/EP-4 levels were mainly associated with microvascular endothelial cells. Conclusions. This study shows that elements of the PGE2 pathway, which play an important role in AAA development, are increased in CS. These results provide insight into the relevance of tobacco smoking in AAA development and reinforce the potential of mPGES-1 and EP-4 as targets for therapy in AAA patients.

  12. Active smoking increases microsomal PGE2-synthase-1/PGE-receptor-4 axis in human abdominal aortic aneurysms.

    Science.gov (United States)

    Dilmé, Jaime-Félix; Solà-Villà, David; Bellmunt, Sergi; Romero, José-María; Escudero, José-Román; Camacho, Mercedes; Vila, Luis

    2014-01-01

    The cyclooxygenase- (COX-) 2/microsomal PGE-synthase- (mPGES-) 1/PGE-receptor- (EP-) 4 axis could play a key role in the physiopathology of abdominal aortic aneurysm (AAA) in humans. In this study, we investigated the influence of cardiovascular risk factors on the expression of the PGE2 pathway in human AAA. Aortic (n = 89) and plasma (n = 79) samples from patients who underwent AAA repair were collected. Patients were grouped according to risk factors. COX-isoenzymes, mPGES-1, EPs, α-actin, and CD45 and CD68 transcripts levels were quantified by QRT-PCR and plasma PGE2 metabolites by EIA. Current smoking (CS) patients compared to no-CS had significantly higher local levels of mPGES-1 (P = 0.009), EP-4 (P = 0.007), and PGE2 metabolites plasma levels (P = 0.008). In the multiple linear regression analysis, these parameters remained significantly enhanced in CS after adding confounding factors. Results from association studies with cell type markers suggested that the increased mPGES-1/EP-4 levels were mainly associated with microvascular endothelial cells. This study shows that elements of the PGE2 pathway, which play an important role in AAA development, are increased in CS. These results provide insight into the relevance of tobacco smoking in AAA development and reinforce the potential of mPGES-1 and EP-4 as targets for therapy in AAA patients.

  13. Pseudouridine synthases.

    Science.gov (United States)

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  14. Glycogen Synthase Kinase-3β, NF-κB Signaling, and Tumorigenesis of Human Osteosarcoma

    Science.gov (United States)

    Tang, Qing-Lian; Xie, Xian-Biao; Wang, Jin; Chen, Qiong; Han, An-Jia; Zou, Chang-Ye; Yin, Jun-Qiang; Liu, Da-Wei; Liang, Yi; Zhao, Zhi-Qiang; Yong, Bi-Cheng; Zhang, Ru-Hua; Feng, Qi-Sheng; Deng, Wu-Guo; Zhu, Xiao-Feng; Zhou, Binhua P.; Zeng, Yi-Xin

    2012-01-01

    Background Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3β in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent. Methods We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3β expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5–8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3β in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3β activity with overall survival. Results Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3β formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3β had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3β resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3β resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3β inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3β, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3β and NF-κB (109.2 months). Conclusion GSK

  15. Structure of human farnesyl pyrophosphate synthase in complex with an aminopyridine bisphosphonate and two molecules of inorganic phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jaeok [McGill University, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6 (Canada); Lin, Yih-Shyan [McGill University, 801 Rue Sherbrooke Ouest, Montreal, QC H3A 0B8 (Canada); Tsantrizos, Youla S. [McGill University, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6 (Canada); McGill University, 801 Rue Sherbrooke Ouest, Montreal, QC H3A 0B8 (Canada); McGill University, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1 (Canada); Berghuis, Albert M., E-mail: albert.berghuis@mcgill.ca [McGill University, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6 (Canada); McGill University, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1 (Canada); McGill University, 3775 Rue University, Montreal, QC H3A 2B4 (Canada)

    2014-02-19

    A co-crystal structure of human farnesyl pyrophosphate synthase in complex with an aminopyridine bisphosphonate, YS0470, and two molecules of inorganic phosphate has been determined. The identity of the phosphate ligands was confirmed by anomalous diffraction data. Human farnesyl pyrophosphate synthase (hFPPS) produces farnesyl pyrophos@@phate, an isoprenoid essential for a variety of cellular processes. The enzyme has been well established as the molecular target of the nitrogen-containing bisphosphonates (N-BPs), which are best known for their antiresorptive effects in bone but are also known for their anticancer properties. Crystal structures of hFPPS in ternary complexes with a novel bisphosphonate, YS0470, and the secondary ligands inorganic phosphate (P{sub i}), inorganic pyrophosphate (PP{sub i}) and isopentenyl pyrophosphate (IPP) have recently been reported. Only the co-binding of the bisphosphonate with either PP{sub i} or IPP resulted in the full closure of the C-@@terminal tail of the enzyme, a conformational change that is required for catalysis and that is also responsible for the potent in vivo efficacy of N-BPs. In the present communication, a co-crystal structure of hFPPS in complex with YS0470 and two molecules of P{sub i} is reported. The unusually close proximity between these ligands, which was confirmed by anomalous diffraction data, suggests that they interact with one another, with their anionic charges neutralized in their bound state. The structure also showed the tail of the enzyme to be fully disordered, indicating that simultaneous binding of two P{sub i} molecules with a bisphosphonate cannot induce the tail-closing conformational change in hFPPS. Examination of homologous FPPSs suggested that this ligand-dependent tail closure is only conserved in the mammalian proteins. The prevalence of P{sub i}-bound hFPPS structures in the PDB raises a question regarding the in vivo relevance of P{sub i} binding to the function of the enzyme.

  16. Broadband Yellowish-Green Emitting Ba4Gd3Na3(PO4)6F2:Eu(2+) Phosphor: Structure Refinement, Energy Transfer, and Thermal Stability.

    Science.gov (United States)

    Fu, Xiaopeng; Lü, Wei; Jiao, Mengmeng; You, Hongpeng

    2016-06-20

    A series of Ba4Gd3Na3(PO4)6F2:Eu(2+) phosphors with a broad emitting band have been synthesized by a traditional solid state reaction. The crystal structural and photoluminescence properties of Ba4Gd3Na3(PO4)6F2:Eu(2+) are investigated. The different crystallographic sites of Eu(2+) in Ba4Gd3Na3(PO4)6F2:Eu(2+) phosphors have been verified by means of their photoluminescence (PL) properties and decay times. Energy transfer between Eu(2+) ions, analyzed by excitation, emission, and PL decay behavior, has been indicated to be a dipole-dipole mechanism. Moreover, the luminescence quantum yield as well as the thermal stability of the Ba4Gd3Na3(PO4)6F2:Eu(2+) phosphor have been investigated systematically. The as-prepared Ba4Gd3Na3(PO4)6F2:Eu(2+) phosphor can act as a promising candidate for n-UV convertible white LEDs.

  17. Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2----q26.3.

    Science.gov (United States)

    Astrin, K H; Warner, C A; Yoo, H W; Goodfellow, P J; Tsai, S F; Desnick, R J

    1991-05-01

    Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%-53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2----q26.3.

  18. Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

    Science.gov (United States)

    Cailleteau, C; Liagre, B; Battu, S; Jayat-Vignoles, C; Beneytout, J L

    2008-09-01

    Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

  19. Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins.

    Science.gov (United States)

    Torchetti, Enza M; Bonomi, Francesco; Galluccio, Michele; Gianazza, Elisabetta; Giancaspero, Teresa A; Iametti, Stefania; Indiveri, Cesare; Barile, Maria

    2011-11-01

    A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

  20. Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain.

    Science.gov (United States)

    Giancaspero, Teresa Anna; Galluccio, Michele; Miccolis, Angelica; Leone, Piero; Eberini, Ivano; Iametti, Stefania; Indiveri, Cesare; Barile, Maria

    2015-09-25

    FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.

  1. Fabrication of Ce3+ doped Gd3Ga3Al2O12 ceramics by reactive sintering method

    Science.gov (United States)

    Ye, Yong; Liu, Peng; Yan, Dongyue; Xu, Xiaodong; Zhang, Jian

    2017-09-01

    Ce3+ doped Gd3Ga3Al2O12 (Ce:GGAG) ceramics were fabricated by solid state reactive sintering method in this study. The ceramics were pre-sintered in normal muffle furnace in air at various temperature range from 1410 °C to 1550 °C for 10 h and post-treated by hot isostatic press at 1400 °C/2 h in 200 MPa Ar. The phase and microstructure evolution of Ce: GGAG samples during the densification process were investigated by X-ray diffraction and scanning electron microscope. Pure GGAG phase appeared with the temperature increased to 1200 °C. The fully dense and translucent GGAG ceramics were fabricated by pre-sintering at 1450 °C and followed by HIP treatment.

  2. The Exchange Coupling of Gd3+- and Cr3+-Ions in Paramagnetic GdCrO3 (In German)

    Science.gov (United States)

    Dräger, K.

    1986-03-01

    Polycrystalline samples of stoichiometric GdCrO3 have been investigated by ESR at 9.4 GHz. In the temperature range between 175 K and 520 K one broad absorption with a Lorentzian line shape and a g-factor of 2.024 has been observed. Attributing the absorption exclusively to the Gd -ion it can be shown that the temperature dependence of the ESR-intensity follows the predictions of a cluster-model. The energy describing the coupling of a single Gd3+-ion to the surrounding Cr3+-ions is found to be ΔE(Gd) = 170 cm-1. Taking advantage of the similarity within the orthochromites it is possible to determine at the same time the exchange - coupling of Cr3+-ions to their identical nearest neighbours as ΔE(Cr) = 293 cm-1. The relative strength of these couplings given by 0.60 is compatible with other experimental issues.

  3. Continuous-wave laser operation of diode-pumped Tm-doped Gd3Ga5O12 crystal

    Science.gov (United States)

    Wang, Yi; Lan, Jinglong; Zhou, Zhiyong; Guan, Xiaofeng; Xu, Bin; Xu, Huiying; Cai, Zhiping; Wang, Yan; Tu, Chaoyang

    2017-04-01

    We report on a diode-pumped Tm:Gd3Ga5O12 (GGG) laser at 2004 nm operated in continuous-wave mode with two-mirror linear cavity configuration. The maximum output power reaches 0.58 W with laser threshold absorbed pump power of about 0.39 W and overall slope efficiency of about 18.4%, which is believed to be the highest output power for Tm:GGG laser up to now. The Tm:GGG laser shows obvious thermally induced saturation of the output power, which indicated that power and efficiency scaling could be furtherly realized by more efficient thermal removal of the laser crystal.

  4. Gd3+ doped Mn-Zn soft ferrite nanoparticles: Superparamagnetism and its correlation with other physical properties

    Science.gov (United States)

    Thakur, Prashant; Sharma, Rohit; Sharma, Vineet; Barman, P. B.; Kumar, Manoj; Barman, Dipto; Katyal, S. C.; Sharma, Pankaj

    2017-06-01

    Superparamagnetic nanoparticles are very important in biomedicine due to their various applications like drug delivery, gene delivery in the body and also used for hyperthermia. In the present work, superparamagnetic nanoparticles of Mn0.5Zn0.5GdxFe2-xO4 (x = 0, 0.025, 0.050, 0.075, 0.1) ferrites have been prepared by co-precipitation method. Thorough characterizations (XRD, FTIR, FE-SEM, EDS, VSM and fluorescence spectroscopy) have proved the formation of cubical spinel superparamagnetic nanoparticles of soft ferrites. A cation distribution has been proposed for the determination of various important theoretical parameters for these samples. With the addition of Gd3+ nanoparticles have shown the superparamagnetism at room temperature confirmed by VSM analysis. Photoluminescence (PL) spectra shows a blue shift (for x = 0.025, 0.075) which may be due to quantum confinement.

  5. Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat, and mouse tissues

    NARCIS (Netherlands)

    Coers, W; Timens, W; Kempinga, C; Klok, PA; Moshage, H

    1998-01-01

    Ten commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several an

  6. Impact of the Xba1-polymorphism of the human muscle glycogen synthase gene on parameters of the insulin resistance syndrome in a Danish twin population

    DEFF Research Database (Denmark)

    Fenger, M; Poulsen, P; Beck-Nielsen, H;

    2000-01-01

    : The Xba1-polymorphism of the human muscle glycogen synthase gene is correlated to insulin resistance and to diastolic blood pressure. The polymorphism does not involve any known transcription factor or any structural change in GYS1, and these correlations are therefore most probably caused by linkage......AIMS: To establish the impact on the insulin resistance syndrome of the intron 14 Xba1-polymorphism in human muscle glycogen synthase (GYS1). METHODS: Parameters related to the insulin resistance syndrome were measured in 244 monozygotic twins and 322 dizygotic twins with or without impaired...... and the remainder had the genotype A1A2. No A2A2-genotypes were detected. In 11 genotypic discordant dizygotic twin pairs the insulin resistance was significantly increased in the twins carrying the A1A2 genotype regardless of sex (HOMA index 1.81 (A1A1) vs. 2.57 (A1A2), P

  7. Specific features of Eu3+ and Tb3+ magnetooptics in gadolinium-gallium garnet (Gd3Ga5O12)

    Institute of Scientific and Technical Information of China (English)

    Uygun V. Valiev; John B. Gruber; FU Dejun; Vasiliy O. Pelenovich; Gary W. Burdick; Mariya E. Malysheva

    2011-01-01

    We reported magnetooptical properties of Eu3+(4t(6)) and Tb3+(4f(8)) in single crystals of Gd3GasO12 (GGG),Y3Ga5O12 (YGG),and Eu3+(4f(6)) in Eu3Ga5O12 (EuGG) for both ions occupying sites of D2 symmetry in the garnet structure.Absorption,luminescence,and magnetic circular polarization of luminescence (MCPL) spectra of Tb3+ in GGG and YGG and absorption and magnetic circular dichroism (MCD) of Eu3+ in EuGG were studied.The data were obtained at 85 K and room temperature (RT).Magnetic susceptibility of Eu3+ in EuGG was also measured between 85 K and RT.The magnetooptical and magnetic susceptibility data were modeled using the wavefunctions of the crystal-field split energy (Stark) levels of Eu3+ and Tb3+ occupying D2 sites in the same garnets.The results reported gave a precise determination of these Stark level assignments and confirmed the symmetry labels (irreducible representations) of the closely-spaced Stark levels (quasi-doublets) found in the 5D1 (Eu3+) and 5D4 (Tb3+) multiplets.Ultraviolet (UV) excitation (<300 nm) of the 6PJ and 6IJ states of Gd3+ in the doped GGG crystals led to emission from 5D4 (Tb3+) and 5D1 and 5D0 (Eu3+) through radiationless energy transfer to the 4f(n-l)Sdband of Tb3+ and to UV quintet states of Eu3+.The temperature-dependent emission line shapes and line shifts of the magnetooptical transitions excited by UV radiation suggested a novel way to explore energy transfer mechanisms in this rare-earth doped garnet system.

  8. Yellow phosphors doping with Gd3+, Tb3+ and Lu3+ in MTiO3 (M = Mg and Sr) luminescence properties

    Indian Academy of Sciences (India)

    Esra Korkmaz; Nilgun Ozpozan Kalaycioglu; V Emir Kafadar

    2013-11-01

    This paper reports Gd3+, Tb3+ and Lu3+ doped MTiO3 (M = Mg and Sr)-based phosphors which were synthesized by the conventional solid-state reaction method, their crystal structures and luminescence properties were investigated. X-ray diffraction patterns (XRD) showed that phosphors sintered at 1000 °C for 2 h were the pure SrTiO3 and MgTiO3 phases. The optimization of reaction conditions was carried out by thermogravimetry and differential thermal analysis (DTA/TG) methods. Surface and elemental analyses were performed by using SEM instrument. The excitation and emission spectra were recorded by a photoluminescence spectrophotometer (PL). The thermoluminescence (TL) properties of MgTiO3:RE (RE = Gd3+, Tb3+, Lu3+) and SrTiO3:RE (RE = Gd3+, Tb3+, Lu3+) were investigated.

  9. Effect of doping Gd3+ on crystal structure and luminescent properties of Sr2SiO4:Eu2+ phosphor

    Institute of Scientific and Technical Information of China (English)

    赵聪; 朱达川; 高唯; 韩涛; 彭玲玲; 涂铭旌

    2015-01-01

    Sr1.995–1.5xGdxSiO4:0.005Eu2+ phosphor series withx=0–0.08 molfor near-ultraviolet white light-emitting diodes (NUV w-LEDs) were synthesized via solid-state reaction method. XRD profile pattern and refinement results demonstrated that doping Gd3+ ions resulted in the phase transformation (β-Sr2SiO4→α’-Sr2SiO4). The photoluminescence spectrum of the sample withx=0 mol dis-played two emission peaks centered at 470 and 525 nm. The two-peak spectra became one-peak spectra with the Gd3+ concentration increasing. Actually, the fitting results demonstrated that the one-peak spectra were still composed of two single emission spectra. The photoluminescence intensity was improved and the CIE chromaticity coordinates were adjusted via doping Gd3+.

  10. Role of nitric oxide and inducible nitric oxide synthase in human abdominal aortic aneurysms: a preliminary study

    Institute of Scientific and Technical Information of China (English)

    LIAO Ming-fang; LI Xiao-yan; JING Zai-ping; BAO Jun-min; ZHAO Zhi-qing; MEI Zhi-jun; LU Qing-shen; Feng Xiang; FENG Rui; ZHANG Su-zen

    2006-01-01

    Background Nitric oxide (NO) is an important mediator in the pathophysiology of many vascular diseases. However, the definite role of NO in human abdominal aortic aneurysm (AAA) formation is unclear. The aim of this study was to investigate production of NO and expression of inducible nitric oxide synthase (iNOS), and their possible role in AAA.Methods A total of 28 patients with AAA, 10 healthy controls, and 8 patients with arterial occlusive disease were enrolled into this study. Standard colorimetric assay was used to examine NO concentration in plasma from patients with AAA and normal controls, and in cultured smooth muscle cells (SMCs). Expression of iNOS in aortas and cultured SMCs were detected by immunochemistry. The correlation of iNOS expression with age of the patient, size of aneurysm, and degree of inflammation was also investigated by Cochran-Mantel-Haenszelχ2 test and Kendall' Tau correlation.Results Expression of iNOS increased significantly in the wall of aneurism in the patients with AAA compared to the healthy controls (P<0.05) and the patients with occlusive arteries (P<0.05). iNOS protein and media NOx (nitrite+nitrate) also increased in cultured SMCs from human AAA (n=4, P<0.05), while plasma NOx decreased in patients with AAA (n=25) compared to the healthy controls (n=20). There was a positive correlation between iNOS protein and degree of inflammation in aneurismal wall (Kendall coefficient=0.5032, P=0.0029)Conclusions SMCs and inflammatory cells were main cellular sources of increased iNOS in AAA, and NO may play a part in pathogenesis in AAA through inflammation.

  11. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    Science.gov (United States)

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

  12. Synthesis and luminescence properties of BaTiO3:RE (RE = Gd3+, Dy3+, Tb3+, Lu3+) phosphors

    Indian Academy of Sciences (India)

    Esra Korkmaz; Nilgun Ozpozan Kalaycioglu

    2012-11-01

    Gd3+, Dy3+, Tb3+ and Lu3+ doped BaTiO3-based phosphors were synthesized with modified solidstate technique at 1000 °C. The optimization of reaction conditions were carried out by thermogravimetry and differential thermal analysis methods (DTA/TG). The reaction products obtained in an air atmosphere were characterized by X-ray powder diffraction (XRD). Surface and elemental analyses were performed by using an SEM instrument. The excitation and emission spectra were recorded by photoluminescence spectrophotometer (PL). The thermoluminescence (TL) properties of BaTiO3 samples doped with Gd3+, Dy3+, Tb3+ and Lu3+ were investigated.

  13. Estudo espectroscópico de complexos de Eu3+, Tb3+ E Gd3+ com ligantes derivados de ácidos dicarboxílicos

    Directory of Open Access Journals (Sweden)

    Lima Patrícia P.

    2005-01-01

    Full Text Available Complexes of Eu3+, Tb3+ and Gd3+ with dipicolinic acid, chelidamic acid and chelidonic acid have been synthesized in order to study the effect of the substituent groups on the luminescence of the lanthanide complexes. The luminescence of the Eu3+ and Tb3+ complexes was quantified by quantum yield measurements. The complexes of Gd3+ have been used to determine the energies of the triplet states of the ligands. The Tb3+ complex synthesized with dipicolinic acid presented the highest quantum yield due to the energy difference between the triplet state of the dipicolinic acid and the emitting level of the Tb3+ ion.

  14. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  15. Mg-12Gd-3Y-0.5Zr镁合金的不同疲劳行为%Different Fatigue Behaviors of Mg-12Gd-3Y-0.5Zr Magnesium Alloy

    Institute of Scientific and Technical Information of China (English)

    杨晓明; 杨帆; 尹树明; 王渠东; 吴世丁; 张哲峰; 李守新

    2011-01-01

    Room temperature low cycle fatigue, very high cycle fatigue, high temperature isothermal fatigue and thermo-mechanical fatigue properties of the as-rolled Mg-12Gd-3Y-0.5Zr magnesium alloy were investigated.The fatigue failure mechanism was analyzed.The results show that for room temperature low cycle fatigue and very high cycle fatigue, the failure was caused by the fatigue cracks resulted from the inclusions or large-sized secondary phases.The cracks initiated from the surface or sub-surface for low cycle fatigue, however, for very high cycle fatigue the internal cracking was the initiation.Both high temperaturei sothermal fatigue crack and thermomechanical fatigue crack originated from the surface.The interaction of cyclic slip and oxide inclusions was the main fatigue mechanism.The alloy had excellent tensile strength and fatigue strength from room temperature to 200 ℃.The opposed phase thermo-mechanical fatigue lifetime was longer than the in-phase one thermo-mechanical fatigue lifetime.%对轧制态Mg-12Gd-3Y-0.5Zr镁合金的室温低周、超高周疲劳,高温等温疲劳以及热机械疲劳性能进行了研究,并对其疲劳失效机制进行了分析.结果表明:对室温低周疲劳、超高周疲劳来说,其失效机制主要是夹杂或大的第二相引起的疲劳开裂;对于低周疲劳,裂纹萌生于表面或亚表面,而对于超高周疲劳,裂纹起源于内部;该合金的高温等温疲劳与热机械疲劳断裂裂纹都萌生于表面,其疲劳机制为循环滑移和氧化物夹杂共同作用;该合金在室温到200℃有良好的抗拉强度与疲劳强度;反相位热机械疲劳寿命比同相位的高.

  16. Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

    Science.gov (United States)

    Foertsch, Franziska; Ilchenko, Mykhailo; Heitkamp, Thomas; Noßmann, Silke; Hoffmann, Birgit; Starke, Ilka; Mrowka, Ralf; Biskup, Christoph; Börsch, Michael

    2017-02-01

    Cytochrome C oxidase and FoF1-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent electron cryotomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.

  17. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yong-Geun [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of); Park, Chin-Ju [Gwangju Institute of Science and Technology, Division of Liberal Arts and Sciences and Department of Chemistry (Korea, Republic of); Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa, E-mail: joonhwa@gnu.ac.kr [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of)

    2015-02-15

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3{sub 10}-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

  18. Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.

    Science.gov (United States)

    Xiong, S; Chirala, S S; Wakil, S J

    2000-04-11

    To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.

  19. Analysis of polymorphisms and haplotype structure of the human thymidylate synthase genetic region: a tool for pharmacogenetic studies.

    Directory of Open Access Journals (Sweden)

    Soma Ghosh

    Full Text Available 5-Fluorouracil (5FU, a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms. Prior studies implicated a VNTR (variable numbers of tandem repeats polymorphism in the 5'-untranslated region (5'-UTR of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T repeats and novel single nucleotide polymorphisms (SNPs flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt, polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.

  20. Synthesis and luminescent properties of Gd3Ga2Al3O12 phosphors doped with Eu3+ or Ce3+

    Science.gov (United States)

    Oh, M. J.; Kim, H. J.

    2016-09-01

    Eu3+-or Ce3+-doped gadolinium gallium aluminum garnet (GGAG), Gd3Ga2Al3O12, phosphors are fabricated using solid-state reactions with Gd2O3, Ga2O3, Al2O3, CeO2 and Eu2O3 powders. The Eu3+-or Ce3+-doped Gd3Ga2Al3O12 phosphors are sintered at 1300 °C or 1600 °C for 5 hours by using an electric furnace under normal atmosphere. X-ray diffraction and field-emission scanning electron microscopy studies are carried out in order to analyze the physical properties of these materials, and their luminescence properties are also measured by using UV and X-ray sources. The Eu3+-or Ce3+-doped Gd3Ga2Al3O12 phosphors show higher light yields in comparison to commercial phosphors such as Gd2O2S:Tb (gadox). This indicates that Gd3Ga2Al3O12:Eu3+ phosphors are promising materials for use in X-ray imaging and dose monitoring at proton beamlines.

  1. Effect of Ga(3+) and Gd(3+) ions substitution on the structural and optical properties of Ce(3+) -doped yttrium aluminium garnet phosphor nanopowders.

    Science.gov (United States)

    Wako, A H; Dejene, F B; Swart, H C

    2016-11-01

    The structural and optical properties of commercially obtained Y3 Al5 O12 :Ce(3)(+) phosphor were investigated by replacing Al(3)(+) with Ga(3)(+) and Y(3)(+) with Gd(3)(+) in the Y3 Al5 O12 :Ce(3)(+) structure to form Y3 (Al,Ga)5 O12 :Ce(3)(+) and (Y,Gd)3 Al5 O12 :Ce(3)(+) . X-Ray diffraction (XRD) results showed slight 2-theta peak shifts to lower angles when Ga(3)(+) was used and to higher angles when Gd(3)(+) was used, with respect to peaks from Y3 Al5 O12 :Ce(3)(+) and JCPDS card no. 73-1370. This could be attributed to induced crystal-field effects due to the different ionic sizes of Ga(3)(+) and Gd(3)(+) compared with Al(3)(+) and Y(3)(+) . The photoluminescence (PL) spectra showed broad excitation from 350 to 550 nm with a maximum at 472 nm, and broad emission bands from 500 to 650 nm, centred at 578 nm for Y3 Al5 O12 :Ce(3)(+) arising from the 5d → 4f transition of Ce(3)(+) . PL revealed a blue shift for Ga(3)(+) substitution and a red shift for Gd(3)(+) substitution. UV-Vis showed two absorption peaks at 357 and 457 nm for Y3 Al5 O12 :Ce(3)(+) , with peaks shifting to 432 nm for Ga(3)(+) and 460 nm for Gd(3)(+) substitutions. Changes in the trap levels or in the depth and number of traps due to Ce(3)(+) were analysed using thermoluminescence (TL) spectroscopy. This revealed the existence of shallow and deep traps. It was observed that Ga(3)(+) substitution contributes to the shallowest traps at 74 °C and fewer deep traps at 163 °C, followed by Gd(3)(+) with shallow traps at 87 °C and deep traps at 146 °C. Copyright © 2016 John Wiley & Sons, Ltd.

  2. N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

    Directory of Open Access Journals (Sweden)

    Costa Nicola

    2007-12-01

    Full Text Available Abstract Background HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC. Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC, on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS in the imbalanced activity of glutamine synthase (GS, the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. Results Incubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS. This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA. In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM, dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. Conclusion In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered

  3. Structural and optical properties of Eu3+/Gd3+ ions in silica xerogels and powders obtained by sol-gel method

    Science.gov (United States)

    Pawlik, Natalia; Szpikowska-Sroka, Barbara; Swinarew, Andrzej S.; Łężniak, Marta; Pisarski, Wojciech A.

    2016-12-01

    The xerogels and sol-gel powders with different quantitative composition have been studied based on excitation and emission measurements as well as luminescence decay analysis. The structure of prepared silica sol-gel materials was examined using FT-IR spectroscopy technique. The photoluminescence spectra of Eu3+ ions were registered upon two different excitation wavelengths related to direct excitation of Eu3+ ions (λexc = 393 nm) and indirect excitation through energy transfer process from Gd3+ to Eu3+ (λexc = 273 nm). Upon direct excitation of Eu3+ (7F0 → 5L6 transition) the characteristic emission bands assigned to the 5D0 → 7FJ(0-4) electronic transitions were observed. Also, it was found that enhanced luminescence of Eu3+ is a result of change the excitation parameter through energy transfer phenomenon (8S7/2 → 6IJ transition of Gd3+) and occurred in both xerogels and powder samples. In this way, a UV photon absorbed by Gd3+ is converted into visible light emitted by Eu3+ ions. Obtained results clearly indicated that high concentration of Gd3+ ions in powders led to particularly strong enhance the emission in red spectral range and allowed for more than 12-times prolongation of luminescence lifetime for the 5D0 excited state of Eu3+ compared to silica xerogel samples. The values of energy transfer efficiency were also estimated based on luminescence decay times of the 6P7/2 state of Gd3+ ions.

  4. Photoluminescence properties of phosphors based on Lu3+-stabilized Gd3Al5O12:Tb3+/Ce3+ garnet solid solutions

    Science.gov (United States)

    Li, Jinkai; Li, Ji-Guang; Li, Xiaodong; Sun, Xudong

    2016-12-01

    The Gd3Al5O12:Tb/Ce (GdAG:Tb/Ce) garnet solutions effectively stabilized by Lu3+ have been achieved by calcining their precursor at 1300 °C. Detailed characterizations are given to the materials in terms of XRD, FE-SEM, BET, PL/PLE, and fluorescence decay analysis. The occurrence of Gd3+ and Tb3+ transitions from the photoluminescence excitation spectrum monitoring the Ce3+ yellow emission strongly confirmed the efficient Gd3+ → Ce3+ and Tb3+ → Ce3+ energy transfer. The [(Gd0.8Lu0.2)0.99-xCe0.01Tbx]AG (x = 0-0.1) phosphors with good dispersion and uniform particle size exhibit various luminescent properties under different excitation wavelength of 275, 338, and 457 nm, respectively. The photoluminescence comparison indicated that owing to the Gd3+ → Ce3+ and Tb3+ → Ce3+ energy transfer, the best luminescent phosphor [(Gd0.8Lu0.2)0.89Ce0.01 Tb0.1]AG is almost identical to the well-known YAG:Ce, higher than LuAG:Ce in emission intensity, and has a substantially red-shifted emission band that is desired for warm-white lighting. The Tb3+ → Ce3+ energy transfer was suggested to be electric multipolar interactions, and the processes of energy migration among the optically active Gd3+, Tb3+, and Ce3+ ions were discussed in detail. Fluorescence decay analysis found the lifetime for the Ce3+ emission hardly changes with the Tb3+ incorporation. The [(Gd0.8Lu0.2)0.99-xCe0.01Tbx]AG garnets developed in this work may serve as a new type of phosphor that hopefully meets the requirements of various lighting, optical display, and scintillation applications.

  5. Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C.

    Science.gov (United States)

    Kabala, Anna Magdalena; Lasserre, Jean-Paul; Ackerman, Sharon H; di Rago, Jean-Paul; Kucharczyk, Roza

    2014-05-01

    Mutations in the human mitochondrial ATP6 gene encoding ATP synthase subunit a/6 (referred to as Atp6p in yeast) are at the base of neurodegenerative disorders like Neurogenic Ataxia and Retinitis Pigmentosa (NARP), Leigh syndrome (LS), Charcot-Marie-Tooth (CMT), and ataxia telangiectasia. In previous studies, using the yeast Saccharomyces cerevisiae as a model we were able to better define how several of these mutations impact the ATP synthase. Here we report the construction of yeast models of two other ATP6 pathogenic mutations, T9185C and T9191C. The first one was reported as conferring a mild, sometimes reversible, CMT clinical phenotype; the second one has been described in a patient presenting with severe LS. We found that an equivalent of the T9185C mutation partially impaired the functioning of yeast ATP synthase, with only a 30% deficit in mitochondrial ATP production. An equivalent of the mutation T9191C had much more severe effects, with a nearly complete block in yeast Atp6p assembly and an >95% drop in the rate of ATP synthesis. These findings provide a molecular basis for the relative severities of the diseases induced by T9185C and T9191C.

  6. Synthesis of visible light active Gd3+-substituted ZnFe2O4 nanoparticles for photocatalytic and antibacterial activities

    Science.gov (United States)

    Patil, S. B.; Bhojya Naik, H. S.; Nagaraju, G.; Viswanath, R.; Rashmi, S. K.

    2017-08-01

    In the present analysis, we study the assembly of a low-cost and visible light active ZnFe2-xGdxO4 ( x = 0 , 0.3, 0.5 and 0.7) nanoparticles (NPs) photocatalyst. The synthesized samples were characterized by several physicochemical techniques, such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), UV-Visible absorption spectroscopy (UV-Vis), scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM). The phase transition from cubic to orthorhombic was confirmed by XRD patterns. The increase in the concentration of Gd3+ substitution in ZnFe2O4 NPs enhances the redshift of absorption in the visible region of UV-Vis absorption spectra and reduces the band gap. In the photo-Fenton-type reaction, the gadolinium-substituted zinc ferrite (ZGF) NPs exhibit a significant catalytic activity for the degradation of methylene blue (99% in 90 minutes) under visible light (500W xenon lamp) with respect to bare samples (95% in 240 minutes) and they also show an excellent reusability nature. These materials were also screened for antibacterial activity against Gram-negative bacteria strains ( Pseudomonas aeruginosa and Escherichia coli).

  7. Gd3+ and calcium sensitive, sodium leak currents are features of weak membrane-glass seals in patch clamp recordings.

    Science.gov (United States)

    Boone, Adrienne N; Senatore, Adriano; Chemin, Jean; Monteil, Arnaud; Spafford, J David

    2014-01-01

    The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE) and calcium (EEEE) selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM) was lowered to 0.1 mM and were inhibited (>40% to >90%) with 10 µM Gd3+, 100 µM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (Sylgard rubber.

  8. HRTEM studies of aging precipitate phases in the Mg-10Gd-3Y-0.4Zr alloy

    Institute of Scientific and Technical Information of China (English)

    王冬舒; 李德江; 谢艳才; 曾小勤

    2016-01-01

    Rare-earth (RE) element addition can remarkably improve the mechanical properties of magnesium alloys through pre-cipitation hardening. The morphology, distribution and crystal structure of precipitates are regarded as major strengthening mecha-nisms in the Mg-RE alloys. In order to understand the formation of precipitates during aging at 225 ºC in a Mg-10Gd-3Y-0.4Zr alloy (GW103K) with high strength and heat resistance, a high-resolution transmission electron microscopy (HRTEM) was employed to characterize the microstructural evolution. It was found that three types of precipitates were observed in the alloy at the early stage, named as: single layer D019 structure, one single layer D019 structure and one layer of Mg, two parallel single layers (containing RE) and Mg layer in between, which was regarded as ordered segregation of RE, precursors to formβ′ andβ′ phase, respectively. Both ofβ′ andβ′ phase were transformed from the precursors. It was also found that large size ofβ′ phase and the small size ofβ′ phase were constantly existent in the whole aging process.β′ phase played a major role in the strengthening of the GW103K alloys and the de-crease of the hardness was caused by the coarsening ofβ′ phase.

  9. Preparation and Characterization of Gd3Sc2Ga3O12 Polycrystalline Material by Co-Precipitation Method

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Gd3Sc2Ga3O12 polycrystalline material for single crystal growth was prepared with Ga, Gd2O3 and Sc2O3 as starting materials and aqueous ammonia as the precipitator by co-precipitation method. The precursors sintered at various temperatures were characterized by infrared spectra (IR), X-ray diffractometry (XRD) and transmitted electron microscopy (TEM). The results showed that pure GSGG phase could be obtained at 900 ℃. The sintered powders were well-dispersed and less-aggregated in the sintered temperature range of 900~1000 ℃. XRD and TEM show that the polycrystalline particle sizes of the polycrystalline powders were about 20~50 nm. Compared with the method that Ga2O3, Gd2O3 and Sc2O3 were mixed directly and sintered to get polycrystalline materials, the synthesized temperature was lower and sintered time was shorter. Thus co-precipitation was a good method to synthesize GSGG polycrystalline material.

  10. Temperature and pressure dependence of the optical properties of Cr3 + -doped Gd3Ga5O12 nanoparticles

    Science.gov (United States)

    Martín-Rodríguez, R.; Valiente, R.; Rodríguez, F.; Bettinelli, M.

    2011-07-01

    Since the crystal-field strength at the Cr3 + site is very close to the excited-state crossover (ESCO), this work investigates the optical properties of Cr3 + -doped Gd3Ga5O12 (GGG) nanoparticles as a function of temperature and pressure in order to establish the effect of the ESCO on the optical behaviour of nanocrystalline GGG. Luminescence, time-resolved emission and lifetime measurements have been performed on GGG:0.5% Cr3 + nanoparticles in the 25-300 K temperature range, as well as under hydrostatic pressure up to 20 GPa. We show how low temperature and high pressure progressively transforms {Cr}^{3+}~^{4} {T}_{2} \\to {}^{4} {A}_{2} broadband emission into a ruby-like ^{2} {E} \\to {}^{4} {A}_{2} luminescence. This behaviour together with the lifetime dependence on pressure and temperature are explained on the basis of the spin-orbit interaction between the 4T2 and 2E states of Cr3 + .

  11. Performance test of the Gd3Al2Ga3O12 (GAGG) scintillator for the nuclear astrophysics experiment

    Science.gov (United States)

    Koshikawa, Ami; Adachi, Satoshi; Furuno, Tatsuya; Kawabata, Takahiro; Tsumura, Miho; Kurosawa, Shunsuke

    2014-09-01

    The γ-decay widths of the excited states in 12C are very important quantities to understand the nucleosynthesis in the universe, but the γ-decay widths for the 31- and 22+ states in 12C have never been measured. To determine the γ-decay widths of the 31- state, we propose to measure the 1H(<12C, 12C*p) reaction under the inverse kinematics condition. The energies and emission angles of the scattered 12C and the recoil proton will be measured by the magnetic spectrometer Grand Raiden and the Si-CsI counter telescope, respectively. We carried out a test experiment at RCNP, Osaka and found that the energy resolution of the CsI detector is poorer than expected due to the high counting rate. To solve the pile-up problem in the recoil proton detector, we have started the performance test of the Ce-doped Gd3Al2Ga3O12 (GAGG) scintillator. The Ce-doped GAGG was recently developed and it has better light output and shorter scintillation decay time than CsI. In the present talk, the results of the performance test of the Ce-doped GAGG scintillator will be reported.

  12. Evidence for Fumonisin inhibition of ceramide synthase in humans: validation in follow-up studies in Guatemala

    Science.gov (United States)

    Fumonisins (FB) are mycotoxins found in corn. FB1 is the most common FB. It is the cause of farm animal diseases and is carcinogenic in rodents. The mode of action is the inhibition of ceramide synthase (CerS). Inhibition of CerS in mice causes a dose-dependent accumulation of sphinganine 1-phosphat...

  13. Pronounced between-subject and circadian variability in thymidylate synthase and dihydropyrimidine dehydrogenase enzyme activity in human volunteers

    NARCIS (Netherlands)

    Jacobs, Bart A W; Deenen, Maarten J; Pluim, Dick; van Hasselt, J G Coen; Krähenbühl, Martin D; van Geel, Robin M J M; de Vries, Niels; Rosing, Hilde; Meulendijks, Didier; Burylo, Artur M; Cats, Annemieke; Beijnen, Jos H; Huitema, Alwin D R; Schellens, Jan H M

    2016-01-01

    AIMS: The enzymatic activity of dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are important for the tolerability and efficacy of the fluoropyrimidine drugs. In the present study, we explored between-subject variability (BSV) and circadian rhythmicity in DPD and TS activity in h

  14. Prostacyclin Synthase: Upregulation during Renal Development and in Glomerular Disease as well as Its Constitutive Expression in Cultured Human Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Thomas Klein

    2015-01-01

    Full Text Available Prostacyclin (PGI2 plays a critical role in nephrogenesis and renal physiology. However, our understanding of how prostacyclin release in the kidney is regulated remains poorly defined. We studied expression of prostacyclin synthase (PGIS in developing and adult human kidneys, and also in selected pediatric renal diseases. We also examined PGI2 formation in human mesangial cells in vitro. We observed abundant expression of PGIS in the nephrogenic cortex in humans and in situ hybridization revealed an identical pattern in mice. In the normal adult kidney, PGIS-immunoreactive protein and mRNA appear to localize to mesangial fields and endothelial and smooth muscle cells of arteries and peritubular capillaries. In kidney biopsies taken from pediatric patients, enhanced expression of PGIS-immunoreactive protein was noted mainly in endothelial cells of patients with IgA-nephropathy. Cultured human mesangial cells produce primarily PGI2 and prostaglandin E2, followed by prostaglandin F2α Cytokine stimulation increased PGI2 formation 24-fold. Under these conditions expression of PGIS mRNA and protein remained unaltered whereas mRNA for cyclooxygenase-2 was markedly induced. In contrast to its constitutive expression in vitro, renal expression of prostacyclin-synthase appears to be regulated both during development and in glomerular disease. Further research is needed to identify the factors involved in regulation of PGIS-expression.

  15. Valproic acid-mediated transcriptional regulation of human GM3 synthase (hST3Gal V) in SK-N-BE(2)-C human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Haw-young KWON; Nam-young KANG; Hyun-mi DAE; Kyoung-sook KIM; Cheorl-ho KIM; Su-il DO; Young-choon LEE

    2008-01-01

    Aim:To investigate whether valproic acid (VPA) modulates human GM3 syn-thase (hST3Gal V) mRNA expression, as a part of ganglioside GM3 biosynthe-sis, in human neuroblastoma cells. Methods: Using RT-PCR and immunofluo-rescent confocal microscopy, we examined hST3Gal V mRNA and GM3 levels during VPA-induced differentiation of human neuroblastoma SK-N-BE(2)-C cells. We characterized the VPA-inducible promoter region within the hST3-Gal V gene using luciferase constructs carrying 5'-deletions of the hST3Gal V promoter. Results: RT-PCR indicated that VPA-mediated hST3Gal V induction is transcriptionally regulated. Functional analysis of the 5'-flanking region of the hST3Gal V gene demonstrated that the -177 to -83 region, which contains a cAMP-responsive element (CRE) at -143, functions as the VPA-inducible promoter by actively binding CRE binding protein (CREB). In addition, site-directed mutagenesis and electrophoretic mobility shift assay indicated that the CRE at -143 is crucial for the VPA-induced expression of hST3Gal V in SK-N-BE(2)-C cells. Conclusion: Our results isolated the core promoter region in the hST3Gal V promoter, a CRE at -143, and demonstrated that it is essential for transcriptional activation of hST3Gal V in VPA-induced SK-N-BE(2)-C cells. Subsequent CREB binding to this CRE mediates VPA-dependent upregulation of hST3Gal V gene expression.

  16. Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Károly Palatka; István Altorjay; Zoltán Serf(o)z(o); Zoltán Veréb; Róbert Bátori; Beáta Lontay; Zoltán Hargitay; Zoltán Nemes; Miklós Udvardy; Ferenc Erd(o)di

    2006-01-01

    AIM: To study the expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) and their role in inflammatory bowel disease (IBD).METHODS: We examined the effect of sera obtained from patients with active Crohn's disease (CD) and ulcerative colitis (UC) on the function and viability of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence,and quantified by the densitometry of Western blots.Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10-4 mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-Ⅴ-biotin method and by propidium iodide staining, respectively.RESULTS: In HUVEC immediately after exposure to UC,serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control (18% ± 16% vs 23% ± 15% P<0.01). UC or CD serum caused a significant increase in iNOS compared to control (UC: 300%±21%; CD:275%±27% vs 108%± 14%, P<0.01). Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum.CONCLUSION: IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinflammatory protection of endothelial cells.

  17. Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin.

    Science.gov (United States)

    Bruning, Rebecca S; Santhanam, Lakshmi; Stanhewicz, Anna E; Smith, Caroline J; Berkowitz, Dan E; Kenney, W Larry; Holowatz, Lacy A

    2012-06-01

    Local skin heating is used to assess microvascular function in clinical populations because NO is required for full expression of the response; however, controversy exists as to the precise NO synthase (NOS) isoform producing NO. Human aging is associated with attenuated cutaneous vasodilation but little is known about the middle aged, an age cohort used for comparison with clinical populations. We hypothesized that endothelial NOS (eNOS) is the primary isoform mediating NO production during local heating, and eNOS-dependent vasodilation would be reduced in middle-aged skin. Vasodilation was induced by local heating (42°C) and during acetylcholine dose-response (ACh-DR: 0.01, 0.1, 1.0, 5.0, 10.0, 50.0, 100.0 mmol/l) protocols. Four microdialysis fibers were placed in the skin of 24 men and women; age cohorts were 12 middle-aged (53 ± 1 yr) and 12 young (23 ± 1 yr). Sites served as control, nonselective NOS inhibited [N(G)-nitro-l-arginine methyl ester (l-NAME)], inducible NOS (iNOS) inhibited (1400W), and neuronal NOS (nNOS) inhibited (N(ω)-propyl-l-arginine). After full expression of the local heating response, l-NAME was perfused at all sites. Cutaneous vascular conductance was measured and normalized to maximum (%CVC(max): Nitropress). l-NAME reduced %CVCmax at baseline, all phases of the local heating response, and at all ACh concentrations compared with all other sites. iNOS inhibition reduced the initial peak (53 ± 2 vs. 60 ± 2%CVC(max); P vasodilation during local heating (52 ± 6 vs. 68 ± 4%CVC(max); P = 0.013) and ACh perfusion (50 mmol/l: 83 ± 3 vs. 93 ± 2%CVC(max); 100 mmol/l: 83 ± 4 vs. 92 ± 3%CVC(max); both P = 0.03) were reduced in middle-aged skin. There were no differences in NOS isoform expression obtained from skin biopsy samples between groups (all P > 0.05). These data suggest that eNOS mediates the production of NO during local heating and that cutaneous vasodilation is attenuated in middle-aged skin.

  18. Oxidized extracellular DNA suppresses nitric oxide production by endothelial NO synthase (eNOS) in human endothelial cells (HUVEC).

    Science.gov (United States)

    Kostyuk, S V; Alekseeva, A Yu; Kon'kova, M S; Glebova, K V; Smirnova, T D; Kameneva, L V; Izhevskaya, V L; Veiko, N N

    2014-06-01

    Circulating DNA from patients with cardiovascular diseases reduce the synthesis of NO in endothelial cells, which is probably related to oxidative modification of DNA. To test this hypothesis, HUVEC cells were cultured in the presence of DNA containing ~1 (nonoxidized DNA), 700, or 2100 8-oxodG/10(6) nucleosides. Nonoxidized DNA stimulated the synthesis of NO, which was associated with an increase in the expression of endothelial NO synthase. Oxidized NO decreased the amount of mRNA and protein for endothelial NO synthase, but increased the relative content of its low active form. These changes were accompanied by reduction of NO production. These findings suggest that oxidative modification of circulating extracellular DNA contributes to endothelial dysfunction manifested in suppression of NO production.

  19. All trans retinoic acid depresses the content and activity of the mitochondrial ATP synthase in human keratinocytes.

    Science.gov (United States)

    Papa, F; Lippolis, R; Sardaro, N; Gnoni, A; Scacco, S

    2017-01-08

    Proteomic analysis shows that treatment of keratinocytes cultures with all trans retinoic acid (ATRA), under condition in which it inhibits cell growth, results in marked decrease of the level of the F1-β subunit of the catalytic sector of the mitochondrial FoF1 ATP synthase complex. Enzymatic analysis shows in ATRA-treated keratinocytes a consistent depression of the ATPase activity, with decreased olygomycin sensitivity, indicating an overall alteration of the ATP synthase complex. These findings, together with the previously reported inhibition of respiratory complex I, show that depression of the activity of oxidative phosphorylation enzymes is involved in the cell growth inhibitory action of ATRA. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Magnetic behavior of Gd3Ru4Al12, a layered compound with distorted kagomé net

    Science.gov (United States)

    Chandragiri, Venkatesh; Iyer, Kartik K.; Sampathkumaran, E. V.

    2016-07-01

    The magnetic behavior of the compound, Gd3Ru4Al12, which was reported about two decades ago to crystallize in a hexagonal structure (space group P63/mmc), has not been investigated in the past literature despite interesting structural features (that is, magnetic layers and triangular as well as kagomé-lattice features favoring frustrated magnetism) characterizing this compound. We report here the results of studies of magnetization, heat capacity and magnetoresistance in the temperature range T  =  1.8-300 K. The results establish that there is a long-range magnetic order of antiferromagnetic type below (T N  =) 18.5 K, despite a much larger value (~80 K) of paramagnetic Curie temperature with a positive sign characteristic of ferromagnetic interaction. We attribute this to geometric frustration. The most interesting finding is that there is an additional magnetic anomaly below ~55 K before the onset of long-range order in the magnetic susceptibility data. Concurrent with this observation, the sign of isothermal change in entropy, ΔS  =  S(0)  -  S(H), where H is the externally applied magnetic field, remains positive above T N, with a broad peak. This observation indicates the presence of ferromagnetic clusters before the onset of long-range magnetic order. Thus, this compound may serve as an example of a situation in which magnetic frustration due to geometrical reasons faces competition from such magnetic precursor effects. There is also a reversal of the sign of  -ΔS in the curves for lower final fields (H  behavior is consistent with the above conclusions.

  1. Gd3+ and calcium sensitive, sodium leak currents are features of weak membrane-glass seals in patch clamp recordings.

    Directory of Open Access Journals (Sweden)

    Adrienne N Boone

    Full Text Available The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE and calcium (EEEE selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM was lowered to 0.1 mM and were inhibited (>40% to >90% with 10 µM Gd3+, 100 µM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (<30% to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 µM mibefradil or 100 µM nifedipine. We hypothesize that the rapid changes in leak current size in response to changing external cations or drugs relates to their influences on the membrane seal adherence and the electro-osmotic flow of mobile cations channeling in crevices of a particular pore size in the interface between the negatively charged patch electrode and the lipid membrane. Observed sodium leak conductance currents in weak patch seals are reproducible between the electrode glass interface with cell membranes, artificial lipid or Sylgard rubber.

  2. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat...

  3. Spectral investigation of, Ce:YAG (:Pr3+, Eu3+, Gd3+) single crystals and their applications in white LEDs

    Institute of Scientific and Technical Information of China (English)

    杜勇; 邵冲云; 董永军; 杨秋红; 华伟

    2015-01-01

    Eu3+, Pr3+, or Gd3+ codoped Ce:YAG single crystals were grown by using the Czochralski method. The pho-toluminescence (PL) emission and excitation spectra and transmittance were measured and investigated. The additional red-emitting bands were observed in the PL emission spectra of Eu,Ce:YAG and Pr,Ce:YAG single crystals and the forma-tion of noticeable peaks was studied with reference to the schematic energy level diagrams. A red-shifted phenomenon was observed in the PL emission spectrum of Gd,Ce:YAG. With codoped Eu3+, Pr3+, or Gd3+ ions, warmer white light was achieved for the white light emitting diodes and the color rendering index became higher.

  4. Optical properties of silica sol-gel materials singly-and doubly-doped with Eu3+and Gd3+ions

    Institute of Scientific and Technical Information of China (English)

    Natalia Pawlik; Barbara Szpikowska-Sroka; Marta Sołtys; Wojciech A.Pisarski

    2016-01-01

    In present work, the optical and structural properties of silica sol-gel glasses and glass-ceramic materials singly-and dou-bly-doped with Eu3+and Gd3+ions were investigated. The optical properties of studied systems were determined based on absorption, excitation and emission spectra as well as luminescence decay analysis. Conducted studies clearly indicated a significant enhance-ment of visible emission originated from Eu3+ions as a result of changing the excitation mechanism, via Gd3+→Eu3+energy transfer. The luminescence intensity R-ratio was analyzed before and after heat-treatment process upon excitation atλex=393 nm andλex=273 nm. Moreover, the influence of excitation wavelength on luminescence decay time of the 5D0 excited state was also analyzed. The Gd3+→Eu3+energy transfer efficiencies for precursor and annealed samples were calculated based on luminescence lifetime of the 6P7/2 level of Gd3+ions. The X-ray diffraction measurements were conducted to verify the nature of obtained sol-gel materials. In re-sult, the formation of orthorhombic GdF3 nanocrystal phase dispersed in amorphous silica glass host was identified after annealing. Obtained results clearly indicated an incorporation of Eu3+ activators into formed GdF3 nanocrystals. Thus, conducted heat-treatment process led to considerable changes in surrounding environment around Eu3+ions. Actually, it was found that en-ergy transfer phenomenon and heat-treatment process were responsible for significant improvement of Eu3+luminescence in stud-ied sol-gel samples.

  5. Growth and scintillation properties of 3 in. diameter Ce doped Gd3Ga3Al2O12 scintillation single crystal

    Science.gov (United States)

    Kamada, Kei; Shoji, Yasuhiro; Kochurikhin, Vladimir V.; Okumura, Satoshi; Yamamoto, Seiichi; Nagura, Aya; Yeom, Jung Yeol; Kurosawa, Shunsuke; Yokota, Yuui; Ohashi, Yuji; Nikl, Martin; Yoshikawa, Akira

    2016-10-01

    The 3 in. size Ce1%:Gd3Al2Ga3O12 single crystals were prepared by the Czochralski (Cz) method. Optical constants were measured. Chemical composition analysis and uniformity of scintillation decay and light yield along growth direction were evaluated. The timing resolution measurement for a pair of 3 mm×3 mm×3 mm size Ce:GAGG scintillator crystals was performed using Si-PMs.

  6. Rare-Earth Ion-Host Lattice Interactions: 15. Analysis of the Spectra of Nd3+ in Gd3Sc2Ga3O12.

    Science.gov (United States)

    1984-05-01

    Kh. S. Bagdasarov, G. A. Bogomolova, M. M. Gritsenko , A. M. Kevorkov, and S. E. Sarkisov, Luminescence and Stimulated Emission of Nd3+ Ions in Gd3 Sc2...Eyring, Jr., eds., North Holland, New York (1982), p 632. 3A. A. Kaminskii, Kh. S. Bagdasarov, G. A. Bogomolova, M. M. Gritsenko , A. M. Kevorkov, and...Eyring, Jr., eds., North Holland, New York (1982), p 632. (3) A. A. Kaminskii, Kh. S. Bagdasarov, G. A. Bogomolova, M. M. Gritsenko , A. M. Kevorkov

  7. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  8. The Gadolinium (Gd3+) and Tin (Sn4+) Co-doped BiFeO3 Nanoparticles as New Solar Light Active Photocatalyst

    Science.gov (United States)

    Irfan, Syed; Rizwan, Syed; Shen, Yang; Li, Liangliang; Asfandiyar; Butt, Sajid; Nan, Ce-Wen

    2017-02-01

    The process of photocatalysis is appealing to huge interest motivated by the great promise of addressing current energy and environmental issues through converting solar light directly into chemical energy. However, an efficient solar energy harvesting for photocatalysis remains a critical challenge. Here, we reported a new full solar spectrum driven photocatalyst by co-doping of Gd3+ and Sn4+ into A and B-sites of BiFeO3 simultaneously. The co-doping of Gd3+ and Sn4+ played a key role in hampering the recombination of electron-hole pairs and shifted the band-gap of BiFeO3 from 2.10 eV to 2.03 eV. The Brunauer-Emmett-Teller (BET) measurement confirmed that the co-doping of Gd3+ and Sn4+ into BiFeO3 increased the surface area and porosity, and thus the photocatalytic activity of the Bi0.90Gd0.10Fe0.95Sn0.05O3 system was significantly improved. Our work proposed a new photocatalyst that could degrade various organic dyes like Congo red, Methylene blue, and Methyl violet under irradiation with different light wavelengths and gave guidance for designing more efficient photocatalysts.

  9. Facile preparation of Gd3+ doped carbon quantum dots: Photoluminescence materials with magnetic resonance response as magnetic resonance/fluorescence bimodal probes

    Science.gov (United States)

    Ren, X. Y.; Yuan, X. X.; Wang, Y. P.; Liu, C. L.; Qin, Y.; Guo, L. P.; Liu, L. H.

    2016-07-01

    There are a few bimodal molecular imaging probes constructed by gadolinium (3+) ions in combination with carbon quantum dots (CQDs), and the reported ones show such obvious drawbacks as low luminous efficiency and weak MRI contrast. In the paper, a kind of CQDs photoluminescence materials with magnetic resonance response was prepared by hydrothermal method and employing gadopentetate monomeglumine (GdPM) as a precusor. Here, the GdPM plays a role of not only carbon source, but also gadolinium (3+) sources. When the GdPM aqueous solution with a concentration of 4 mg mL-1 was pyrolyzed under 220 °C and 2.0 MPa for 8 h, an optimal CQDs was obtained which are doped with gadolinium (3+) ions in both chelates and Gd2O3 (named as Gd3+-CQDs). The average diameter of the Gd3+-CQDs is about 1.6 nm, which show a high photoluminescence quantum yield of 7.1%, as well as high longitudinal relaxivity (r1) of 9.87 mM-1 s-1. And owing to the unconspicuous cell toxicity, the Gd3+-CQDs show big possibility for clinical application in magnetic resonance/fluorescence bimodal molecular imaging.

  10. A novel long lasting phosphor Sr5 (PO4)3FxCl1-x:Eu2+,Gd3+prepared in air condition

    Institute of Scientific and Technical Information of China (English)

    邓耀敏; 易双萍; 黄军; 赵韦人; 方夏冰

    2013-01-01

    A series of blue long afterglow mixed halide-phosphate phosphors Sr5 (PO4)3 FxCl1-x:Eu2+,Gd3+were synthesized in air by traditional solid-state reaction route. The crystal structures, photoluminescence, thermoluminescence properties and afterglow proper-ties of the phosphors were characterized systematically using X-ray diffraction (XRD), luminescence spectrophotometer, microcom-puter thermoluminescence dosimeter and single photon counter, respectively. Under 280 nm excitation, the broadband emissions of Eu2+ions were observed at 445 nm (blue) due to the 4f7→4f65d transition. It was demonstrated that there existed the self-reduction of the Eu3+to Eu2+ions in this special halide-phosphate matrix in air condition. The addition of Gd3+ions obviously enhanced the after-glow properties of the single doped Eu2+ions in the halide-phosphate phosphors. And the content of the fluoride anions also had sig-nificant influence on the afterglow properties. All results indicated that Sr5 (PO4)3 FxCl1-x:Eu2+,Gd3+might be potential phosphors for long lasting phosphorescence (LLP) materials.

  11. GD3 expression in CHO-K1 cells increases growth rate, induces morphological changes, and affects cell-substrate interactions.

    Science.gov (United States)

    Daniotti, Jose L; Zurita, Adolfo R; Trindade, Vera M T; Maccioni, Hugo J F

    2002-11-01

    We have generated a panel of CHO-K1 cell clones with different glycolipid compositions by stable transfection of appropriate glycosyltransferases and studied the morphological and growth phenotype of a clone stably expressing Sial-T2. Compared with the GM3 expressing parental cells, Sial-T2 transfectants show low expression of GM3 and neo expression of GD3 and GT3. These cells show about 60% reduction of the mean cell area, and about 2-fold increase of the mean colony area and growth rate. Cells over expressing Sial-T2 showed a flattened appearance, and with time in culture they detached from the substrate leaving adhered material that was GD3 immunoreactive. No apoptotic or proteome differences could be detected in the Sial-T2 transfectants. Thus, increased expression of GD3 and GT3 influence parameters of growth and social behavior of CHO-K1 cells. However, the molecular and cellular basis underlying these influences requires further investigation.

  12. Enhanced UVB emission and analysis of chemical states of Ca5(PO4)3OH:Gd3+,Pr3+ phosphor prepared by co-precipitation

    Science.gov (United States)

    Mokoena, P. P.; Nagpure, I. M.; Kumar, Vinay; Kroon, R. E.; Olivier, E. J.; Neethling, J. H.; Swart, H. C.; Ntwaeaborwa, O. M.

    2014-08-01

    Hydroxyapatite (Ca5(PO4)3OH) is a well-known bioceramic material used in medical applications because of its ability to form direct chemical bonds with living tissues. This mineral is currently used as a host for rare-earth ions (e.g. Gd3+, Pr3+, Tb3+, etc.) to prepare phosphors that can be used in light emitting devices of different types. In this study Ca5(PO4)3OH:Gd3+,Pr3+ phosphors were prepared by the co-precipitation method and were characterised by x-ray diffraction, x-ray photoelectron spectroscopy, scanning electron microscopy, high resolution transmission electron microscopy, energy dispersive x-ray spectroscopy and photoluminescence spectroscopy. The x-ray diffraction pattern was consistent with the hexagonal phase of Ca5(PO4)3OH referenced in JCPDS card number 73-0293. The x-ray photoelectron spectroscopy data indicated that Ca2+ occupied two different lattice sites, referred to as Ca1 and Ca2. The photoluminescence data exhibited a narrowband emission located at 313 nm, which is associated with the 6P7/2→8S7/2 transition of the Gd3+ ion. This emission is classified as ultraviolet B and it is suitable for use in phototherapy lamps to treat various skin diseases. The photoluminescence intensity of the 313 nm emission was enhanced considerably by Pr3+ co-doping.

  13. EPR detected polarization transfer between Gd3+ and protons at low temperature and 3.3 T: The first step of dynamic nuclear polarization

    Science.gov (United States)

    Nagarajan, Vijayasarathi; Hovav, Yonatan; Feintuch, Akiva; Vega, Shimon; Goldfarb, Daniella

    2010-06-01

    Electron-electron double resonance pulsed electron paramagnetic resonance (EPR) at 95 GHz (3.3 T) is used to follow the dynamics of the electron spin polarization during the first stages of dynamic nuclear polarization in solids. The experiments were performed on a frozen solution of Gd+3 (S =7/2) in water/glycerol. Focusing on the central |-1/2⟩→|+1/2⟩ transition we measured the polarization transfer from the Gd3+ electron spin to the adjacent H1 protons. The dependence of the echo detected EPR signal on the length of the microwave irradiation at the EPR "forbidden" transition corresponding to an electron and a proton spin flip is measured for different powers, showing dynamics on the microsecond to millisecond time scales. A theoretical model based on the spin density matrix formalism is suggested to account for this dynamics. The central transition of the Gd3+ ion is considered as an effective S =1/2 system and is coupled to H1 (I =1/2) nuclei. Simulations based on a single electron-single nucleus four level system are shown to deviate from the experimental results and an alternative approach taking into account the more realistic multinuclei picture is shown to agree qualitatively with the experiments.

  14. Pulse shape discrimination properties of Gd3Ga3Al2O12:Ce,B single crystal in comparison with CsI:Tl

    Science.gov (United States)

    Rawat, S.; Tyagi, Mohit; Netrakanti, P. K.; Kashyap, V. K. S.; Mitra, A.; Singh, A. K.; Desai, D. G.; Kumar, G. Anil; Gadkari, S. C.

    2016-12-01

    Single crystals of Gd3Ga3Al2O12:Ce,B and CsI:Tl were grown by Czochralski and Bridgman techniques, respectively. While both the crystals exhibited similar emission at about 550 nm, their scintillation decay times showed significantly different characteristics. The average scintillation decay time of Gd3Ga3Al2O12:Ce,B crystal was found to be about 284 ns for alpha excitation compared to 108 ns measured for a gamma source. On the other hand in CsI:Tl crystals, the alpha excitation resulted in a lower average decay time of 600 ns compared to 1200 ns with gamma excitation. Their pulse shape discrimination (PSD) for gamma and alpha radiations were studied by coupling the scintillators with photomultiplier tube or SiPM and employing an advanced digitizer as well as a conventional zero-crossing setup. In spite of having a poor α/γ light yield ratio, the PSD figure of merit and the difference of zero-crossing time in Gd3Ga3Al2O12:Ce,B crystals were found to be superior in comparison to CsI:Tl crystals.

  15. Binary and Ternary Heterometallic (La3+, Gd3+, Y3+–Eu3+ Functionalized SBA-15 Mesoporous Hybrids: Chemically Bonded Assembly and Photoluminescence

    Directory of Open Access Journals (Sweden)

    Yan Bing

    2010-01-01

    Full Text Available Abstract A novel kind of organic–inorganic monomer SUASi has been achieved by modifying 5-sulfosalicylic acid (SUA with 3-aminopropyltrimethoxysilane (APS, subsequently binary and ternary Eu3+ mesoporous hybrid materials with 5-sulfosalicylic acid (SUA-functionalized SBA-15 and 1,10-phenanthroline (phen are synthesized by co-condensation of SUASi and TEOS in the presence of Eu3+ complex and Pluronic P123 as a template. Finally, luminescent hybrid mesoporous materials consisting of active rare earth ions (Eu3+—inert rare earth ions (Y3+, La3+, Gd3+ complex covalently bonded to the mesoporous materials network have been obtained via this sol–gel approach. The physical characterization and photoluminescence of all these resulting materials are studied in detail. Especially the luminescent behavior has been studied with the different ratios of Eu3+–(Y3+, La3+, Gd3+, which suggests that the existence of inert rare earth ions can enhance the luminescence intensity of Eu3+. This may be due to the intramolecular energy transfer between Y3+, La3+, Gd3+, and Eu3+ through the covalently bonded mesoporous framework.

  16. The Gadolinium (Gd3+) and Tin (Sn4+) Co-doped BiFeO3 Nanoparticles as New Solar Light Active Photocatalyst

    Science.gov (United States)

    Irfan, Syed; Rizwan, Syed; Shen, Yang; Li, Liangliang; Asfandiyar, A; Butt, Sajid; Nan, Ce-Wen

    2017-01-01

    The process of photocatalysis is appealing to huge interest motivated by the great promise of addressing current energy and environmental issues through converting solar light directly into chemical energy. However, an efficient solar energy harvesting for photocatalysis remains a critical challenge. Here, we reported a new full solar spectrum driven photocatalyst by co-doping of Gd3+ and Sn4+ into A and B-sites of BiFeO3 simultaneously. The co-doping of Gd3+ and Sn4+ played a key role in hampering the recombination of electron-hole pairs and shifted the band-gap of BiFeO3 from 2.10 eV to 2.03 eV. The Brunauer-Emmett-Teller (BET) measurement confirmed that the co-doping of Gd3+ and Sn4+ into BiFeO3 increased the surface area and porosity, and thus the photocatalytic activity of the Bi0.90Gd0.10Fe0.95Sn0.05O3 system was significantly improved. Our work proposed a new photocatalyst that could degrade various organic dyes like Congo red, Methylene blue, and Methyl violet under irradiation with different light wavelengths and gave guidance for designing more efficient photocatalysts. PMID:28195198

  17. Effect of Mg$^{2+}$ ions co-doping on timing performance and radiation tolerance of Cerium doped Gd$_{3}$Al$_{2}$Ga$_{3}$O$_{12}$ crystals

    CERN Document Server

    Lucchini, M.T.; Bohacek, P.; Gundacker, S.; Kamada, K.; Nikl, M.; Petrosyan, A.; Yoshikawa, A.; Auffray, E.

    2016-01-01

    Inorganic scintillators with high density and high light yield are of major interest for applications in medical imaging and high energy physics detectors. In this work, the optical and scintillation properties of Mg co-doped Ce:Gd3Al2Ga3O12 crystals, grown using Czochralski technique, have been investigated and compared with Ce:Gd3Al2Ga3O12 ones prepared with identical technology. Improvements in the timing performance of the Mg co-doped samples with respect to Ce:Gd3Al2Ga3O12 ones have been measured, namely a substantial shortening of the rise time and scintillation decay components and lower afterglow were achieved. In particular, a significantly better coincidence time resolution of 233 ps FWHM, being a fundamental parameter for TOF-PET devices, has been observed in Mg co-doped crystals. The samples have also shown a good radiation tolerance under high doses of γ-rays, making them suitable candidates for applications in harsh radiation environments, such as detectors at future collider experiments.

  18. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hye Jin [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Lee, Dong-Hyung [Department of Obstetrics and Gynecology, Medical Research Institute, Pusan National University, Busan (Korea, Republic of); Park, Seong-Hwan; Kim, Juil; Do, Kee Hun [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); An, Tae Jin; Ahn, Young Sup; Park, Chung Berm [Department of Herbal Crop Research, NIHHS, RDA, Eumseong (Korea, Republic of); Moon, Yuseok, E-mail: moon@pnu.edu [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Medical Research Institute and Research Institute for Basic Sciences, Pusan National University, Busan (Korea, Republic of)

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  19. The prostaglandin F synthase activity of the human aldose reductase AKR1B1 brings new lenses to look at pathologic conditions.

    Directory of Open Access Journals (Sweden)

    Eva eBresson

    2012-05-01

    Full Text Available Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs, lead us to the discovery that AKR1B5 and later AKR1B1 were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2 and PGF2α may constitute a functional dyad with physiological relevance at least as important as the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231 also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region (MSRR containing two putative antioxidant response elements (ARE adjacent to TonE and AP1.We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors (ARIs like alrestatin, statil (ponalrestat and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human

  20. Changes in cardiac aldosterone and its synthase in rats with chronic heart failure: an intervention study of long-term treatment with recombinant human brain natriuretic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, X.Q. [Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Department of Cardiology, The Central Hospital of Enshi Autonomous Prefecture, Enshi, Hubei (China); Hong, H.S. [Department of Geriatrics, Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Lin, X.H. [Department of Emergency Medicine, Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Chen, L.L. [Department of Cardiology, Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Li, Y.H. [Department of Cardiology, The Central Hospital of Enshi Autonomous Prefecture, Enshi, Hubei (China)

    2014-07-11

    The physiological mechanisms involved in isoproterenol (ISO)-induced chronic heart failure (CHF) are not fully understood. In this study, we investigated local changes in cardiac aldosterone and its synthase in rats with ISO-induced CHF, and evaluated the effects of treatment with recombinant human brain natriuretic peptide (rhBNP). Sprague-Dawley rats were divided into 4 different groups. Fifty rats received subcutaneous ISO injections to induce CHF and the control group (n=10) received equal volumes of saline. After establishing the rat model, 9 CHF rats received no further treatment, rats in the low-dose group (n=8) received 22.5 μg/kg rhBNP and those in the high-dose group (n=8) received 45 μg/kg rhBNP daily for 1 month. Cardiac function was assessed by echocardiographic and hemodynamic analysis. Collagen volume fraction (CVF) was determined. Plasma and myocardial aldosterone concentrations were determined using radioimmunoassay. Myocardial aldosterone synthase (CYP11B2) was detected by quantitative real-time PCR. Cardiac function was significantly lower in the CHF group than in the control group (P<0.01), whereas CVF, plasma and myocardial aldosterone, and CYP11B2 transcription were significantly higher than in the control group (P<0.05). Low and high doses of rhBNP significantly improved hemodynamics (P<0.01) and cardiac function (P<0.05) and reduced CVF, plasma and myocardial aldosterone, and CYP11B2 transcription (P<0.05). There were no significant differences between the rhBNP dose groups (P>0.05). Elevated cardiac aldosterone and upregulation of aldosterone synthase expression were detected in rats with ISO-induced CHF. Administration of rhBNP improved hemodynamics and ventricular remodeling and reduced myocardial fibrosis, possibly by downregulating CYP11B2 transcription and reducing myocardial aldosterone synthesis.

  1. Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway.

    Science.gov (United States)

    Puisac, Beatriz; Ramos, Mónica; Arnedo, María; Menao, Sebastián; Gil-Rodríguez, María Concepción; Teresa-Rodrigo, María Esperanza; Pié, Angeles; de Karam, Juan Carlos; Wesselink, Jan-Jaap; Giménez, Ignacio; Ramos, Feliciano J; Casals, Nuria; Gómez-Puertas, Paulino; Hegardt, Fausto G; Pié, Juan

    2012-04-01

    The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues.

  2. Synthesis, Crystal Structure, Magnetism, and Optical Properties of Gd 3[SiON 3]O—An Oxonitridosilicate Oxide with Noncondensed SiON 3 Tetrahedra

    Science.gov (United States)

    Höppe, Henning A.; Kotzyba, Gunter; Pöttgen, Rainer; Schnick, Wolfgang

    2002-09-01

    The novel oxonitridosilicate oxide (sion oxide) Gd 3[SiON 3]O was obtained by the reaction of gadolinium metal with its carbonate oxide and silicon diimide in a radiofrequency (r.f.) furnace at a temperature of 1400°C. The crystal structure of Gd 3[SiON 3]O ( I4/ mcm, a=649.1(2) pm, c=1078.8(6) pm, Z=4, R1=0.0411, w R2=0.0769, 405 F2 values, 19 parameters, 123 K) is isotypic with that of Ba 3[SiO 4]O and Cs 3[CoCl 4]Cl. It can be derived from the perovskite structure type by a hierarchical substitution: Ti 4+→O 2-, O 2-→Gd 3+, Ca 2+→[SiON 3] 7- resulting in the formation of large [OGd 6] 16+ octahedra, which are twisted by ξ=16.47(1)° around [001]. The low-temperature single-crystal data investigation led to a crystallographic splitting of the central O atom which could not be resolved at room temperature. The UV-Vis absorption spectra in reflection geometry of the yellow title compound revealed two overlaying broad bands, one peaking at almost the same wavelength as observed in gadolinium oxide (340 nm) and a second red-shifted band at approximately 400 nm indicating a strong influence of nitrogen on the ligand field splitting of the 5 d states of Gd 3+. Temperature-dependent magnetic susceptibility measurements of Gd 3[SiON 3]O show Curie-Weiss behavior from 2 to 300 K with an experimental magnetic moment of 7.68(5) μB/Gd, indicating trivalent gadolinium. There is no evidence for magnetic ordering down to 2 K. According to the paramagnetic Curie temperature of -7(1) K, the exchange between the gadolinium magnetic moments is supposed to be only weak. The vibrational spectroscopic data (IR and Raman) are reported.

  3. Steroid receptor RNA activator (SRA modification by the human pseudouridine synthase 1 (hPus1p: RNA binding, activity, and atomic model.

    Directory of Open Access Journals (Sweden)

    Tiphaine Huet

    Full Text Available The most abundant of the modified nucleosides, and once considered as the "fifth" nucleotide in RNA, is pseudouridine, which results from the action of pseudouridine synthases. Recently, the mammalian pseudouridine synthase 1 (hPus1p has been reported to modulate class I and class II nuclear receptor responses through its ability to modify the Steroid receptor RNA Activator (SRA. These findings highlight a new level of regulation in nuclear receptor (NR-mediated transcriptional responses. We have characterised the RNA association and activity of the human Pus1p enzyme with its unusual SRA substrate. We validate that the minimal RNA fragment within SRA, named H7, is necessary for both the association and modification by hPus1p. Furthermore, we have determined the crystal structure of the catalytic domain of hPus1p at 2.0 Å resolution, alone and in a complex with several molecules present during crystallisation. This model shows an extended C-terminal helix specifically found in the eukaryotic protein, which may prevent the enzyme from forming a homodimer, both in the crystal lattice and in solution. Our biochemical and structural data help to understand the hPus1p active site architecture, and detail its particular requirements with regard to one of its nuclear substrates, the non-coding RNA SRA.

  4. Steroid receptor RNA activator (SRA) modification by the human pseudouridine synthase 1 (hPus1p): RNA binding, activity, and atomic model.

    Science.gov (United States)

    Huet, Tiphaine; Miannay, François-Alexandre; Patton, Jeffrey R; Thore, Stéphane

    2014-01-01

    The most abundant of the modified nucleosides, and once considered as the "fifth" nucleotide in RNA, is pseudouridine, which results from the action of pseudouridine synthases. Recently, the mammalian pseudouridine synthase 1 (hPus1p) has been reported to modulate class I and class II nuclear receptor responses through its ability to modify the Steroid receptor RNA Activator (SRA). These findings highlight a new level of regulation in nuclear receptor (NR)-mediated transcriptional responses. We have characterised the RNA association and activity of the human Pus1p enzyme with its unusual SRA substrate. We validate that the minimal RNA fragment within SRA, named H7, is necessary for both the association and modification by hPus1p. Furthermore, we have determined the crystal structure of the catalytic domain of hPus1p at 2.0 Å resolution, alone and in a complex with several molecules present during crystallisation. This model shows an extended C-terminal helix specifically found in the eukaryotic protein, which may prevent the enzyme from forming a homodimer, both in the crystal lattice and in solution. Our biochemical and structural data help to understand the hPus1p active site architecture, and detail its particular requirements with regard to one of its nuclear substrates, the non-coding RNA SRA.

  5. Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT cell development and transplantation.

    Directory of Open Access Journals (Sweden)

    Dale Christiansen

    2008-07-01

    Full Text Available The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3, is believed to be critical for natural killer T (NKT cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S. We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3Gal xenoantigen, with obvious significance in the field of xenotransplantation.

  6. DOWN-REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION BY INOSITOL HEXAPHOSPHATE IN HUMAN COLON CANCER CELLS.

    Science.gov (United States)

    Kapral, Małgorzata; Wawszczyk, Joanna; Sośnicki, Stanisław; Węglarz, Ludmiła

    2015-01-01

    Inflammatory bowel disease (IBD) is chronic inflammatory condition associated with increased risk of developing colorectal cancer. A number of mediators of inflammation, such as pro-inflammatory cytokines, prostaglandins and nitric oxide have been involved in carcinogenesis, especially in the promotion and progression stages. NO is synthesized from L-arginine by constitutively expressed endothelial and neuronal nitric oxide synthases (eNOS and nNOS, respectively) and an inducible NOS (iNOS) isoform expressed under inflammatory conditions. A selective inhibitors of iNOS could be, therefore, considered to be good candidates as chemopreventive agents against colon cancer. In this study, the effect of inositol hexaphosphate (IP6), dietary phytochemical, on the mRNA expression of iNOS stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium) and IL-1β in intestinal cells Caco-2 for 6 and 12 h was investigated. A transcription level of iNOS with the use real time QRT-PCR technique was determined in cells treated with 1 and 2.5 mM IP6. Stimulation of Caco-2 with pro-inflammatory factors (LPS and IL-1β) resulted in an up-expression of iNOS mRNA at 6 and 12 h. Cells exposed to IP6 only revealed significant reduction in iNOS gene transcription after 12 h. A decrease in iNOS transcription by IP6 following the gene induction by proinflammatory agents in 6 and 12 h lasting cultures was also determined. The findings of this study suggest that one of the anti-cancer and anti-inflammatory abilities of IP6 can be realized by suppressing the expression of gene encoding inducible nitric oxide synthase isoform at the transcriptional level.

  7. Impact of the Xba1-polymorphism of the human muscle glycogen synthase gene on parameters of the insulin resistance syndrome in a Danish twin population

    DEFF Research Database (Denmark)

    Fenger, M; Poulsen, P; Beck-Nielsen, H;

    2000-01-01

    was increased in female carriers of the A2-allele with impaired glucose tolerance or Type 2 diabetes mellitus (79 +/- 1 vs. 94 +/- 4 mmHg, P insulin resistance syndrome were associated with the polymorphism. CONCLUSIONS......AIMS: To establish the impact on the insulin resistance syndrome of the intron 14 Xba1-polymorphism in human muscle glycogen synthase (GYS1). METHODS: Parameters related to the insulin resistance syndrome were measured in 244 monozygotic twins and 322 dizygotic twins with or without impaired...... and the remainder had the genotype A1A2. No A2A2-genotypes were detected. In 11 genotypic discordant dizygotic twin pairs the insulin resistance was significantly increased in the twins carrying the A1A2 genotype regardless of sex (HOMA index 1.81 (A1A1) vs. 2.57 (A1A2), P

  8. Effect of Gd3+ doping on structural, optical and frequency-dependent dielectric response properties of pseudo-cubic BaTiO3 nanostructures

    Science.gov (United States)

    Borah, Manjit; Mohanta, Dambarudhar

    2014-06-01

    We report on the structural, optical and dielectric characterization of solid state derived, pseudo-cubic nanoscale barium titanates (BTs) with gadolinium (Gd3+) as substitutional dopant. Referring to X-ray diffractograms, apart from the BT peaks related to perovskite structure, the non-existence of any additional peaks due to byproducts has revealed that Gd3+ has undergone substitutional doping into the BT host lattice. The well-separated BT nanoparticles of typical size ˜10-15 nm were observed through electron microscopy studies. Following a direct, allowed type carrier transition ( n=1/2), a reduction in the optical band gap value (from 3.28 to 3.255 eV) was observed when the Gd-doping level was varied within 0-7 %. Conversely, the Urbach energy followed an increasing trend, from a value of 0.741 to 1.879 eV. Furthermore, the dielectric constant showed a decreasing tendency with doping content and with increasing frequency. However, in the low-frequency region, the loss tangent (tan δ), which is the combined result of orientational polarization and electrical conduction, was found to be quite high in the doped samples as compared to their un-doped counterpart. The frequency-dependent electrical data were also analyzed in the framework of conductivity and impedance formalisms. In particular, the ac conductivity which varies as ˜ ω s approaches ideal Debye behavior ( s→1) for a low Gd level and a higher doping concentration did not show improved dielectric feature of the host. The incorporation of rare-earth (Gd3+) ions into the BT host system could greatly manifest dielectric relaxation and carrier conduction mechanisms, in a given frequency range, and thus can find immense scope in miniaturized nanoelectronic elements including ceramic capacitors and transducers.

  9. Effect of heat treatment on corrosion behavior of low pressure sand cast Mg-10Gd-3Y-0.5Zr alloys

    OpenAIRE

    2016-01-01

    The corrosion behaviors of low-pressure sand cast Mg-10Gd-3Y-0.5Zr (wt.%) alloys in as-cast, solution treated (T4) and aged (T6) conditions were studied by means of immersion test and electrochemical measurements in 5wt.% NaCl solution saturated with Mg(OH)2. It was observed that the corrosion rate in the T4 condition was lower than that of the as-cast and T6 conditions by both sand casting and permanent mold casting with the same order of as-cast>T6>T4; while the corrosion resistance of the ...

  10. Study the effect of Gd3+ incorporation into nanocrystalline (Ni-Ti) substituted Mn-Zn ferrites on its structure and functional properties

    Science.gov (United States)

    Rady, K. E.; Shams, M. S.

    2017-03-01

    Ferrite samples with general chemical formula Mn0.9Zn0.1Ni0.05Ti0.05GdtFe1.9-tO4; (0.0≤ t≤0.05; step 0.01) were prepared using solid state reaction technique and the effect of Gd3+ ions incorporation on its physical properties has been studied. From the obtained results, XRD analysis reveals that the samples have a cubic spinel single phase structure for 0.0≤ t≤0.02; while for t≥0.03 a small peak of secondary phase (Gd3Fe5O12) appears and becomes more noticeable with increasing Gd content. The lattice parameter (a) of the prepared samples was found to be initially increases and then decreases with increasing Gd content which may be attributed to the difference in the ionic radii of the cations involved and the solubility limit of Gd3+ ions. The crystallite size of the samples was estimated using Scherrer's equation and ranged from 96 nm to 107 nm. A vibrating sample magnetometer (VSM) was used at room temperature in order to study the effect of Gd content on the magnetic hysteresis parameters of the prepared ferrites such as saturation magnetization and coercivity. DC molar magnetic susceptibility (χM) for the prepared samples was measured using Faraday's method as a function of temperature and the Curie temperature was calculated from the magnetic susceptibility measurements. Also the DC resistivity of the samples was measured at room temperature. The obtained results show that, the substitution by Gd3+ ions improves the electrical properties of the samples by increasing it DC electrical resistivity by 118% and consequently decreases it eddy current loss while the saturation magnetization slightly decreased by 14% only. The sample of t=0.01 shows a high dc magnetic susceptibility, high saturation magnetization (43.1 emu/g), high electric resistivity 12×103 Ω.m and high Curie temperature (496 K), which is useful in some technological applications such as transformer and inductor cores.

  11. The development of new phosphors of Tb3+/Eu3+ co-doped Gd3Al5O12 with tunable emission

    Science.gov (United States)

    Teng, Xin; Wang, Wenzhi; Cao, Zhentao; Li, Jinkai; Duan, Guangbin; Liu, Zongming

    2017-07-01

    The gadolinium aluminum garnets Gd3Al5O12 (GdAG) activated with Tb3+/Eu3+ were successfully prepared via co-precipitation method at 1500 °C in this work. The crystal structure stabilization, elements analysis, microphotograph, PL/PLE spectra, decay behavior and quantum efficiency were discussed in detail. The metastable GdAG compounds been effectively stabilized by doping with smaller 10 at.% Tb3+, which then allows the development of new phosphors of (Gd0.9-xTb0.1Eux)3Al5O12 (GdAG:Tb3+/Eu3+, x = 0-0.03) for opto-functionality explorations. The PLE/PL spectra displays that the strongest PLE peak was located at ∼276 nm, which overlaps the 8S7/2 → 6IJ transition of Gd3+. Under 276 nm excitation, the phosphors exhibited both Tb3+ and Eu3+ emissions at 548 nm (green, 5D4 → 7F5 transition of Tb3+) and 592 nm (orange-red, 5D0 → 7F1 transition of Eu3+), respectively. The emission intensities of Tb3+ and Eu3+ remarkably varied with the Eu3+ incorporation. As a consequence, the emission color can be readily tuned from approximately green to orange-red. Fluorescence decay analysis found that the lifetime for the Tb3+ emission rapidly decreased conforming to the Tb3+ → Eu3+ energy transfer, and the energy transfer efficiency was calculated. Owing to the Gd3+ → Eu3+ and Gd3+ → Tb3+ energy transfer, the emission intensities of Tb3+ and Eu3+ in (Gd0.9-xTb0.1Eux)AG phosphor were higher than (Y0.87Tb0.1Eu0.03)AG and (Lu0.87Tb0.1Eu0.03)AG system. The (Gd0.9-xTb0.1Eux)AG garnet phosphors developed in this work may serve as a new type of phosphor which hopefully meets the requirements of various lighting and optical display applications.

  12. Synthesis and Photophysical Properties of Dy3+ and Gd3+ Polymeric Complexes with Functionalized Polybenzimidazole Containing β-Diketone Side Group

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The polymeric ligand PBIa (functionalized polybenzimidazole containing β-diketone side group) was successfully synthesized via the reaction of polybenzimidazole (PBI) with 3-Br-acetylacetone in DMSO solvent using NaH as the deprotonation reagent. Its corresponding polymeric complexes of Dy3+ and Gd3+ were prepared and characterized by FT-IR, 1H NMR, molar conductance measurements, and thermal analysis. The photoluminescence properties and the probable mechanism of the Dy and Gd complexes were studied. The measurement and analysis of the thermal properties showed that these were thermal stable.

  13. Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

    Science.gov (United States)

    Newton, Derek C; Bevan, Sian C; Choi, Stephen; Robb, G Brett; Millar, Adam; Wang, Yang; Marsden, Philip A

    2003-01-03

    Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

  14. Modeling human Coenzyme A synthase mutation in yeast reveals altered mitochondrial function, lipid content and iron metabolism

    Directory of Open Access Journals (Sweden)

    Camilla Ceccatelli Berti

    2015-04-01

    Full Text Available Mutations in nuclear genes associated with defective coenzyme A biosynthesis have been identified as responsible for some forms of neurodegeneration with brain iron accumulation (NBIA, namely PKAN and CoPAN. PKAN are defined by mutations in PANK2, encoding the pantothenate kinase 2 enzyme, that account for about 50% of cases of NBIA, whereas mutations in CoA synthase COASY have been recently reported as the second inborn error of CoA synthesis leading to CoPAN. As reported previously, yeast cells expressing the pathogenic mutation exhibited a temperature-sensitive growth defect in the absence of pantothenate and a reduced CoA content. Additional characterization revealed decreased oxygen consumption, reduced activities of mitochondrial respiratory complexes, higher iron content, increased sensitivity to oxidative stress and reduced amount of lipid droplets, thus partially recapitulating the phenotypes found in patients and establishing yeast as a potential model to clarify the pathogenesis underlying PKAN and CoPAN diseases.

  15. Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.

    Science.gov (United States)

    ben Bdira, Fredj; González, Esperanza; Pluta, Paula; Laín, Ana; Sanz-Parra, Arantza; Falcon-Perez, Juan Manuel; Millet, Oscar

    2014-11-01

    Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.

  16. Er3+/Yb3+共掺Gd3Sc2Ga3O12晶体的上转换发光%Up-conversion luminescence in Er3+/Yb3+-codoped Gd3Sc2Ga3O12 laser crystals

    Institute of Scientific and Technical Information of China (English)

    罗建乔; 孙敦陆; 张庆礼; 刘文鹏; 谷长江; 吴路生; 殷绍唐

    2008-01-01

    研究了提拉法生长的Er3+/Yb3+:Gd3Sc2Ga3O12和Er3+:Gd3Sc2Ga3O12晶体在室温下320-1700 nm范围的吸收光谱和500-750 nm范围内的上转换荧光谱,同时对其上转换荧光的可能发生机制、途径以及上转换过程可能对Er3+的2.8 μm波段激光振荡产生的影响进行了分析和讨论.结果表明:Yb3+的敏化显著地增强了晶体在966 nm附近的吸收能力,大幅度加宽了晶体在该处的吸收带宽.在940 nm激光的激发下,Er3+/Yb3+:Gd3Sc2Ga3O12中的上转换荧光强度明显强于Er3+:Gd3Sc2Ga3O12中的上转换荧光强度,表明Yb3+与Er3+之间存在高效率的能量传递,其主要上转换机制可能为Yb3+-Er3+,Er3+-Er3+能量传递.

  17. Hydrothermal Synthesis and Characterization of Pyrochlore Titanate R2 Ti2O7(R=Gd3+, Tb3+, Dy3+)

    Institute of Scientific and Technical Information of China (English)

    PENG Wen; HU Bin; CHEN Yan; HU Wei-wei; GUO Li; YUAN Hong-ming; FENG Shou-hua

    2011-01-01

    Pyrochlore titanate oxides, R2Ti2O7(R=Gd3+, Tb3+, Dy3+), were synthesized under mild hydrothermal conditions. The crystal growth of pyrochlore titanate oxides and taking place of chemical reaction in the hydrothermal processing were sensitive to the alkalinity, temperature, reaction time, the nature of the rare earth ion and the composition of initial reaction mixture. The as-prepared samples were characterized by powder X-ray diffraction, scanning electron microscopy, Raman spectrum and variable temperature dc magnetic susceptibility(Superconductivity quantum interference device, SQUIDS). The magnetic studies gave 7.29× 10-23 A·m2/Gd3+ and -8.28 K, 8.75 × 10-23 A·m2/ Tb3+ and -19.7 K, and 8.85×10-23 A·m2/Dy3+ and 0.84 K effective moments and Weiss constants for Gd2Ti2O7,Tb2Ti2O7 and Dy2Ti2O7, respectively.

  18. Study on the Grain Refinement Behavior of Mg-Zr Master Alloy and Zr Containing Compounds in Mg-10Gd-3Y Magnesium Alloy

    Science.gov (United States)

    Wu, Guohua; Sun, Ming; Dai, Jichun; Ding, Wenjiang

    The effects of Mg-Zr master alloy and a potassium fluozirconate (K2ZrF6) salt mixture (KSM) on the grain refinement behavior of Mg-10Gd-3Y magnesium alloy were studied. The results show that the Mg-10Gd-3Y alloy is well refined by Mg-Zr or KSM. The characteristic micro structure feature of the alloy refined by Mg-Zr master alloy is the Zr-rich cores that exist in most grains, while the Zr-rich cores are not observed in the alloy refined by KSM. It is suggested that the grain refinement mechanisms of zirconium in the two cases are different: the Zr released from Mg-Zr master alloy works by adding heterogeneous nucleants, while the Zr produced from the in-situ reaction between Mg melt and K2ZrF6 works by restricting grain growth. Compared with the Mg-30.wt%Zr master alloy, the KSM refiner shows much longer fading time during melting.

  19. Effect of Mg2+ ions co-doping on luminescence and defects formation processes in Gd3(Ga,Al)5O12:Ce single crystals

    Science.gov (United States)

    Babin, V.; Bohacek, P.; Grigorjeva, L.; Kučera, M.; Nikl, M.; Zazubovich, S.; Zolotarjovs, A.

    2017-04-01

    Photo- and radioluminescence and thermally stimulated luminescence characteristics of Ce3+ - doped and Ce3+, Mg2+ co-doped Gd3(Ga,Al)5O12 (GAGG) single crystals of similar composition are investigated in the 9-500 K temperature range. The Ce3+ - related luminescence spectra and the photoluminescence decay kinetics in these crystals are found to be similar. Under photoexcitation in the Ce3+ - and Gd3+ - related absorption bands, no prominent rise of the photoluminescence intensity in time is observed neither in GAGG:Ce,Mg nor in GAGG:Ce crystals. The afterglow is strongly reduced in GAGG:Ce,Mg as compared to GAGG:Ce, and the afterglow decay kinetics is much faster. Co-doping with Mg2+ results in a drastic decrease of the thermally stimulated luminescence (TSL) intensity in the whole investigated temperature range and in the appearance of a new complex Mg2+ - related TSL glow curve peak around 285 K. After irradiation in the Ce3+ - related 3.6 eV absorption band, the TSL intensity in GAGG:Ce,Mg is found to be comparable with that in the GAGG:Ce epitaxial film of similar composition. The Mg2+ - induced changes in the concentration, origin and structure of the crystal lattice defects and their influence on the scintillation characteristics of GAGG:Ce,Mg are discussed.

  20. Investigation of cyano-bridged coordination nanoparticles Gd3+/[Fe(CN)6]3-/d-mannitol as T1-weighted MRI contrast agents

    Science.gov (United States)

    Perrier, M.; Gallud, A.; Ayadi, A.; Kennouche, S.; Porredon, C.; Gary-Bobo, M.; Larionova, J.; Goze-Bac, Ch.; Zanca, M.; Garcia, M.; Basile, I.; Long, J.; de Lapuente, J.; Borras, M.; Guari, Y.

    2015-07-01

    Cyano-bridged Gd3+/[Fe(CN)6]3- coordination polymer nanoparticles of 3-4 nm stabilized with d-mannitol presenting a high r1 relaxivity value of 11.4 mM-1 s-1 were investigated in vivo as contrast agents (CA) for Magnetic Resonance Imaging (MRI). They allow an increase of the MR image contrast and can act as an efficient intravascular T1 CA with a relatively long blood-circulation lifetime (60 min) without specific toxicity.Cyano-bridged Gd3+/[Fe(CN)6]3- coordination polymer nanoparticles of 3-4 nm stabilized with d-mannitol presenting a high r1 relaxivity value of 11.4 mM-1 s-1 were investigated in vivo as contrast agents (CA) for Magnetic Resonance Imaging (MRI). They allow an increase of the MR image contrast and can act as an efficient intravascular T1 CA with a relatively long blood-circulation lifetime (60 min) without specific toxicity. Electronic supplementary information (ESI) available: Experimental details and procedures, toxicological data, physical characterization. See DOI: 10.1039/c5nr01557j

  1. 5 7 Fe Emission Mössbauer Study on Gd 3 Ga 5 O 1 2 implanted with dilute 5 7 Mn

    Science.gov (United States)

    Krastev, P. B.; Gunnlaugsson, H. P.; Nomura, K.; Adoons, V.; Gerami, A. M.; Johnston, K.; Ncube, M.; Mantovan, R.; Masenda, H.; Matveyev, Y. A.; Mølholt, T. E.; Unzueta, I.; Bharuth-Ram, K.; Gislason, H.; Langouche, G.; Naidoo, D.; Ólafsson, S.

    2016-12-01

    57Fe emission Mössbauer spectroscopy has been applied to study the lattice location and properties of Fe in gadolinium gallium garnet Gd3Ga5 O 12 (GGG) single crystals in the temperature interval 300 - 563 K within the extremely dilute (CERN. These results are compared with earlier Mössbauer spectroscopy study of Fe-doped gadolinium gallium garnet Gd3Ga5 O 12(GGG), with implantation fluences between 8×1015 and 6×1016 atoms cm-2. Three Fe components are observed in the emission Mössbauer spectra: (i) high spin Fe2+ located at damage sites due to the implantation process, (ii) high spin Fe3+ at substitutional tetrahedral Ga sites, and (iii) interstitial Fe, probably due to the recoil imparted on the daughter57∗Fe nucleus in the β - decay of57Mn. In contrast to high fluence57Fe implantation studies the Fe3+ ions are found to prefer the tetrahedral Ga site over the octahedral Ga site. No annealing stages are evident in the temperature range investigated. Despite the very low concentration, high-spin Fe3+ shows fast spin relaxation, presumably due to an indirect interaction between nearby gadolinium atoms.

  2. Charge deformation and orbital hybridization: intrinsic mechanisms on tunable chromaticity of Y3Al5O12:Ce3+ luminescence by doping Gd3+ for warm white LEDs

    Science.gov (United States)

    Chen, Lei; Chen, Xiuling; Liu, Fayong; Chen, Haohong; Wang, Hui; Zhao, Erlong; Jiang, Yang; Chan, Ting-Shan; Wang, Chia-Hsin; Zhang, Wenhua; Wang, Yu; Chen, Shifu

    2015-07-01

    The deficiency of Y3Al5O12:Ce (YAG:Ce) luminescence in red component can be compensated by doping Gd3+, thus lead to it being widely used for packaging warm white light-emitting diode devices. This article presents a systematic study on the photoluminescence properties, crystal structures and electronic band structures of (Y1-xGdx)3Al5O12: Ce3+ using powerful experimental techniques of thermally stimulated luminescence, X-ray diffraction, X-ray absorption near edge structure (XANES), extended X-ray absorption fine structure (EXAFS) and ultraviolet photoelectron spectra (UPS) of the valence band, assisted with theoretical calculations on the band structure, density of states (DOS), and charge deformation density (CDD). A new interpretation from the viewpoint of compression deformation of electron cloud in a rigid structure by combining orbital hybridization with solid-state energy band theory together is put forward to illustrate the intrinsic mechanisms that cause the emission spectral shift, thermal quenching, and luminescence intensity decrease of YAG: Ce upon substitution of Y3+ by Gd3+, which are out of the explanation of the classic configuration coordinate model. The results indicate that in a rigid structure, the charge deformation provides an efficient way to tune chromaticity, but the band gaps and crystal defects must be controlled by comprehensively accounting for luminescence thermal stability and efficiency.

  3. Charge deformation and orbital hybridization: intrinsic mechanisms on tunable chromaticity of Y3Al5O12:Ce3+ luminescence by doping Gd3+ for warm white LEDs.

    Science.gov (United States)

    Chen, Lei; Chen, Xiuling; Liu, Fayong; Chen, Haohong; Wang, Hui; Zhao, Erlong; Jiang, Yang; Chan, Ting-Shan; Wang, Chia-Hsin; Zhang, Wenhua; Wang, Yu; Chen, Shifu

    2015-07-15

    The deficiency of Y3Al5O12:Ce (YAG:Ce) luminescence in red component can be compensated by doping Gd(3+), thus lead to it being widely used for packaging warm white light-emitting diode devices. This article presents a systematic study on the photoluminescence properties, crystal structures and electronic band structures of (Y1-xGdx)3Al5O12: Ce(3+) using powerful experimental techniques of thermally stimulated luminescence, X-ray diffraction, X-ray absorption near edge structure (XANES), extended X-ray absorption fine structure (EXAFS) and ultraviolet photoelectron spectra (UPS) of the valence band, assisted with theoretical calculations on the band structure, density of states (DOS), and charge deformation density (CDD). A new interpretation from the viewpoint of compression deformation of electron cloud in a rigid structure by combining orbital hybridization with solid-state energy band theory together is put forward to illustrate the intrinsic mechanisms that cause the emission spectral shift, thermal quenching, and luminescence intensity decrease of YAG: Ce upon substitution of Y(3+) by Gd(3+), which are out of the explanation of the classic configuration coordinate model. The results indicate that in a rigid structure, the charge deformation provides an efficient way to tune chromaticity, but the band gaps and crystal defects must be controlled by comprehensively accounting for luminescence thermal stability and efficiency.

  4. Effect of Gd3+ substitution on the structural and electrical properties of Mg0.75Zn0.25Fe2O4 nanoparticles

    Science.gov (United States)

    Sebastian, R. M.; Mohammed, E. M.

    2016-12-01

    Nanoparticles of gadolinium substituted Mg-Zn ferrite, Mg0.75Zn0.25GdxFe2-xO4 (where x = 0.00, 0.05, 0.10, 0.15) have been prepared by the sol-gel technique. The lattice constant is found to increase with increase in the Gd3+ ion concentration. The observed variation of dielectric constant with frequency is explained on the basis of the Maxwell-Wagner two layer model and the electron hopping mechanism. The dielectric constant and dielectric loss decrease with Gd3+ ion substitution. The variation of AC conductivity with frequency suggests that the conduction is due to small polaron hopping. It is also noticed that the dielectric constant and the AC conductivity increase with temperature and it is explained in terms of the thermally enhanced drift mobility of the charge carriers. The observed low value of dielectric constant and also the dielectric loss factor obtained for gadolinium substituted ferrites make them desirable for high frequency applications.

  5. (1)H relaxivity of water in aqueous suspensions of Gd(3+)-loaded NaY nanozeolites and AlTUD-1 mesoporous material: the influence of Si/Al ratio and pore size.

    Science.gov (United States)

    Norek, Małgorzata; Neves, Isabel C; Peters, Joop A

    2007-07-23

    The results of a (1)H nuclear magnetic relaxation dispersion (NMRD) and EPR study on aqueous suspensions of Gd(3+)-loaded NaY nanozeolites and AlTUD-1 mesoporous material are described. Upon increase of the Si/Al ratio from 1.7 to 4.0 in the Gd(3+)-loaded zeolites, the relaxation rate per mM Gd(3+) (r1) at 40 MHz and 25 degrees C increases from 14 to 27 s(-)1 mM(-1). The NMRD and EPR data were fitted with a previously developed two-step model that considers the system as a concentrated aqueous solution of Gd(3+) in the interior of the zeolite that is in exchange with the bulk water outside the zeolite. The results show that the observed increase in relaxivity can mainly be attributed to the residence lifetime of the water protons in the interior of the material, which decreased from 0.3 to 0.2 micros, upon the increase of the Si/Al ratio. This can be explained by the decreased interaction of water with the zeolite walls as a result of the increased hydrophobicity. The importance of the exchange rate of water between the inside and the outside of the material was further demonstrated by the relatively high relaxivity (33 s(-1) mM(-1) at 40 MHz, 25 degrees C) observed for a suspension of the Gd(3+)-loaded mesoporous material AlTUD-1. Unfortunately, Gd(3+) leaches rather easily from that material, but not from the Gd(3+)-loaded NaY zeolites, which may have potential as contrast agents for magnetic resonance imaging.

  6. Novel alleles of 31-bp VNTR polymorphism in the human cystathionine -synthase (CBS) gene were detected in healthy Asians

    Indian Academy of Sciences (India)

    Yik-Yuen Gan; Chuan-Fei Chen

    2010-12-01

    A 31-bp variable number of tandem repeats (VNTR) polymorphism of the cystathionine -synthase (CBS) gene was earlier reported in Caucasians of predominantly European descent and Indo–Caucasoid populations.We report here for the first time, the detection of allele 20, which was absent in Caucasian and Indo–Caucasoid populations, as a common allele present in Singaporean Chinese (6.25%), Indians (11.7%), and Malays (11.5%). Hence, allele 20 might be a specific allele for Asian populations. A relatively common allele 19 found in the Caucasian and Indo–Caucasoid populations (10.4%–10.6%) was absent in the Asian samples of this study. Therefore, allele 19 might be a specific allele for the Caucasian populations. A novel and rare allele 13, which was not reported before in the Caucasian and Indo–Caucasoid populations, was found in 0.5% of Singaporean Chinese as genotype 13/17 heterozygotes. The presence of alleles 13 and 20 were verified by DNA sequencing. There were five new genotypes (13/17, 16/20, 17/20, 18/20 and 20/20) not reported before in the Caucasian and Indo–Caucasoid populations, detected in this study. Nine genotypes (15/18, 16/18, 16/21, 17/19, 18/19, 18/21, 19/19, 19/21 and 21/21) which were present in the Caucasian and/or Indo–Caucasoid populations were absent in this study. Our results showed that CBS 31-bp VNTR polymorphism has a distinct genetic difference in allele and genotype frequencies between the European Caucasians, Indo–Caucasoid and Asian populations.

  7. EFFECT OF TNF-( AND IFN-( ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

    Institute of Scientific and Technical Information of China (English)

    庞希宁; 王芸庆; 宋今丹

    2002-01-01

    Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes. 

  8. Up-regulation of miR-31 in human atrial fibrillation begets the arrhythmia by depleting dystrophin and neuronal nitric oxide synthase

    Science.gov (United States)

    Carnicer, Ricardo; Recalde, Alice; Muszkiewicz, Anna; Jayaram, Raja; Carena, Maria Cristina; Wijesurendra, Rohan; Stefanini, Matilde; Surdo, Nicoletta C.; Lomas, Oliver; Ratnatunga, Chandana; Sayeed, Rana; Krasopoulos, George; Rajakumar, Timothy; Bueno-Orovio, Alfonso; Verheule, Sander; Fulga, Tudor A.; Rodriguez, Blanca; Schotten, Ulrich

    2016-01-01

    Atrial fibrillation (AF) is a growing public health burden, and its treatment remains a challenge. AF leads to electrical remodeling of the atria, which in turn promotes AF maintenance and resistance to treatment. Although remodeling has long been a therapeutic target in AF, its causes remain poorly understood. We show that atrial-specific up-regulation of microRNA-31 (miR-31) in goat and human AF depletes neuronal nitric oxide synthase (nNOS) by accelerating mRNA decay and alters nNOS subcellular localization by repressing dystrophin translation. By shortening action potential duration and abolishing rate-dependent adaptation of the action potential duration, miR-31 overexpression and/or disruption of nNOS signaling recapitulates features of AF-induced remodeling and significantly increases AF inducibility in mice in vivo. By contrast, silencing miR-31 in atrial myocytes from patients with AF restores dystrophin and nNOS and normalizes action potential duration and its rate dependency. These findings identify atrial-specific up-regulation of miR-31 in human AF as a key mechanism causing atrial dystrophin and nNOS depletion, which in turn contributes to the atrial phenotype begetting this arrhythmia. miR-31 may therefore represent a potential therapeutic target in AF. PMID:27225184

  9. Mutation of Glu-361 in Human Endothelial Nitric-oxide Synthase Selectively Abolishes L-Arginine Binding without Perturbing the Behavior of Heme and Other Redox Centers

    Science.gov (United States)

    Chen, Pei-Feng; Tsai, Ah-Lim; Berka, Vladimir; Wu, Kenneth K.

    2010-01-01

    Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics. PMID:9045621

  10. Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression.

    Science.gov (United States)

    Ko, Jen-Chung; Zheng, Hao-Yu; Chen, Wen-Ching; Peng, Yi-Shuan; Wu, Chia-Hung; Wei, Chia-Li; Chen, Jyh-Cheng; Lin, Yun-Wei

    2016-12-15

    Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2μg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment.

  11. Human endogenous retrovirus W env increases nitric oxide production and enhances the migration ability of microglia by regulating the expression of inducible nitric oxide synthase

    Institute of Scientific and Technical Information of China (English)

    Ran Xiao; Shan Li; Qian Cao; Xiuling Wang; Qiujin Yan; Xiaoning Tu; Ying Zhu; Fan Zhu

    2017-01-01

    Human endogenous retrovirus W env (HERV-W env) plays a critical role in many neuropsychological diseases such as schizophrenia and multiple sclerosis (MS).These diseases are accompanied by immunological reactions in the central nervous system (CNS).Microglia are important immunocytes in brain inflammation that can produce a gasotransmitter-nitric oxide (NO).NO not only plays a role in the function of neuronal cells but also participates in the pathogenesis of various neuropsychological diseases.In this study,we reported increased NO production in CHME-5 microglia cells after they were transfected with HERV-W env.Moreover,HERV-W env increased the expression and function of human inducible nitric oxide synthase (hiNOS) and enhanced the promoter activity of hiNOS.Microglial migration was also enhanced.These data revealed that HERV-W env might contribute to increase NO production and microglial migration ability in neuropsychological disorders by regulating the expression of inducible NOS.Results from this study might lead to the identification of novel targets for the treatment of neuropsychological diseases,including neuroinflammatory diseases,stroke,and neurodegenerative diseases.

  12. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

    Full Text Available Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  13. Luminescent Properties of Phosphor Na3GdSi2O7 ∶ Tb3 + and Gd3 +→Tb3 + Energy Transfer%Na3GdSi2O7∶Tb3+荧光粉发光特性及Gd3+→Tb3+之间的能量传递

    Institute of Scientific and Technical Information of China (English)

    倪海勇; 梁宏斌; 王灵利; 张秋红

    2013-01-01

    Tb3+ doped sodium gadolinium pyrosilicate phosphors Na3Gd1-xTbxSi2O7 were prepared using a high-temperature solid-state reaction technique.The spectroscopic properties of Na3Gd1-xTbxSi2O7 in VUV-UV-Vis range and fluorescent lifetimes were investigated.The lowest 5d orbitals of Tb3 + in two sites both 6-fold distorted octahedron coordination (trigonal antiprism tap) (Gd3+ (1)) and 6-fold regular trigonal prism coordination (tp) (Gd3 + (2)) were discussed.The lowest 5d orbitals of Tb3 + in Gd3+ (1) is 235 nm for allowed transition,and 280 nm for forbidden transition.The lowest 5d orbitals of Tb3+ in Gd3+ (2) is 224 nm for allowed transition,and 256 nm for forbidden transition.The nonradiative energy-transfer rate K and energy-transfer efficiency ηwere also investigated.The results show that there is efficient Gd3+-Tb3+ energy-transfer in Na3Gd1-xTbxSi2O7,for K and ηincreasing with the increasing of Tb3+ concentration.%采用高温固相法合成了Na3Gd1-xTbxSi2O7(x=0.01,0.02,0.04,0.06,0.08,0.1)系列荧光粉.研究了荧光粉的真空紫外-可见发光光谱和荧光寿命,讨论了Tb3+在扭曲八面体结构(标示为Gd(1)3+)和正三棱柱构型(标示为Gd(2)3+)两种格位中的最低5d轨道能级.同时研究了Gd3+→Tb3+之间无辐射能量传递速率K和无辐射能量传递效率η.研究结果表明:Tb3+在Gd(1)3+格位中的最低允许跃迁和禁戒跃迁的5d轨道能级分别位于235 nm和280 nm,在Gd(2)3+格位中的最低允许跃迁和禁戒跃迁的5d轨道能级分别位于224nm和256 nm.随着Tb3+浓度的增加,能量传递效率及速率显著增大,说明在Na3Gd1-xTbxSi2O7中存在有效的Gd3+-Tb3+能量传递.

  14. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

    Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

  15. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    Science.gov (United States)

    Krijt, Jakub; Kopecká, Jana; Hnízda, Aleš; Moat, Stuart; Kluijtmans, Leo A J; Mayne, Philip; Kožich, Viktor

    2011-02-01

    Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

  16. Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression.

    Directory of Open Access Journals (Sweden)

    Alessio Ligabue

    Full Text Available BACKGROUND: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS. Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G(1 phase and hTS is localized in the nuclei during S and G(2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. CONCLUSIONS/SIGNIFICANCE: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5

  17. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiming [Geneva Univ. Medical School (Switzerland); Morris, M.A.; Rossier, C. [Cantonal Hospital, Geneva (Switzerland)] [and others

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  18. Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro.

    Science.gov (United States)

    Cioccoloni, Giorgia; Bonmassar, Laura; Pagani, Elena; Caporali, Simona; Fuggetta, Maria Pia; Bonmassar, Enzo; D'Atri, Stefania; Aquino, Angelo

    2015-08-01

    Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis.

  19. Potent inhibition of human 5-lipoxygenase and microsomal prostaglandin E₂ synthase-1 by the anti-carcinogenic and anti-inflammatory agent embelin.

    Science.gov (United States)

    Schaible, Anja M; Traber, Heidi; Temml, Veronika; Noha, Stefan M; Filosa, Rosanna; Peduto, Antonella; Weinigel, Christina; Barz, Dagmar; Schuster, Daniela; Werz, Oliver

    2013-08-15

    Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) possesses anti-inflammatory and anti-carcinogenic properties in vivo, and these features have been related to interference with multiple targets including XIAPs, NFκB, STAT-3, Akt and mTOR. However, interference with these proteins requires relatively high concentrations of embelin (IC₅₀>4 μM) and cannot fully explain its bioactivity observed in several functional studies. Here we reveal human 5-lipoxygenase (5-LO) and microsomal prostaglandin E₂ synthase (mPGES)-1 as direct molecular targets of embelin. Thus, embelin potently suppressed the biosynthesis of eicosanoids by selective inhibition of 5-LO and mPGES-1 with IC₅₀=0.06 and 0.2 μM, respectively. In intact human polymorphonuclear leukocytes and monocytes, embelin consistently blocked the biosynthesis of various 5-LO products regardless of the stimulus (fMLP or A23187) with IC₅₀=0.8-2 μM. Neither the related human 12- and 15-LO nor the cyclooxygenases-1 and -2 or cytosolic phospholipase A₂ were significantly affected by 10 μM embelin. Inhibition of 5-LO and mPGES-1 by embelin was (I) essentially reversible after wash-out, (II) not impaired at higher substrate concentrations, (III) unaffected by inclusion of Triton X-100, and (IV) did not correlate to its proposed antioxidant properties. Docking simulations suggest concrete binding poses in the active sites of both 5-LO and mPGES-1. Because 5-LO- and mPGES-1-derived eicosanoids play roles in inflammation and cancer, the interference of embelin with these enzymes may contribute to its biological effects and suggests embelin as novel chemotype for development of dual 5-LO/mPGES-1 inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Age hardening characteristics and mechanical properties of Mg-3.5Dy-4.0Gd-3.1Nd-0.4Zr alloy

    Institute of Scientific and Technical Information of China (English)

    LI De-hui; DONG Jie; ZENG Xiao-qin; LU Chen; DING Wen-jiang

    2006-01-01

    Age hardening characteristic and tensile property of Mg-3.5Dy-4.0Gd-3.1Nd-0.4Zr alloy were investigated. The alloy exhibits a considerable age hardening effect up to 250 ℃. Increasing the aging temperature leads to a shorter aging time to reach the peak hardness and a lower peak hardness. The tensile results show that the peak-aged specimens have higher tensile strength at the temperature below 200℃. However, with the increase of temperature further, the tensile strength decreases dramatically and elongation increases drastically. The loss in tensile strength and increase in elongation at high temperature are possibly associated with the instability of secondary precipitates. The fracture mechanism of alloy transfers from intergranular to transgranular with the increase of experimental temperature.

  1. High-efficiency Watt-level picosecond pulse generation based on Yb:Gd3AlxGa5-xO12 crystal

    Science.gov (United States)

    Li, Yanbin; Jia, Zhitai; Lou, Fei; Zhang, Baitao; He, Jingliang; Tao, Xutang

    2015-07-01

    A diode-pumped passively mode-locked Yb3+:Gd3Al0.5Ga4.5O12 (Yb:GAGG) laser has been investigated by using a semiconductor saturable absorber mirror (SESAM) for the first time. Pulses with duration of 1.6 ps were produced at the central wavelength of 1027.8 nm. At the absorbed pump power of 8.4 W, the maximum average output power of 1.02 W was obtained with the repetition rate of 45 MHz. The corresponding single pulse energy and the peak power were calculated to be 22.7 nJ and 14.2 kW, respectively.

  2. Luminescence rise time in self-activated PbWO4 and Ce-doped Gd3Al2Ga3O12 scintillation crystals

    CERN Document Server

    E. Auffray; A. Borisevich; V. Gulbinas; A. Fedorov; M. Korjik; M.T. Lucchini; V. Mechinsky; S. Nargelas; E. Songaila; G. Tamulaitis; A. Vaitkevičius; S. Zazubovich

    2016-01-01

    The time resolution of scintillation detectors of ionizing radiation is one of the key parameters sought for in the current and future high-energy physics experiments. This study is encouraged by the necessity to find novel detection methods enabling a sub-10-ps time resolution in scintillation detectors and is focused on the exploitation of fast luminescence rise front. Time-resolved photoluminescence (PL) spectroscopy and thermally stimulated luminescence techniques have been used to study two promising scintillators: self-activated lead tungstate (PWO, PbWO4) and Ce-doped gadolinium aluminum gallium garnet (GAGG, Gd3Al2Ga3O12). A sub-picosecond PL rise time is observed in PWO, while longer processes in the PL response in GAGG:Ce are detected and studied. The mechanisms responsible for the PL rise time in self-activated and doped scintillators are under discussion.

  3. Human cystathionine β-synthase (CBS) contains two classes of binding sites for S-adenosylmethionine (SAM): complex regulation of CBS activity and stability by SAM.

    Science.gov (United States)

    Pey, Angel L; Majtan, Tomas; Sanchez-Ruiz, Jose M; Kraus, Jan P

    2013-01-01

    CBS (cystathionine β-synthase) is a multidomain tetrameric enzyme essential in the regulation of homocysteine metabolism, whose activity is enhanced by the allosteric regulator SAM (S-adenosylmethionine). Missense mutations in CBS are the major cause of inherited HCU (homocystinuria). In the present study we apply a novel approach based on a combination of calorimetric methods, functional assays and kinetic modelling to provide structural and energetic insight into the effects of SAM on the stability and activity of WT (wild-type) CBS and seven HCU-causing mutants. We found two sets of SAM-binding sites in the C-terminal regulatory domain with different structural and energetic features: a high affinity set of two sites, probably involved in kinetic stabilization of the regulatory domain, and a low affinity set of four sites, which are involved in the enzyme activation. We show that the regulatory domain displays a low kinetic stability in WT CBS, which is further decreased in many HCU-causing mutants. We propose that the SAM-induced stabilization may play a key role in modulating steady-state levels of WT and mutant CBS in vivo. Our strategy may be valuable for understanding ligand effects on proteins with a complex architecture and their role in human genetic diseases and for the development of novel pharmacological strategies.

  4. Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, Lydia M.; Celeste, Lesa R.; Lovelace, Leslie L.; Lebioda, Lukasz (SC)

    2012-02-21

    Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.

  5. Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis.

    Science.gov (United States)

    Assrir, Nadine; Richez, Celine; Durand, Philippe; Guittet, Eric; Badet, Bernard; Lescop, Ewen; Badet-Denisot, Marie-Ange

    2014-02-01

    The enzyme glucosamine-6P Synthase (Gfat, L-glutamine:D-fructose-6P amidotransferase) is involved in the hexosamine biosynthetic pathway and catalyzes the formation of glucosamine-6P from the substrates d-fructose-6-phosphate and l-glutamine. In eukaryotic cells, Gfat is inhibited by UDPGlcNAc, the end product of the biochemical pathway. In this work we present the dissection of the binding and inhibition properties of this feedback inhibitor and of its fragments by a combination of STD-NMR experiments and inhibition measurements on the wild type human enzyme (hGfat) as well as on site-directed mutants. We demonstrate that the UDPGlcNAc binding site is located in the isomerase domain of hGfat. Two amino acid residues (G445 and G461) located at the bottom of the binding site are identified to play a key role in the specificity of UDPGlcNAc inhibition of hGfat activity vs its bacterial Escherichia coli counterpart. We also show that UDPGlcNAc subcomponents have distinct features: the nucleotidic moiety is entirely responsible for binding whereas the N-acetyl group is mandatory for inhibition but not for binding, and the sugar moiety acts as a linker between the nucleotidic and N-acetyl groups. Combining these structural recognition determinants therefore appears as a promising strategy to selectively inhibit hGfat, which may for example help reduce complications in diabetes.

  6. Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding.

    Science.gov (United States)

    Gibson, Lydia M; Celeste, Lesa R; Lovelace, Leslie L; Lebioda, Lukasz

    2011-01-01

    Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.

  7. Evidence for a spatial and temporal regulation of prostaglandin-endoperoxide synthase 2 expression in human amnion in term and preterm parturition.

    Science.gov (United States)

    Lee, Deug-Chan; Romero, Roberto; Kim, Jung-Sun; Yoo, Wonsuk; Lee, JoonHo; Mittal, Pooja; Kusanovic, Juan Pedro; Hassan, Sonia S; Yoon, Bo Hyun; Kim, Chong Jai

    2010-09-01

    Prostaglandin-endoperoxide synthase 2 (PTGS2) is a key enzyme involved in parturition. PTGS2 mRNA was found to be differentially expressed between placental amnion (amnion overlying the placental disc) and reflected amnion (amnion of the extraplacental chorioamniotic membranes) in term placentas. The aim was to evaluate the spatial and temporal regulation of PTGS2 expression in the amnion and the chorion-decidua. PTGS2 expression was analyzed in the amnion and chorion-decidua obtained from 32 women: term not in labor (n = 12), term in labor (n = 12), and preterm labor (n = 8), by immunoblotting and densitometry. Prostaglandin E(2) (PGE(2)) in the amnion and chorion-decidua was measured by a specific immunoassay. Compared to preterm labor cases, PTGS2 expression increased at term before the onset of labor far more prominently in placental amnion (4.5-fold; P = 0.002) than in reflected amnion (1.4-fold; P = 0.007). There was a significant increase in PTGS2 expression in reflected amnion (2.9-fold; P human parturition.

  8. Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization.

    Science.gov (United States)

    Boppana, Nithin B; Kodiha, Mohamed; Stochaj, Ursula; Lin, Ho-sheng; Haimovitz-Friedman, Adriana; Bielawska, Alicja; Bielawski, Jacek; Divine, George W; Boyd, John A; Korbelik, Mladen; Separovic, Duska

    2014-11-01

    The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.

  9. The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein.

    Science.gov (United States)

    Pey, Angel L; Majtan, Tomas; Kraus, Jan P

    2014-09-01

    Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.

  10. Complexation of N'-[1-(3-aminophenyl)ethylidine]isonicotinohydrazide with La3+, Pr3+, Nd3+, Sm3+, Eu3+ and Gd3+ ions and associated thermodynamics

    Institute of Scientific and Technical Information of China (English)

    Sharmeli Yumnam; Lonibala Rajkumari

    2012-01-01

    A novel Schiff base N'-[1-(3-aminophenyl)ethylidine]isonicotinohydrazide was prepared and its complexation behavior towards some selected lanthanides had been studied employing pH-metric and calorimetric titration and spectral techniques.pH-metric studies were carried out for the trivalent La,Pr,Nd,Sr,Eu,and Gd complexes in 30% aqueous-dioxane medium at constant ionic strength of 0.05 mol/L NaClO4 and at different temperatures of 293,303 and 313 K.The proton-ligand formation constants of the ligand indicated the presence of only one dissociable proton while the metal-ligand formation constants were compatible with the formation of 1∶1 Ln(Ⅲ) complexes.The stability of the complexes followed the order:La3+<Pr3+<Nd3+<Sm3+<Eu3+>Gd3+,showing a break at gadolirium.The thermodynamic parameters,△G,△H and AS associated with protonation and complexation reactions were negative which suggested that all reactions were exothermic and enthalpy-driven.Isothermal calorimetric studies of Gd3+-aeINH systems at 303 K also showed exothermic nature of the complexation reaction and formation of 1∶1 complex in agreement with the pH-metric data.Formation of 1∶1 complexes was confirmed by the characterization of Nd(Ⅲ) complex.A seven coordinated geometry was assigned for the complex based on its elemental and spectral data.

  11. Effect of heat treatment on corrosion behavior of low pressure sand cast Mg-10Gd-3Y-0.5Zr alloys

    Institute of Scientific and Technical Information of China (English)

    Qian-qian Wu; Guang-ling Wei; Guo-hua Wu; Wen-cai Liu; Tian-peng Xuan; Wen-jiang Ding

    2016-01-01

    The corrosion behaviors of low-pressure sand cast Mg-10Gd-3Y-0.5Zr (wt.%) aloys in as-cast, solution treated (T4) and aged (T6) conditions were studied by means of immersion test and electrochemical measurements in 5wt.% NaCl solution saturated with Mg(OH)2. It was observed that the corrosion rate in the T4 condition was lower than that of the as-cast and T6 conditions by both sand casting and permanent mold casting with the same order of as-cast>T6>T4; while the corrosion resistance of the permanent mold casting is superior to the sand casting. The morphologies of the corrosion products are similar porous structures consisting of tiny erect lfakes perpendicular to the corroded surface of the aloy, irrespective of the heat treatment conditions. Especialy, the corrosion iflm in T4 condition is more compact than that in the other two conditions. In addition, the severer corrosion happening to the as-cast condition is correlated with the galvanic corrosion between the matrix and the eutectic compounds; while improved corrosion resistance for the T4 and T6 conditions is ascribed to the dissolution of the secondary eutectic compounds. The measured corrosion current densities of Mg-10Gd-3Y-0.5Zr aloys in as-cast, T4, and T6 conditions are 36 μA·cm-2, 10 μA·cm-2, and 33 μA·cm-2, respectively. The proposed equivalent circuit [Rs(CPE1(Rt(RfCPE2))] by Zview software matches wel with the tested electrochemical impedance spectra (EIS) data.

  12. Effect of heat treatment on corrosion behavior of low pressure sand cast Mg-10Gd-3Y-0.5Zr alloys

    Directory of Open Access Journals (Sweden)

    Qian-qian Wu

    2016-07-01

    Full Text Available The corrosion behaviors of low-pressure sand cast Mg-10Gd-3Y-0.5Zr (wt.% alloys in as-cast, solution treated (T4 and aged (T6 conditions were studied by means of immersion test and electrochemical measurements in 5wt.% NaCl solution saturated with Mg(OH2. It was observed that the corrosion rate in the T4 condition was lower than that of the as-cast and T6 conditions by both sand casting and permanent mold casting with the same order of as-cast>T6>T4; while the corrosion resistance of the permanent mold casting is superior to the sand casting. The morphologies of the corrosion products are similar porous structures consisting of tiny erect flakes perpendicular to the corroded surface of the alloy, irrespective of the heat treatment conditions. Especially, the corrosion film in T4 condition is more compact than that in the other two conditions. In addition, the severer corrosion happening to the as-cast condition is correlated with the galvanic corrosion between the matrix and the eutectic compounds; while improved corrosion resistance for the T4 and T6 conditions is ascribed to the dissolution of the secondary eutectic compounds. The measured corrosion current densities of Mg-10Gd-3Y-0.5Zr alloys in as-cast, T4, and T6 conditions are 36 μA·cm-2, 10 μA·cm-2, and 33 μA·cm-2, respectively. The proposed equivalent circuit [Rs(CPE1(Rt(RfCPE2] by Zview software matches well with the tested electrochemical impedance spectra (EIS data.

  13. Synthesis and luminescent properties of Eu3+, Eu3+/Bi3+ and Gd3+ codoped YAG:Ce3+ phosphors and their potential applications in warm white light-emitting diodes

    Science.gov (United States)

    Yang, Yuguo; Li, Jing; Liu, Bing; Zhang, Yuanyuan; Lv, Xianshun; Wei, Lei; Wang, Xuping; Xu, Jianhua; Yu, Huajian; Hu, Yanyan; Zhang, Huadi; Ma, Ling; Wang, Jiyang

    2017-10-01

    A series of YAG:Ce3+/Eu3+, YAG:Ce3+/Eu3+/Bi3+ and YAG:Ce3+/Gd3+ phosphors were synthesized by a co-precipitation method. The results suggest that all of phosphors have the cubic phase and nearly spherical morphology. However, the red emission can be produced by codoping Eu3+ and Eu3+/Bi3+ ions in YAG:Ce3+, and the codoped Gd3+ ions can induce the red-shift of Ce3+ emission. These results suggest that the Eu3+, Eu3+/Bi3+ and Gd3+ ions can be used to decrease the correlated color temperature and increase the color-rendering index of white light-emitting diodes based on InGaN blue chip and YAG:Ce3+ phosphors.

  14. Effect of Gd3+ doping on phase structure,magnetic and electrical properties of double perovskite Sr2FeMoO6%Gd3+掺杂对Sr2FeMoO6结构及电磁性能的影响

    Institute of Scientific and Technical Information of China (English)

    翟永清; 乔静; 张张; 霍国燕; 温静娟

    2011-01-01

    采用微波固相烧结法合成了双钙钛矿Sr2-xGdxFeMoO6(x=0.0、0.1、0.2、0.3)。用XRD和VSM对样品的物相结构和电磁性能进行了研究。结果表明所得Sr2FeMoO6为四方晶系结构,空间群为I4/mmm,随Gd3+掺杂量的增加,Fe、Mo排列的有序度逐渐降低,同时伴有少量Gd2O3杂相生成。样品Sr2-xGdxFeMoO6均表现为铁磁性,磁转变温度均高于室温,Gd3+掺杂使得样品的室温饱和磁化强度降低,但适量Gd3+的掺杂(x=0.3)可明显提高样品的室温磁电阻变化率。样品Sr2-xGdxFeMoO6均呈现典型的半导体行为,当x=0.0、0.1、0.2时,在100~300K,其电输运行为服从小极化子变程跃迁导电机制;当x=0.3时,在150~300K,服从小极化子变程跃迁导电机制;在100~150K,则属于绝热小极化子导电机制。%Double perovskite Sr2-xGdxFeMoO6(x=0.0,0.1,0.2,0.3) were synthesized by microwave sintering method.The phase structure,magnetic and electrical properties of all the samples were investigated by X-ray powder diffraction(XRD) and vibrating-sample magnetometer(VSM) respectively.The results show that the as-synthesized sample is Sr2FeMoO6 with tetragonal crystal structure and I4/mmm space group.With the increase of doped Gd3+,the ordering degree of the Fe and Mo cations decreases gradually,accompanied by a small amount of impurity phase Gd2O3.The samples Sr2-xGdxFeMoO6 are ferromagnetic,and the magnetic transition temperature is above room temperature.The saturation magnetization is decreased with the doping of Gd3+ at room temperature,but magnetoresistance ratio can be improved significantly when the doping concernt of Gd3+ x=0.3.The samples Sr2-xGdxFeMoO6 exhibit typical semiconductor behavior.When x=0.0,0.1,0.2,the electrical transport behavior follows the small polaron variable-range transition model in the range of 100-300K.When x=0.3,it follows the small polaron variable-range transition model in the range of 150-300K;but it is

  15. An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS and human dihydrofolate reductase (hDHFR. These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs.

  16. Structure of the Varicella Zoster Virus Thymidylate Synthase Establishes Functional and Structural Similarities as the Human Enzyme and Potentiates Itself as a Target of Brivudine.

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    Kelly Hew

    Full Text Available Varicella zoster virus (VZV is a highly infectious human herpesvirus that is the causative agent for chicken pox and shingles. VZV encodes a functional thymidylate synthase (TS, which is the sole enzyme that produces dTMP from dUMP de novo. To study substrate binding, the complex structure of TSVZV with dUMP was determined to a resolution of 2.9 Å. In the absence of a folate co-substrate, dUMP binds in the conserved TS active site and is coordinated similarly as in the human encoded TS (TSHS in an open conformation. The interactions between TSVZV with dUMP and a cofactor analog, raltitrexed, were also studied using differential scanning fluorimetry (DSF, suggesting that TSVZV binds dUMP and raltitrexed in a sequential binding mode like other TS. The DSF also revealed interactions between TSVZV and in vitro phosphorylated brivudine (BVDUP, a highly potent anti-herpesvirus drug against VZV infections. The binding of BVDUP to TSVZV was further confirmed by the complex structure of TSVZV and BVDUP solved at a resolution of 2.9 Å. BVDUP binds similarly as dUMP in the TSHS but it induces a closed conformation of the active site. The structure supports that the 5-bromovinyl substituent on BVDUP is likely to inhibit TSVZV by preventing the transfer of a methylene group from its cofactor and the subsequent formation of dTMP. The interactions between TSVZV and BVDUP are consistent with that TSVZV is indeed a target of brivudine in vivo. The work also provided the structural basis for rational design of more specific TSVZV inhibitors.

  17. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

    Directory of Open Access Journals (Sweden)

    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  18. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, Peter S., E-mail: Peter.Gilmour@astrazeneca.com [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); O' Shea, Patrick J.; Fagura, Malbinder [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Pilling, James E. [Discovery Sciences, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Sanganee, Hitesh [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Wada, Hiroki [R and I IMed, AstraZeneca R and D, Molndal (Sweden); Courtney, Paul F. [DMPK, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Kavanagh, Stefan; Hall, Peter A. [Safety Assessment, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Escott, K. Jane [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom)

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  19. Expression of the prostaglandin F synthase AKR1B1 and the prostaglandin transporter SLCO2A1 in human fetal membranes in relation to spontaneous term and preterm labour

    Directory of Open Access Journals (Sweden)

    Hana A Alzamil

    2014-07-01

    Full Text Available Background: Human labour is a complex series of cellular and molecular events that occur at the materno-fetal and uterine levels. Many hypotheses have been proposed for the initiation of human labour, one hypothesis suggests that maturation of the fetus releases a signal in the amniotic fluid that will be transmitted to myometrium via the fetal membranes and initiate uterine contractions. There is strong evidence that prostaglandins (PGs play a central role in initiation and progression of human labour. Objectives: In this study we intended to investigate the expression of prostaglandin F synthase and the prostaglandin transporter in the human fetal membranes and to explore the relationship between cytokines and PGs in the mechanism of human labour. Methods: We used fetal membranes obtained before labour at term and after spontaneous labour at term or preterm to identify the changes in prostaglandin F synthase (AKR1B1 and human prostaglandin transporter (SLCO2A1 proteins in relation to parturition. Using fetal membranes explants we tested the effect of cytokines (interleukin-1 and tumour necrosis factor alpha on PG production and the concomitant changes in cyclooxygenase-2 (PTGS2, AKR1B1 and SLCO2A1 expression. Results: Expression of PTGS2 and AKR1B1 was upregulated in the fetal membranes in association with term labour while SLCO2A1 was downregulated with advancing gestation and during term labour. Before labour, IL-1 increased the expression of PTGS2, however during labour TNF upregulated PTGS2 and AKR1B1 proteins. Conclusions: The prostaglandin F synthase AKR1B1 is upregulated while prostaglandin transporter is downregulated during term labour. The amnion is more responsive than choriodecidua to stimulation with pro-inflammatory cytokines. The mechanisms of term and preterm labour are different.

  20. Expression of the prostaglandin F synthase AKR1B1 and the prostaglandin transporter SLCO2A1 in human fetal membranes in relation to spontaneous term and preterm labor.

    Science.gov (United States)

    Alzamil, Hana A; Pawade, Joya; Fortier, Michel A; Bernal, A López

    2014-01-01

    Human labor is a complex series of cellular and molecular events that occur at the materno-fetal and uterine levels. Many hypotheses have been proposed for the initiation of human labor, one hypothesis suggests that maturation of the fetus releases a signal in the amniotic fluid that will be transmitted to myometrium via the fetal membranes and initiate uterine contractions. There is strong evidence that prostaglandins (PGs) play a central role in initiation and progression of human labor. In this study we intended to investigate the expression of prostaglandin F synthase and the prostaglandin transporter in the human fetal membranes and to explore the relationship between cytokines and PGs in the mechanism of human labor. We used fetal membranes obtained before labor at term and after spontaneous labor at term or preterm to identify the changes in prostaglandin F synthase (AKR1B1) and human prostaglandin transporter (SLCO2A1) proteins in relation to parturition. Using fetal membranes explants we tested the effect of cytokines (interleukin-1 and tumor necrosis factor alpha) on PG production and the concomitant changes in cyclooxygenase-2 (PTGS2), AKR1B1 and SLCO2A1 expression. Expression of PTGS2 and AKR1B1 was upregulated in the fetal membranes in association with term labor while SLCO2A1 was downregulated with advancing gestation and during term labor. Before labor, IL-1 increased the expression of PTGS2, however during labor TNF upregulated PTGS2 and AKR1B1 proteins. The prostaglandin F synthase AKR1B1 is upregulated while prostaglandin transporter is downregulated during term labor. The amnion is more responsive than choriodecidua to stimulation with pro-inflammatory cytokines. The mechanisms of term and preterm labor are different.

  1. Nitric oxide production and inducible nitric oxide synthase protein expression in human abdominal aortic aneurysms and cultured aneurismal smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    LIAO Ming-fang; JING Zai-ping; BAO Jun-min; ZHAO Zhi-qing; MEI Zhi-jun; LU Qing-sheng; CUI Jia-sen; QU Le-feng; ZHANG Su-zhen

    2006-01-01

    Objective:To investigate the production of nitric oxide(NO) and the expression of inducible nitric oxide synthase (iNOS), and their possible role in abdominal aortic aneurysm (AAA). Methods: A total of 28 patients with AAA, 10 healthy controls, and 8 patients with arterial occlusive disease were enrolled into this study. Standard colorimetric assay was used to examine NO concentration in plasma from patients with AAA and normal controls, and in cultured smooth muscle cells (SMCs). Expression of iNOS in aortas and cultured SMCs were detected by immunochemistry. The correlation of iNOS expression with age of the patient, size of aneurysm, and degree of inflammation was also investigated by CochranMantel-Haenszel x2 test and Kendall correlation. Results: Expression of iNOS increased significantly in the wall of aneurism in the patients with AAA compared to the healthy controls (P<0.05) and the patients with occlusive arteries (P<0. 05). iNOS protein and media NOx (nitrite+nitrate) also increased in cultured SMCs from human AAA (n=4, P<0.05), while plasma NOx decreased in patients with AAA (n=25) compared to the healthy controls (n= 20). There was a positive correlation between iNOS protein and the degree of inflammation in aneurismal wall (Kendall coefficient = 0. 5032, P = 0. 0029). Conclusion:SMCs and inflammatory cells are main cellular sources of increased iNOS in AAA, and NO may play a part in pathogenesis in AAA through inflammation, SMCs and oxidative stress.

  2. Human Lipocalin-Type Prostaglandin D Synthase-Based Drug Delivery System for Poorly Water-Soluble Anti-Cancer Drug SN-38.

    Science.gov (United States)

    Nakatsuji, Masatoshi; Inoue, Haruka; Kohno, Masaki; Saito, Mayu; Tsuge, Syogo; Shimizu, Shota; Ishida, Atsuko; Ishibashi, Osamu; Inui, Takashi

    2015-01-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily, which is composed of secretory transporter proteins, and binds a wide variety of small hydrophobic molecules. Using this function, we have reported the feasibility of using L-PGDS as a novel drug delivery vehicle for poorly water-soluble drugs. In this study, we show the development of a drug delivery system using L-PGDS, one that enables the direct clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), a poorly water-soluble anti-cancer drug. In the presence of 2 mM L-PGDS, the concentration of SN-38 in PBS increased 1,130-fold as compared with that in PBS. Calorimetric experiments revealed that L-PGDS bound SN-38 at a molecular ratio of 1:3 with a dissociation constant value of 60 μM. The results of an in vitro growth inhibition assay revealed that the SN-38/L-PGDS complexes showed high anti-tumor activity against 3 human cancer cell lines, i.e., Colo201, MDA-MB-231, and PC-3 with a potency similar to that of SN-38 used alone. The intravenous administration of SN-38/L-PGDS complexes to mice bearing Colo201 tumors showed a pronounced anti-tumor effect. Intestinal mucositis, which is one of the side effects of this drug, was not observed in mice administered SN-38/L-PGDS complexes. Taken together, L-PGDS enables the direct usage of SN-38 with reduced side effects.

  3. Human Lipocalin-Type Prostaglandin D Synthase-Based Drug Delivery System for Poorly Water-Soluble Anti-Cancer Drug SN-38.

    Directory of Open Access Journals (Sweden)

    Masatoshi Nakatsuji

    Full Text Available Lipocalin-type prostaglandin D synthase (L-PGDS is a member of the lipocalin superfamily, which is composed of secretory transporter proteins, and binds a wide variety of small hydrophobic molecules. Using this function, we have reported the feasibility of using L-PGDS as a novel drug delivery vehicle for poorly water-soluble drugs. In this study, we show the development of a drug delivery system using L-PGDS, one that enables the direct clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38, a poorly water-soluble anti-cancer drug. In the presence of 2 mM L-PGDS, the concentration of SN-38 in PBS increased 1,130-fold as compared with that in PBS. Calorimetric experiments revealed that L-PGDS bound SN-38 at a molecular ratio of 1:3 with a dissociation constant value of 60 μM. The results of an in vitro growth inhibition assay revealed that the SN-38/L-PGDS complexes showed high anti-tumor activity against 3 human cancer cell lines, i.e., Colo201, MDA-MB-231, and PC-3 with a potency similar to that of SN-38 used alone. The intravenous administration of SN-38/L-PGDS complexes to mice bearing Colo201 tumors showed a pronounced anti-tumor effect. Intestinal mucositis, which is one of the side effects of this drug, was not observed in mice administered SN-38/L-PGDS complexes. Taken together, L-PGDS enables the direct usage of SN-38 with reduced side effects.

  4. Overexpression of fatty acid synthase in human gliomas correlates with the WHO tumor grade and inhibition with Orlistat reduces cell viability and triggers apoptosis.

    Science.gov (United States)

    Grube, Susanne; Dünisch, Pedro; Freitag, Diana; Klausnitzer, Maren; Sakr, Yasser; Walter, Jan; Kalff, Rolf; Ewald, Christian

    2014-06-01

    Fatty acid synthase (FASN), catalyzing the de novo synthesis of fatty acids, is known to be deregulated in several cancers. Inhibition of this enzyme reduces tumor cell proliferation. Unfortunately, adverse effects and chemical instability prevent the in vivo use of the best-known inhibitors, Cerulenin and C75. Orlistat, a drug used for obesity treatment, is also considered as a potential FASN inhibitor, but its impact on glioma cell biology has not yet been described. In this study, we analyzed FASN expression in human glioma samples and primary glioblastoma cell cultures and the effects of FASN inhibition with Orlistat, Cerulenin and C75. Immunohistochemistry followed by densitometric analysis of 20 glioma samples revealed overexpression of FASN that correlated with the WHO tumor grade. Treatment of glioblastoma cells with these inhibitors resulted in a significant, dose-dependent reduction in tumor cell viability and fatty acid synthesis. Compared to Cerulenin and C75, Orlistat was a more potent inhibitor in cell cultures and cell lines. In LN229, cell-growth was reduced by 63.9 ± 8.7 % after 48 h and 200 µM Orlistat compared to controls; in LT68, the reduction in cell growth was 76.3 ± 23.7 %. Nuclear fragmentation assay and Western blotting analysis after targeting FASN with Orlistat demonstrated autophagy and apoptosis. Organotypic slice cultures treated with Orlistat showed reduced proliferation after Ki67 staining and increased caspase-3 cleavage. Our results suggest that FASN may be a therapeutic target in malignant gliomas and identify Orlistat as a possible anti-tumor drug in this setting.

  5. Melatonin attenuated adipogenesis through reduction of the CCAAT/enhancer binding protein beta by regulating the glycogen synthase 3 beta in human mesenchymal stem cells.

    Science.gov (United States)

    Rhee, Yun-Hee; Ahn, Jin-Chul

    2016-06-01

    Adipogenic differentiation is characterized by an increase in two major transcription factors: peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT/enhancer binding protein alpha (C/EBPα). These two signals are influenced by C/EBPβ and C/EBPδ and cross-regulate each other's expression during the initial stages of adipogenesis. Melatonin has been known to act as not only a direct scavenger of free radicals but also an inhibitor of glycogen synthase kinase 3β (GSK-3β). Here, we report that melatonin inhibits the adipogenic differentiation of human mesenchymal stem cells (hMSCs) which is due to the regulations of C/EBPβ in the early stage of adipogenic differentiation. Melatonin reduced the lipid accumulation, adiponectin, and lipoprotein lipase (LPL) during the adipogenic differentiation of hMSCs. Since C/EBPβ has been associated with the activation of PPARγ and the consensus site of ERK/GSK-3β, PPARγ and β-catenin were detected by immunofluorescence staining after pretreatment of melatonin. Melatonin blocked the activation of PPARγ which induced the degradation of β-catenin. Melatonin also decreased the levels of cyclic adenosine-3,5-monophosphate (cAMP) and reactive oxygen species (ROS). The cAMP triggered the activity of C/EBPβ which is a critical inducer of PPARγ and C/EBPα activation in the early stage of adipogenic differentiation, and this is further affected by ROS production. The adipogenic marker proteins such as PPARγ, C/EBPα, C/EBPβ, and pERK were also decreased by melatonin. In summary, melatonin inhibited the cAMP synthesis through ROS reduction and the phosphorylation of the ERK/GSK-3β site which is known to be responsible for C/EBPβ activation for adipogenic differentiation in hMSCs.

  6. Effect of Interleukin-10 and Laminar Shear Stress on Endothelial Nitric Oxide Synthase and Nitric Oxide in African American Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Babbitt, Dianne M; Kim, Ji-Seok; Forrester, Steven J; Brown, Michael D; Park, Joon-Young

    2015-11-05

    African Americans have a predisposition to heightened systemic inflammation and a high prevalence of hypertension. The purpose of this study was to evaluate the influence of interleukin-10 (IL-10) and laminar shear stress (LSS) on African American endothelial cells by measuring total endothelial nitric oxide synthase (eNOS) protein expression and its phosphorylated form (p-eNOS) at Serine 1177, and nitric oxide (NO) levels, in response to IL-10 incubation and high physiological levels of LSS, used as an in vitro mimetic for aerobic exercise training (AEXT). Human umbilical vein endothelial cells (HUVEC) from an African American donor were cultured. The experimental conditions included Static, Static with IL-10 Incubation, LSS at 20 dynes/cm², and LSS at 20 dynes/cm² with IL-10 Incubation. Western blotting was used to measure eNOS and p-eNOS protein expression in the cells. A modified Griess assay was used to measure NO metabolites in the cell culture media. There were significant increases in p-eNOS, eNOS, and NO in the LSS at 20 dynes/cm² and LSS at 20 dynes/cm² with IL-10 Incubation experimental conditions when compared to the Static experimental condition. There were no other statistically significant differences demonstrating that IL-10 did not have an additive effect on eNOS activity in our study. The significant increases in p-eNOS, eNOS, and NO as a result of LSS in African American HUVECs suggest that AEXT may be a viable, nonpharmacologic method to improve vascular inflammation status and vasodilation, and thereby contribute to hypertension reduction in the African American population.

  7. Development, characterization, and epitope mapping of a panel of twenty-four monoclonal antibodies specific for human inducible nitric oxide synthase.

    Science.gov (United States)

    Webber, Robert J; Rodriguez, John G; Webber, Douglas S; Dunnebacke, Thelma H

    2005-02-01

    A panel of monoclonal antibodies (MAbs) to human inducible nitric oxide synthase (hiNOS) has been developed. By isotype analysis of the MAbs cloned from the 24 different positive hybridomas, 13 were determined to be mouse IgG1, two were mouse IgG2a, two were mouse IgG2b, and the seven others were mouse IgM antibodies: all contained kappa light chains. The anti-hiNOS MAbs were initially characterized by ELISA, RIA, Western blot, and immunocytochemistry, and then they were epitope mapped using synthetic peptides and a three-step mapping procedure. In the first step, each of the 24 MAbs was tested by indirect ELISA for binding to 96 overlapping 18-amino acid-long peptides that span the entire 1153-amino acid length of hiNOS. Eight IgG class anti-hiNOS MAbs were found to bind to one of five different peptides. In the second step, a series of amino terminal and carboxyl terminal truncated peptides were synthesized for each of the five peptides to which one or more of the MAbs bound. Each of the eight anti-hiNOS MAbs was found to bind to the truncated peptides with a unique specificity that identified the amino acid segment involved in binding. The third step in the epitope mapping process utilized three series of overlapping 5-, 6-, 7-, 8-, and 9-amino acid-long peptides for each of these segments and identified the exact amino acids of hiNOS involved in antibody binding. Anti-hiNOS MAbs 2A1-F8, 2D2-B2, 21C10-1D10, and 24B10-2C7 were found to be especially useful in different immunoassays.

  8. Bacterial over-expression and purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) reductase domain of human FAD synthase: functional characterization and homology modeling.

    Science.gov (United States)

    Miccolis, Angelica; Galluccio, Michele; Giancaspero, Teresa Anna; Indiveri, Cesare; Barile, Maria

    2012-12-11

    FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3'phosphoadenosine 5'phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble "truncated" form of hFADS lacking the N-terminal domain (Δ(1-328)-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ(1-328)-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ(1-328)-hFADS enzyme shows a slight, but not significant, change of K(m) values (0.24 and 6.23 µM for FMN and ATP, respectively) and of k(cat) (4.2 × 10-2 s-1) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ(1-328)-hFADS versus wild-type protein.

  9. High-resolution structures of mutants of residues that affect access to the ligand-binding cavity of human lipocalin-type prostaglandin D synthase.

    Science.gov (United States)

    Perduca, Massimiliano; Bovi, Michele; Bertinelli, Mattia; Bertini, Edoardo; Destefanis, Laura; Carrizo, Maria E; Capaldi, Stefano; Monaco, Hugo L

    2014-08-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2 (prostaglandin H2) to produce PGD2 (prostaglandin D2) with 9-hydroxy and 11-keto groups. The product of the reaction, PGD2, is the precursor of several metabolites involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as β-trace protein, the second most abundant protein in human cerebrospinal fluid. Previous structural work on the mouse and human proteins has focused on the identification of the amino acids responsible and the proposal of a mechanism for catalysis. In this paper, the X-ray structures of the apo and holo forms (bound to PEG) of the C65A mutant of human L-PGDS at 1.40 Å resolution and of the double mutant C65A/K59A at 1.60 Å resolution are reported. The apo forms of the double mutants C65A/W54F and C65A/W112F and the triple mutant C65A/W54F/W112F have also been studied. Mutation of the lysine residue does not seem to affect the binding of PEG to the ligand-binding cavity, and mutation of a single or both tryptophans appears to have the same effect on the position of these two aromatic residues at the entrance to the cavity. A solvent molecule has also been identified in an invariant position in the cavity of virtually all of the molecules present in the nine asymmetric units of the crystals that have been examined. Taken together, these observations indicate that the residues that have been mutated indeed appear to play a role in the entrance-exit process of the substrate and/or other ligands into/out of the binding cavity of the lipocalin.

  10. The role of regulatory proteins and S-nitrosylation of endothelial nitric oxide synthase in the human clitoris: implications for female sexual function.

    Science.gov (United States)

    Oliver, Janine L; Kavoussi, Parviz K; Smith, Ryan P; Woodson, Robin I; Corbett, Sean T; Costabile, Raymond A; Palmer, Lisa A; Lysiak, Jeffrey J

    2014-08-01

    During female sexual arousal, clitoral blood flow is controlled by endothelial nitric oxide synthase (eNOS) and its product, nitric oxide (NO). The mechanisms regulating eNOS activity and NO bioavailability in the clitoris are largely unknown. To identify proteins involved in regulation of eNOS activity within the clitoris and to evaluate the effects of S-nitrosoglutathione reductase (GSNO-R) and eNOS nitrosylation/denitrosylation on clitoral blood flow. Immunohistochemistry for eNOS, caveolin-1 (Cav1), heat shock protein-90 (Hsp90), phosphodiesterase type 5 (PDE5), GSNO-R, and soluble guanylate cyclase (sGC) was performed on human and murine clitoral tissue. Western blot analysis was performed for eNOS, phosphorylated eNOS (phospho-eNOS, Ser1177), Cav1, Hsp90, sGC, PDE5, phosphoinositide 3-kinase (PI3K), Akt (protein kinase B), and GSNO-R on protein from human clitoral tissue. A biotin switch assay was used to analyze the S-nitrosylation of eNOS, nNOS, and GSNO-R. Clitoral blood flow was measured in wild-type and GSNO-R(-/-) mice at baseline and during cavernous nerve electrical stimulation (CNES). Localization of eNOS regulatory proteins and clitoral blood flow. eNOS and GSNO-R co-localized to the vascular endothelium and sinusoids of human clitoral tissue. Immunohistochemistry also localized Cav1 and Hsp90 to the endothelium and PDE5 and sGC to the trabecular smooth muscle. Expression of S-nitrosylated (SNO)-eNOS and SNO-GSNO-R was detected by biotin switch assays. Wild-type control mice exhibited increased clitoral blood flow with CNES whereas GSNO-R(-/-) animals failed to show an increase in blood flow. Several key eNOS regulatory proteins are present in the clitoral tissue in a cellular specific pattern. S-nitrosylation of eNOS may also represent a key regulatory mechanism governing eNOS activation/deactivation since mice deficient in GSNO-R failed to increase clitoral blood flow. Additional studies are necessary to define the role of S-nitrosylation in the

  11. Functions of chondroitin sulfate/dermatan sulfate chains in brain development. Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7.

    NARCIS (Netherlands)

    Purushothaman, A.; Fukuda, J.; Mizumoto, S.; Dam, G.B. ten; Kuppevelt, A.H.M.S.M. van; Kitagawa, H.; Mikami, T.; Sugahara, K.

    2007-01-01

    Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the s

  12. Effect of Y3+, Gd 3+and La3+dopant ions on structural, optical and electrical properties of o-mullite nanoparticles

    Institute of Scientific and Technical Information of China (English)

    MMS Sanad; MM Rashad; EA Abdel-Aal; MF El-Shahat; K Powers

    2014-01-01

    Dielectric ceramics of M(x)Al6(1-x)Si2O13 doped mullite were synthesized via co-precipitation technique. The X-ray diffrac-tion profiles revealed that these nanoparticles were crystallized well and the volume of mullite unit cell was increased as a function of the ionic radius of dopant ion. TEM images showed regular orthorhombic crystal morphology for the pure mullite sample. Meanwhile, the doped samples exhibited slightly distorted crystal morphology of larger particle sizes. DSC thermograms evinced that the exo-thermic peak temperature of mullite was shifted to the lower value with M3+ion insertion. The photoluminescence spectra were stud-ied for mullite samples, and it was found that the intensity of the emission spectra was affected by the M3+ion type. It was found that, Y3+doped mullite achieved the minimum dielectric loss value of 0.01 in the radio wave frequency region (1 MHz). Meanwhile, Gd3+doped mullite achieved the minimum dielectric loss value of 0.09 in the microwave frequency region (1 GHz).

  13. Effect of Mg co-doping on scintillation properties of Ce:Gd3(Ga, Al)5O12 single crystals with various Ga/Al ratios

    Science.gov (United States)

    Yoshino, Masao; Kamada, Kei; Shoji, Yasuhiro; Yamaji, Akihiro; Kurosawa, Shunsuke; Yokota, Yuui; Ohashi, Yuji; Yoshikawa, Akira; Chani, Valery I.

    2017-06-01

    Mg co-doped Ce1%:Gd3GaxAl5-xO12 (Ce:GAGG) crystals (x=2.4, 2.7, 3.0) were successfully grown by the Czochralski (Cz) method. Effect of Mg co-doping on the scintillation properties of Ce:GAGG was examined. This study covers measurements of solidification fraction and scintillation properties such as light yield, energy resolution and non-proportionality for each crystal. Pulse-height spectra of various gamma and X-ray sources with energies ranged from 30 keV to 662 keV were measured. Regardless of the presence/absence of Mg dopant, the non-proportionality curves with lower content of gallium in the crystal structure tend to improve. Mg co-doped Ce:GAGG samples did not show a significant difference as compared with non co-doped Ce:GAGG. Mg co-doped crystals with x=2.4 and 2.7 showed the promising scintillation properties of faster decay time and higher energy resolution than those with x=3.0.

  14. 2 inch size Czochralski growth and scintillation properties of Li+ co-doped Ce:Gd3Ga3Al2O12

    Science.gov (United States)

    Kamada, Kei; Shoji, Yasuhiro; Kochurikhin, Vladimir V.; Yoshino, Masao; Okumura, Satoshi; Yamamoto, Seiichi; Yeom, Jung Yeol; Kurosawa, Shunsuke; Yokota, Yuui; Ohashi, Yuji; Nikl, Martin; Yoshino, Masao; Yoshikawa, Akira

    2017-03-01

    The 2 inch size Li 0.15 and 1.35 mol% co-doped Ce:Gd3Al2Ga3O12 single crystals were prepared by the Czochralski (Cz) method. Absorption and luminescence spectra were measured together with several other scintillation characteristics, namely the scintillation decay and light yield to reveal the effect of Li co-doping. Ce4+ CT absorption below 350 nm is clearly enhanced by Li co-doping as same as divalent ions co-doping. By 1.35 at.% Li co-doping, light yield was decrease to 88% of the Ce: GAGG standard and decay time was accelerated to 34.3ns 21.0%, 84.6ns 68.7%, 480ns 10.3%. The timing resolution measurement for a pair of 3 × 3 × 3mm3 size Li,Ce:GAGG scintillator crystals was performed using Si-PMs and the timing resolution of the 1.35 at.% Li co-doped Ce:GAGG was 218ps.

  15. Effects of heat treatment on microstructure and mechanical properties of Mg-8Gd-3Y-0.5Zr (wt.% alloy fabricated by semi-continuous casting

    Directory of Open Access Journals (Sweden)

    Jun Yang

    2015-07-01

    Full Text Available The microstucture, mechanical properties and fracture behaviors of semi-continuous cast Mg-8Gd-3Y-0.5Zr (wt.%, GW83K alloy after different heat treatments were investigated. Almost all the eutectic compounds were dissolved into the matrix and there was no evident grain growth after optimum solution treatment at 500 °C for 4 h. Further ageing at low temperatures led to significant precipitation hardening, which strengthened the alloy. Peak-aged at 200 °C, the alloy had the highest ultimate tensile strength (UTS and lowest elongation at 395 MPa and 2.8%, respectively. When aged at 225 °C for 15 h, the alloy exhibited prominent mechanical performance with UTS and elongation of 363 MPa and 5.8 %, respectively. With regard to microstructure and tensile properties, the processes of 500 °C, 4 h + 225 °C, 15 h are selected as the optimal heat treatment conditions. The alloy under different conditions shows different fracture behaviors: in the as-cast alloy, a quasi-cleavage pattern is observed; after solution treatment, the alloy exhibits a trans-granular quasi-cleavage fracture; after being peak-aged at 200 °C and 225 °C, the fracture mode is a mixed mode of trans-granular and inter-granular fracture, in which the inter-granular mode is dominant in the alloy peak-aged at 200 °C.

  16. Real-time tracking of dissociation of hyperpolarized 89Y-DTPA: a model for degradation of open-chain Gd3+ MRI contrast agents

    Science.gov (United States)

    Ferguson, Sarah; Niedbalski, Peter; Parish, Christopher; Kiswandhi, Andhika; Kovacs, Zoltan; Lumata, Lloyd

    Gadolinium (Gd) complexes are widely used relaxation-based clinical contrast agents in magnetic resonance imaging (MRI). Gd-based MRI contrast agents with open-chain ligand such as Gd-DTPA, commercially known as magnevist, are less stable compared to Gd complexes with macrocyclic ligands such as GdDOTA (Dotarem). The dissociation of Gd-DPTA into Gd ion and DTPA ligand under certain biological conditions such as high zinc levels can potentially cause kidney damage. Since Gd is paramagnetic, direct NMR detection of the Gd-DTPA dissociation is quite challenging due to ultra-short relaxation times. In this work, we have investigated Y-DTPA as a model for Gd-DPTA dissociation under high zinc content solutions. Using dissolution dynamic nuclear polarization (DNP), the 89Y NMR signal is amplified by several thousand-fold. Due to the the relatively long T1 relaxation time of 89Y which translates to hyperpolarization lifetime of several minutes, the dissociation of Y-DTPA can be tracked in real-time by hyperpolarized 89Y NMR spectroscopy. Dissociation kinetic rates and implications on the degradation of open-chain Gd3+ MRI contrast agents will be discussed. This work was supported by the U.S. Department of Defense Award Number W81XWH-14-1-0048 and by the Robert A. Welch Foundation research Grant Number AT-1877.

  17. A Eu3+/Gd3+-EDTA-doped structurally controllable hollow mesoporous carbon for improving the oral bioavailability of insoluble drugs and in vivo tracing

    Science.gov (United States)

    Liu, Jia; Zhao, Yating; Cui, Yu; Yue, Yang; Gao, Yikun; Zhao, Qinfu; Liu, Jie; Wang, Siling

    2016-08-01

    A structurally controllable fluorescence-labeled hollow mesoporous carbon (HMC) was simply prepared to improve the oral bioavailability of insoluble drugs and further trace their delivery process in vivo. The hollow structure was derived from an inverse replica process using mesoporous silica as a template and the fluorescent label was prepared by doping the carboxylated HMC with a confinement of Eu3+/Gd3+-EDTA. The physicochemical properties of the composites were systematically characterized by transmission electron microscopy, Fourier transform infrared spectroscopy and photoluminescence spectra tests prior to studying their effects on drug-release behavior and biodistribution. As a result, the thickness of the carrier’s shell was adjusted from 70 nm to 130 nm and the maximum drug loading was up to 73.6%. The model drug carvedilol (CAR) showed sustained release behavior compared to CAR commercial capsules, and the dissolution rate slowed down as the shells got thicker. AUC0-48h and Tmax were enlarged 2.2 and 6.5 fold, respectively, which demonstrated that oral bioavailability was successfully improved. Bioimaging tests showed that the novel carbon vehicle had a long residence time in the gastrointestinal tract. In short, the newly designed HMC is a promising drug carrier for both oral bioavailability improvement and in vivo tracing.

  18. Fabrication of cerium-doped nonstoichiometric (Ce, Lu, Gd)3+δ(Ga, Al)5-δO12 transparent ceramics

    Institute of Scientific and Technical Information of China (English)

    陈先强; 秦海明; 张烨; 罗朝华; 刘永福; 蒋俊; 江浩川

    2015-01-01

    Cerium-doped nonstoichiometric (Ce,Lu,Gd)3+δ(Ga,Al)5–δO12 (LuGGAG) transparent garnet ceramic samples were fabri-cated via a solid state reaction method in this study. The ceramics were prepared via oxygen sintering followed by hot isostatic press-ing (HIP). The phase and microstructure of the samples were analyzed by X-ray diffraction and scanning electron microscopy (SEM), respectively. The excitation, emission and transmission spectra were also measured. The total optical transmittance of the annealed LuGGAG ceramics with thickness of 3 mm reached 47% at the emission wavelength of 555 nm. The decay time was about 60 ns. Compact microstructure of polycrystalline grains with scale around 5μm were gained according to scanning electron microscopy characterization. The successful preparation of the bulk ceramic material and implementation of the combined oxygen sintering-hot isostatic pressing treatment process provided an important method for the exploration of nonstoichiometric scintillator material.

  19. Performance of cerium-doped Gd3Al2Ga3O12 (GAGG:Ce) scintillator in gamma-ray spectrometry

    Science.gov (United States)

    Iwanowska, Joanna; Swiderski, Lukasz; Szczesniak, Tomasz; Sibczynski, Pawel; Moszynski, Marek; Grodzicka, Martyna; Kamada, Kei; Tsutsumi, Kousuke; Usuki, Yoshiyuki; Yanagida, Takayuki; Yoshikawa, Akira

    2013-06-01

    Performance of cerium-doped Gd3Al2Ga3O12 (GAGG:Ce) scintillator in gamma-ray spectrometry has been investigated. The measurements of two samples of GAGG:Ce cover the tests of emission spectra (maximum of emission at about 530 nm), light output, non-proportionality, energy resolution, time resolution and decay time of light pulses. We compare the results with commonly known scintillators, such as NaI(Tl), LSO, LuAG etc. The results show that GAGG:Ce has a high light yield of about 33000 ph/MeV as measured with Hamamatsu S3590-18 Si PiN photodiode [1]. The total energy resolution for 662 keV gamma-rays from 137Cs source is equal to about 6%, whereas intrinsic resolution is equal to 5.2%. Additionally, we made basic measurements of photoelectron yield, non-proportionality and total energy resolution of small sample (5×5×5 mm3) of GAGG:Ce crystal coupled to Hamamatsu MPPC array (6×6 mm2). The results show that the performance of GAGG:Ce measured with the MPPC array are similar to those measured with the PMT.

  20. Studies of Spin-Liquid and Analog to Helium-4 Melting Curve in a 3D Frustrated Magnet: Gd3Ga5O12

    Science.gov (United States)

    Tsui, Y. K.

    2000-03-01

    Gadolinium gallium garnet, Gd_3Ga_5O_12 (GGG) has an extraordinary low temperature phase diagram [1,2]. Although the Curie-Weiss temperature of GGG is ~ -2 K, GGG shows no long range order down to T ~ 0.4 K. At low temperatures, GGG has a spin glass phase at low fields (properties of the intermediate field (IF) state through heat capacity, thermal conductivity, and magnetocaloric measurements [3]. Our results show a sharp high-field phase boundary of the thermal irreversibility of the spin glass phase of GGG implying that the IF phase is distinct from the spin glass. The lower field boundary of the AFM phase is shown to have distinct minimum at T ~ 0.2 K, in analogy to the minimum in the melting curve of ^4He. The existence of such a minimum is confirmed by measurements of the latent heat of the transition below that temperature and is consistent with our heat capacity data. Currently we are measuring the dc magnetization (M) of GGG. Our preliminary data confirm the clear boundary between the spin glass and the IF states. M also shows indications of the reentrance of the IF phase. [1] P. Schiffer et al., Phys. Rev. Lett. 73, 2500 (1994). [2] P. Schiffer et al., Phys. Rev. Lett. 74, 2379 (1995). [3] Y.K. Tsui et al., Phys. Rev. Lett. 82, 3532 (1999).

  1. Atorvastatin prevents hypoxia-induced inhibition of endothelial nitric oxide synthase expression but does not affect heme oxygenase-1 in human microvascular endothelial cells

    NARCIS (Netherlands)

    Loboda, Agnieszka; Jazwa, Agnieszka; Jozkowicz, Alicj A.; Dorosz, Jerzy; Balla, Jozsef; Molema, Grietje; Dulak, Jozef

    2006-01-01

    Beneficial cardiovascular effects of statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are particularly assigned to the modulation of inflammation. Endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) are listed among the crucial protective, anti-i

  2. 白光LED用新型红色荧光粉SrMgSi2O6:Eu3+,M(M=Gd3+,Ti4+)的合成及性质%Preparation of New Red Emitting Phosphor SrMgSi2O6:Eu3+,M(M=Gd3+,Ti4+)for White LEDs and Its Properties

    Institute of Scientific and Technical Information of China (English)

    翟永清; 冯仕华; 张张; 石领娟; 郝晓红

    2010-01-01

    以Na2CO3为电荷补偿剂,采用凝胶-燃烧法合成新型红色硅酸盐发光材料SrMgSi2O6:Eu3+.用X射线粉末衍射仪、荧光分光光度计等对合成产物进行分析和表征.结果表明:SrMgSi2O6:Eu3+的晶体结构与Sr2MgSi2O7相同,同属四方晶系.样品nSrMgSi2O6:Eu3+的激发光谱在220~300 nm出现一宽带吸收,归属于O2--Eu3+之间的电荷迁移,300 nm以后出现的锐线峰为Eu3+的f-f跃迁吸收峰,其最强锐线峰位于400 nm,因而,可以被InGaN管芯产生的紫外辐射有效激发.发射光谱由两个强发射峰组成,位于592和618 nm处,分别属于典型的Eu3+的5D0→7F1和5D0→7F2跃迁.此外,研究发现:共掺Gd3+或Ti4+均能敏化Eu3+的发光,可有效提高样品的红光发射.因此,SrMgSi2O6:Eu3+,M(M=Gd3+,Ti4+)有望成为一种与InGaN管芯匹配的白光LED用红色荧光粉.

  3. An investigation into eukaryotic pseudouridine synthases.

    Science.gov (United States)

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  4. Development of a human mitochondrial oligonucleotide microarray (h-MitoArray and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

    Directory of Open Access Journals (Sweden)

    Hansíková Hana

    2008-01-01

    Full Text Available Abstract Background To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. Results Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate

  5. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  6. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  7. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been...... in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...

  8. Gd3+-1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic-2-hydroxypropyl-β-cyclodextrin/Pluronic Polyrotaxane as a Long Circulating High Relaxivity MRI Contrast Agent.

    Science.gov (United States)

    Zhou, Zhuxian; Mondjinou, Yawo; Hyun, Seok-Hee; Kulkarni, Aditya; Lu, Zheng-Rong; Thompson, David H

    2015-10-14

    A multivalent magnetic resonance imaging agent based on a 2-hydroxypropyl-β-cyclodextrin (HPCD):Pluronic F127 polyrotaxane carrier has been synthesized, and its blood pool contrast properties have been characterized. This Gd3+-DO3A-HPCD/Pluronic polyrotaxane construct is shown to circulate for more than 30 min and provide >100-fold vascular enhancement relative to the monomeric Gd3+-DO3A-HPCD control that is rapidly cleared via the kidney. The high r1 relaxivity at 37 °C (23.83 mM(-1) s(-1) at 1.5 T; 34.08 mM(-1) s(-1) at 0.5 T), extended blood circulation, well-known pharmacology of the polyrotaxane precursors, and absence of acute toxicity make it a highly attractive blood pool contrast agent candidate.

  9. Luminescence properties of an orange-red phosphor GdAl3(BO3)4:Sm3+ under VUV excitation and energy transfer from Gd3+ to Sm3+

    Science.gov (United States)

    He, Jin; Zhang, Su; Zhou, Jianbang; Zhong, Jiuping; Liang, Hongbin; Sun, Shuaishuai; Huang, Yan; Tao, Ye

    2015-01-01

    In order to develop a new warm-color emission phosphor under vacuum ultraviolet (VUV) excitation, trivalent samarium ion (Sm3+) doped GdAl3(BO3)4 was prepared by a solid state reaction technique at high temperature. The VUV excitation and emission spectra of as-synthesized sample GdAl3(BO3)4:Sm3+ were determined in the Beijing Synchrotron Radiation Facilities (BSRF). Compared with the emission of GdAl3(BO3)4:Eu3+, GdAl3(BO3)4:Sm3+ shows an orange-red emission under Xe 172 nm VUV excitation, which indicates that Sm3+ ion can be a possible activator to obtain warm color emission in the field of lighting. Furthermore, for improving the emission of Sm3+ doped in GdAl3(BO3)4, the energy transfer process from Gd3+ in the host to the activator Sm3+ was investigated through the analysis of spectroscopic characteristics and luminescence decay curves of GdAl3(BO3)4:Sm3+. The results reveal that there exists energy transfer from Gd3+ to Sm3+, but the energy transfer from Sm3+ to Gd3+ is inefficient.

  10. Suitability of the rare-earth compounds Dy2Ti2O7 and Gd3Al5O12 for low temperature (4K-20K) magnetic refrigeration cycle

    Science.gov (United States)

    Flood, D. J.

    1973-01-01

    Measurements were made of the magnetic entropy and magnetization of powered samples of the compounds Dy2Ti2O7 and Gd3Al5O12. The magnetization was measured for temperatures at and below 4.2 K, in applied fields ranging to 7.0 tesla. Isothermal changes in magnetic entropy were measured for temperatures from 1.2 to 20 K, in applied fields up to 10 tesla. The results of the measurements are consistent with a doublet ground state for Dy2Ti2O7, and an eight-fold degenerate ground state for Gd3Al5O12. Absolute values of magnetic entropy have been obtained at the lower temperatures, permitting the isotherms to be properly located in the S-H plane with the use of adiabatic magnetization data. The iso-field lines in the S-T plane were determined. The results indicate that Dy2Ti2O7 can absorb a maximum of 71 + or - 4 joules/kg of heat at 4.2 K, while Gd3Al5O12 can absorb 233 + or - joules/kg at the same temperature. The large difference between the two is most likely a result of crystal field interactions in the dysoprosium compound. Both materials can be cycled adiabatically between 4.2 and 20 K.

  11. Chromosomal location of two human genes encoding tetrahydrobiopterin-metabolizing enzymes: 6-pyruvoyl-tetrahydropterin synthase maps to 11q22. 3-q23. 3, and pterin-4[alpha]-carbinolamine dehydratase maps to 10q22

    Energy Technology Data Exchange (ETDEWEB)

    Thoeny, B.; Heizmann, C.W. (Univ. of Zuerich (Switzerland)); Mattei, M.G. (Institut National de la Sante et de la Recherche Medicale, Marseille (France))

    1994-01-15

    Tetrahydrobiopterin (BH[sub 4]) is the redox cofactor for the aromatic amino acid hydroxylases such as phenylalanine hydroxylase. At least five enzymes are known to be involved in BH[sub 4] biosynthesis and regeneration. A deficiency in several of the BH[sub 4] metabolic enzymes causes variant types of hyperphenylalaninemias in man. Recently, the authors cloned and expressed the human cDNAs for two of the BH[sub 4] enzymes, the 6-pyruvoyl-tetrahydropterin synthase and the pterin-4[alpha]-carbinolamine dehydratase (gene symbols PTS and PCD/DCOH, respectively). In this report, they localized the two genes on the human chromsomes by in situ hybridization. The PTS gene was mapped to the chromosomal region 11q22.3-q23.3, and the PCD/DCOH gene was mapped to the 10q22 band of the genome. 18 refs., 2 figs.

  12. Central nervous system lipocalin-type prostaglandin D2-synthase is correlated with orexigenic neuropeptides, visceral adiposity and markers of the hypothalamic-pituitary-adrenal axis in obese humans.

    Science.gov (United States)

    Elias, E; Benrick, A; Behre, C J; Ekman, R; Zetterberg, H; Stenlöf, K; Wallenius, V

    2011-06-01

    Lipocalin-type prostaglandin D2-synthase (L-PGDS) is the main producer of prostaglandin D2 (PGD2) in the central nervous system (CNS). Animal data suggest effects of central nervous L-PGDS in the regulation of food intake and obesity. No human data are available. We hypothesised that a role for CNS L-PGDS in metabolic function in humans would be reflected by correlations with known orexigenic neuropeptides. Cerebrospinal fluid (CSF) and serum samples were retrieved from 26 subjects in a weight loss study, comprising a 3-week dietary lead-in followed by 12-weeks of leptin or placebo treatment. At baseline, CSF L-PGDS was positively correlated with neuropeptide Y (NPY) (ρ = 0.695, P obesity by interaction with the neuroendocrine circuits regulating appetite and fat distribution. Further interventional studies will be needed to characterise these interactions in more detail.

  13. Crystal Structure and Substrate Specificity of Human Thioesterase 2: INSIGHTS INTO THE MOLECULAR BASIS FOR THE MODULATION OF FATTY ACID SYNTHASE*

    OpenAIRE

    Ritchie, Melissa K.; Johnson, Lynnette C.; Clodfelter, Jill E.; Pemble, Charles W.; Fulp, Brian E.; Furdui, Cristina M.; Kridel, Steven J.; Lowther, W. Todd

    2015-01-01

    The type I fatty acid synthase (FASN) is responsible for the de novo synthesis of palmitate. Chain length selection and release is performed by the C-terminal thioesterase domain (TE1). FASN expression is up-regulated in cancer, and its activity levels are controlled by gene dosage and transcriptional and post-translational mechanisms. In addition, the chain length of fatty acids produced by FASN is controlled by a type II thioesterase called TE2 (E.C. 3.1.2.14). TE2 has been implicated in br...

  14. Human platelet nitric oxide synthase activity: an optimized method Atividade da óxido nítrico sintase em plaquetas humanas: um método otimizado

    Directory of Open Access Journals (Sweden)

    Elisa Mitiko Kawamato

    2002-09-01

    Full Text Available We investigated the kinetic analysis of human platelet Nitric Oxide Synthase (NOS activity by the rate of conversion of [³H] arginine to [³H]-citrulline in unstimulated fresh platelets. NOS activity was present in the membrane fraction and cytosol, and was Ca2+- and calmodulin dependent which is a characteristic of endothelial NOS. NOS activity was also dependent of NADPH since the omission of this cofactor induced an important decrease (85,2% in the enzyme activity. The kinetic varied with protein and arginine concentration but optimum concentrations were found up to 60 minutes, and up to 80 µg of protein at 120 nM of arginine and 0.5 µCi of ³H-arginine. NOS activity in the absence of FAD (flavin adenine dinucleotide, FMN (flavin mononucleotide and BH4 (tetrahydrobiopterin was only 2.8% of the activity measured in the presence of these three cofactors. The enzyme activity was completely inhibited by L-NAME (1 mM (98.1 % and EGTA (5 mM (98.8 %. Trifluoperazine (TFP caused 73.2% inhibition of the enzyme activity at 200 µM and 83.8 % at 500 µM. Under basal conditions, NOS Km for L-arginine was 0.84 ± 0.08 µM and mean Vmax values were 0.122 ± 0.025 pmol.mg-1.min-1. Mean human NOS platelet activity was 0.020 ± 0.010 pmol.mg-1.min-1. Results indicate that the eNOS in human platelet can be evaluated by conversion of [³H]-arginine to [³H]citrulline in an optimized method, which provide reproducible and accurate results with good sensitivity to clinical experiments involving neurological and psychiatric diseases.A análise cinética da atividade da óxido nítrico sintase (NOS plaquetária foi avaliada pela conversão de [³H]-arginina em [³H]-citrulina em plaquetas humanas frescas não estimuladas. A atividade da NOS foi detectada na fração citosólica e na membrana, além de ser dependente de Ca2+-calmodulina, que é uma característica da NOS endotelial (eNOS. A omissão de NADPH levou à diminuição da atividade da NOS dependente da

  15. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  16. Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle

    DEFF Research Database (Denmark)

    Højlund, K; Yi, Z; Lefort, N;

    2009-01-01

    AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site......-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-beta) and determine protein abundance of ATPsyn-beta and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. METHODS: Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type...... 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-beta using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic...

  17. Macrocyclic Gd3+ chelates attached to a silsesquioxane core as potential magnetic resonance imaging contrast agents: synthesis, physicochemical characterization, and stability studies.

    Science.gov (United States)

    Henig, Jörg; Tóth, Eva; Engelmann, Jörn; Gottschalk, Sven; Mayer, Hermann A

    2010-07-05

    Two macrocyclic ligands, 1,4,7,10-tetraazacyclododecane-1-glutaric-4,7,10-triacetic acid (H(5)DOTAGA) and the novel 1,4,7,10-tetraazacyclododecane-1-(4-(carboxymethyl)benzoic)-4,7,10-triacetic acid (H(5)DOTABA), were prepared and their lanthanide complexes (Ln = Gd(3+), Y(3+)) attached to an amino-functionalized T(8)-silsesquioxane. The novel compounds Gadoxane G (GG) and Gadoxane B (GB) possess eight monohydrated lanthanide complexes each, as evidenced by multinuclear ((1)H, (13)C, (29)Si) NMR spectroscopy and high resolution mass spectrometry (HR-MS). Pulsed-field gradient spin echo (PGSE) diffusion (1)H NMR measurements revealed hydrodynamic radii of 1.44 nm and global rotational correlation times of about 3.35 ns for both compounds. With regard to potential MRI contrast agent applications, a variable-temperature (17)O NMR and (1)H nuclear magnetic relaxation dispersion (NMRD) study was carried out on aqueous solutions of the gadolinium(III) complexes of the Gadoxanes and the corresponding monomeric ligands to yield relevant physicochemical properties. The water exchange rates of the inner-sphere water molecules are all very similar (k(ex)(298) between (5.3 +/- 0.5) x 10(6) s(-1) and (5.9 +/- 0.3) x 10(6) s(-1)) and only slightly higher than that reported for the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H(4)DOTA) (k(ex)(298) = 4.1 x 10(6) s(-1)). Despite their almost identical size and their similar water exchange rates, GB shows a significantly higher longitudinal relaxivity than GG over nearly the whole range of magnetic fields (e.g., 17.1 mM(-1) s(-1) for GB and 12.1 mM(-1) s(-1) for GG at 20 MHz and 25 degrees C). This difference arises from their different local rotational correlation times (tau(lR)(298) = 240 +/- 10 ps and 380 +/- 20 ps, respectively), because of the higher rigidity of the phenyl ring of GB as compared to the ethylene spacer of GG. A crucial feature of these novel compounds is the lability of the

  18. Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase).

    Science.gov (United States)

    Perrine, Cynthia; Ju, Tongzhong; Cummings, Richard D; Gerken, Thomas A

    2009-03-01

    Mucin-type protein O-glycosylation is initiated by the addition of alpha-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of beta-Gal to GalNAc by the UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase), forming the Core 1 structure (beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr), is a common and biologically significant subsequent step in O-glycan biosynthesis. What dictates the sites of Core 1 glycosylation is poorly understood; however, the peptide sequence and neighboring glycosylation effects have been implicated. To systematically address the role of the peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAG (where X = G, A, P, V, I, F, Y, S, N, D, E, H, R, and K) as a substrate. The Core 1 glycosylated product was isolated on immobilized PNA (Arachis hypogaea) lectin and its composition determined by Edman amino acid sequencing for comparison with the initial substrate composition, from which transferase preferences were obtained. From these studies, elevated preferences for Gly at the +1 position with moderately high preferences for Phe and Tyr in the +3 position relative to the acceptor Thr-O-GalNAc were found. A number of smaller Pro enhancements were also observed. Basic residues, i.e., Lys, Arg, and His, in any position were disfavored, suggesting electrostatic interactions as an additional important component modulating transferase specificity. This work suggests that there are indeed subtle specific and nonspecific protein-targeting sequence motifs for this transferase.

  19. Association of Human Methionine Synthase-A2756G Transition With Prostate Cancer: A Case-Control Study and in Silico Analysis

    Directory of Open Access Journals (Sweden)

    Arezou Ebrahimi

    2017-07-01

    Full Text Available Methionine synthase (MTR is one of the key enzymes of folate pathway, which play a key role in the construction, repair, and methylation of DNA. In this study, an association of MTR A2756G gene transition with prostate cancer in men populations of Kashan-Iran was investigated by a case-control study and an in silico analysis. The 200 samples including 100 patients with prostate cancer, as case group and 100 healthy men, as control group included in this study. MTR-A2756G genotyping was performed by PCR-RFLP technique. Some in silico tools used to evaluate the effects of A2756G transition on the structure and function of MTR. Results showed that the AG genotype (OR: 2.4014, 95% CI: 1.3216-4.3636, P=0.0040, and GG genotype (OR: 3.6324, 95% CI: 1.2629-10.4475, P=0.0167 and G allele (OR: 2.0120, 95% CI: 1.3098-3.0905, P=0.0014 were associated with prostate cancer. In silico analysis showed that polymorphisms of the enzyme protein might change properties of MTR such as relative mutability and flexibility, which leads to alteration of stability and function of the enzyme. Based on the results, an MTR-A2756G polymorphism which changes activity and stability of the methionine synthase associated with prostate cancer in men. It is a preliminary study and is presenting data for future comprehensive study for making a clinical conclusion that this gene transition is a biomarker for susceptibility to prostate cancer.

  20. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  1. Free carrier absorption in self-activated PbWO_4 and Ce-doped Y_3(Al_0.25Ga_0.75)_3O_12 and Gd_3Al_2Ga_3O_12 garnet scintillators

    CERN Document Server

    E. Auffray; M.T. Lucchini; S. Nargelas; O. Sidletskiy; G. Tamulaitis; Y. Tratsiak; A. Vaitkevičius

    2016-01-01

    tungstate (PbWO_4, PWO) ant two garnet crystals, GAGG:Ce and YAGG:Ce. It was shown that free electrons appear in the conduction band of PWO and YAGG:Ce crystals within a sub-picosecond time scale, while the free holes in GAGG:Ce appear due to delocalization from Gd^3+ ground states to the valence band within a few picoseconds after short-pulse excitation. The influence of Gd ions on the nonequilibrium carrier dynamics is discussed on the base of comparison the results of the free carrier absorption in GAGG:Ce containing gadolinium and in YAGG without Gd in the host lattice.

  2. Microstructure and Corrosion Resistance of PEO-EPD Composite Film on Mg-9Gd-3Y Magnesium Alloy%Mg-9Gd-3Y镁合金PEO-EPD复合膜层组织结构与耐蚀性研究

    Institute of Scientific and Technical Information of China (English)

    李建平; 王刚; 王萍; 郭永春; 杨忠

    2014-01-01

    The samples of Mg-9Gd-3Y(GW93) Mg alloy were coated with plasma electrolytic oxidation (PEO) ,electrophoretic deposition (EPD) and PEO-EPD processes respectively .Varius coating surface and cross-section morphologies ,organic functional structure and the corresponded corrosion resistance were studied using scanning electron microscopy (SEM ) ,Fourier transform infrared spectrometer , electrochemical and salt spray tests .The effect of voltage and solids fraction in electrodeposition solution on the thickness ,forming rate and corrosion resistance of PEO-EPD composite coating was discussed and compared with those of PEO and EPD only .The results showed that thickness and film forming rate of PEO-EPD composite coating increased with the increase of voltage and solids ,the corrosion rate increased first ,then decreased with the increase of voltage ;however ,the corrosion rate is decreased with the increase of solids .The PEO-EPD composite film consists of ceramic zone ,which formed during PEO process ,and the organic compound zone ,which formed by EPD process .The organic compound zone is bonded closely with the PEO zone in the optimum EPD condition of voltage 70 V ,solids fraction 19% . Compared with PEO coating only ,the corrosion current density of composite coating significantly decreased by two orders of magnitude ,the corrosion potential moved positively more than 200 mV ,and the corrosion resistance improved up to 13 times .%为了提高镁合金的耐蚀性,本文利用扫描电镜分析了Mg-9Gd-3Y(GW93)镁稀土合金表面等离子电解氧化陶瓷层、等离子电解氧化-电泳复合膜层和电泳膜层的表面与纵截面形貌组织,利用傅立叶红外光谱仪(FT-IR)分析了电泳膜层有机官能团类型,用电化学测试手段和盐雾试验方法测试了该合金不同处理膜层的耐蚀性,讨论了电泳电压和固体粉料分数对等离子电解氧化-电泳复合膜层厚度、成膜速率和耐蚀性的影响。

  3. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

    van Schie, C.C.N.; Haring, M.A.; Schuurink, R.C.; Bach, T.J.; Rohmer, M.

    2013-01-01

    Gibberellins are derived from the diterpene precursor geranylgeranyl diphophosphate (GGPP). GGPP is converted to ent-kaurene, which contains the basic structure of gibberellins, in the plastids by the combined actions of copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Generally, ge

  4. Homo-timeric structural model of human microsomal prostaglandin E synthase-1 and characterization of its substrate/inhibitor binding interactions

    Science.gov (United States)

    Xing, Li; Kurumbail, Ravi G.; Frazier, Ronald B.; Davies, Michael S.; Fujiwara, Hideji; Weinberg, Robin A.; Gierse, James K.; Caspers, Nicole; Carter, Jeffrey S.; McDonald, Joseph J.; Moore, William M.; Vazquez, Michael L.

    2009-01-01

    Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic π-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H2 (PGH2), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH2 in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.

  5. Electrochemical corrosion behavior of diphase ceramic coating prepared by PEO process on Mg-9Gd-3Y alloy%Mg-9Gd-3Y镁合金PEO工艺制备复相陶瓷层电化学腐蚀行为

    Institute of Scientific and Technical Information of China (English)

    王萍; 王刚; 李建平; 杨忠; 郭永春

    2014-01-01

    采用等离子体电解氧化(PEO)工艺在Mg-9Gd-3Y镁合金表面制备了复相陶瓷层.通过微观结构分析及电化学测试技术研究了PEO陶瓷层的微观组织及腐蚀行为.结果表明,偏铝酸盐体系中PEO法制备的陶瓷涂层主要由MgO和MgAl2O4相组成,还有少量MgF2相,其中MgAl2 O4尖晶石相约占陶瓷层的19.87%,且由非贯通的等离子体放电微孔与喷射沉积复相氧化物组成.浸泡初期,PEO陶瓷层表现出较好的耐蚀性;浸泡后期,陶瓷层腐蚀电流密度Icorr逐渐增大,陶瓷层电阻Rct快速减小,144 h后陶瓷层的保护能力迅速下降,且陶瓷层表面出现点蚀及裂纹萌生;浸泡过程中,交流阻抗谱由浸泡0~72 h的两个容抗弧转变为浸泡l44~ 300 h的单容抗弧和感抗弧组成,表明腐蚀介质已渗透整个陶瓷层,并萌生点蚀.腐蚀产物主要由Mg(OH)2相组成.陶瓷层的腐蚀主要由等离子体放电微孔开始,逐渐向四周蔓延并形成放射状裂纹而加速腐蚀.

  6. Mammalian N-acetylglutamate synthase.

    Science.gov (United States)

    Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

    2004-04-01

    N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

  7. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  8. Linking pseudouridine synthases to growth, development and cell competition.

    Science.gov (United States)

    Tortoriello, Giuseppe; de Celis, José F; Furia, Maria

    2010-08-01

    Eukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H/ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA-like molecules. In humans, loss of pseudouridine synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing disc, an advantageous model system for studies of cell growth and differentiation. In this organ, depletion of the mfl-encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a key role in the development of the loss of function mutant phenotype. Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine synthase loss of function phenotype is caused by defects in Notch signalling.

  9. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    Science.gov (United States)

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  10. Human mitochondrial HMG CoA synthase: Liver cDNA and partial genomic cloning, chromosome mapping to 1p12-p13, and possible role in vertebrate evolution

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal (Canada)] [and others

    1994-10-01

    Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholersterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of an HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. the nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistant with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain. 56 refs., 4 figs., 2 tabs.

  11. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    Cranial parasympathetic outflow is mediated through the sphenopalatine ganglion (SPG). The present study was performed to examine the expression of the parasympathetic signaling transmitters and their receptors in human and rat SPG. Indirect immunofluorescence technique was used for the demonstra...

  12. Comparative study of the Mn4+2E → 4A2 luminescence in isostructural RE2Sn2O7:Mn4+ pyrochlores (RE3+ = Y3+, Lu3+ or Gd3+)

    Science.gov (United States)

    Senden, Tim; Broers, Fréderique T. H.; Meijerink, Andries

    2016-10-01

    Red emitting Mn4+-doped crystalline materials have potential for application in light emitting devices and therefore it is important to understand how the optical properties of Mn4+ are influenced by the host lattice the Mn4+ ions are situated in. In this work we investigate the effect of the host cations in the second coordination sphere on the Mn4+ emission by studying the luminescence of Mn4+ ions doped into three isostructural rare earth (RE) stannate RE2Sn2O7 pyrochlores (RE3+ = Y3+, Lu3+ or Gd3+). It is found that the energies of the Mn4+4T1 and 4T2 states significantly increase with decreasing Mn4+-O2- distance, whereas the energy of the 2E level shows a small shift to higher energies from RE3+ = Gd3+ to Lu3+ to Y3+. The observed trend for the 2E level energy is not related to the size of the RE3+ ion and is not in line with theoretical calculations reported previously. Low temperature emission spectra of the RE2Sn2O7:Mn4+ phosphors reveal that only asymmetrical vibronic modes couple to the 2E → 4A2 transition and furthermore show there is significant and unexpected local disorder for Mn4+ in Gd2Sn2O7 that is not observed for Mn4+ in the other hosts. Photoluminescence decay measurements demonstrate that the luminescence of RE2Sn2O7:Mn4+ is strongly quenched below room temperature which is assigned to non-radiative relaxation via a low-lying O2- → Mn4+ charge-transfer state.

  13. Sm~(3+)和Gd~(3+)共掺杂TiO_2粉体的制备和性能表征%Preparation and characterization of Sm~(3+) and Gd~(3+) co-doped TiO_2 powders with high photocatalytic activity

    Institute of Scientific and Technical Information of China (English)

    卢旭东; 姜承志; 侯雪; 董多

    2012-01-01

    Pure TiO2,Sm3+ or Gd3+ single-doped and Sm3+/Gd3+ co-doped TiO2 composite nano-particle were prepared by sol-gel method and characterized by the techniques such as XRD and SEM/EDAX.The photocatalytic degradation of methylene blue(MB) in aqueous solution was used as a probe reaction to evaluate their photocatalytic activity.The mechanisms of effects of Sm3+/Gd3+ co-doped,the initial concentration of methylene blue in water and the concentration of TiO2 in solution on the photocatalytic activity of the TiO2 composite nano-particles were also discussed.The results showed that Sm3+/Gd3+ co-doped can significantly enhance the photocatalytic activity of TiO2 nano-particles.The co-doped of Sm3+/Gd3+ showed a synergistic effect for photocatalytic activity of TiO2 nano-particles,could inhibit the phase transformation from anatase to rutile and decrease the diameter of TiO2 nano-particles,and the matrix distortion of TiO2 nano-particles increased when the Sm3+/Gd3+ have co-doped.When the co-doped amounts for Sm3+/Gd3+ were 0.1% and 0.2%,the initial concentration of methylene blue was 4mg/L,and the concentration of TiO2 was 2g/L the photocatalytic activity of TiO2 could be markedly improved,and its degradation rate reached 99.71%.Kinetic equation of photocatalytic degradation of methylene blue was corresponded to Langmuir-Hinshelwood equation.%采用溶胶-凝胶法制备了纯TiO2,1%Sm3+或2%Gd3+单掺杂和1%Sm3+/2%Gd3+共掺杂TiO2复合粉体,采用XRD和SEM/EDAX等技术进行表征。以对亚甲基蓝的光催化降解为目标反应,评价了TiO2复合粉体的光催化活性,探讨了Sm3+/Gd3+共掺杂、亚甲基蓝初始浓度和粉体投加量对TiO2粉体光催化活性的影响机制。结果表明,Sm3+/Gd3+共掺杂可以显著提高TiO2粉体的光催化活性;Sm3+/Gd3+共掺杂在TiO2粉体中产生协同作用,抑制了TiO2由锐钛矿相向金红石相转变,使TiO2粒径尺寸减小,增大了TiO2的晶格畸变。当

  14. Evidence for fumonisin inhibition of ceramide synthase in humans consuming maize-based foods and living in high exposure communities in Guatemala

    Science.gov (United States)

    Fumonisin B1 (FB1) is a toxic chemical produced by molds. The molds that produce fumonisin are common in corn. Consumption of contaminated corn by farm animals has been shown to be the cause of disease. Fumonisin has been hypothesized to be an environmental risk factor for diseases in humans in c...

  15. Malarial pigment haemozoin, IFN-gamma, TNF-alpha, IL-1beta and LPS do not stimulate expression of inducible nitric oxide synthase and production of nitric oxide in immuno-purified human monocytes

    Directory of Open Access Journals (Sweden)

    Ceretto Monica

    2007-06-01

    Full Text Available Abstract Background Enhanced production of nitric oxide (NO following upmodulation of the inducible isoform of NO synthase (iNOS by haemozoin (HZ, inflammatory cytokines and LPS may provide protection against Plasmodium falciparum malaria by killing hepatic and blood forms of parasites and inhibiting the cytoadherence of parasitized erythrocytes (RBC to endothelial cells. Monocytes and macrophages are considered to contribute importantly to protective upregulation of iNOS and production of NO. Data obtained with murine phagocytes fed with human HZ and synthetic HZ (sHZ indicate that supplemental treatment of those cells with IFN-gamma elicited significant increases in protein and mRNA expression of iNOS and NO production, providing a potential mechanism linking HZ phagocytosis and increased production of NO. Purpose of this study was to analyse the effect of P. falciparum HZ and sHZ supplemental to treatment with IFN-gamma and/or a stimulatory cytokine-LPS mix on iNOS protein and mRNA expression in immuno-purified human monocytes. Methods Adherent immunopurified human monocytes (purity >85%, and murine phagocytic cell lines RAW 264.7, N11 and ANA1 were fed or not with P. falciparum HZ or sHZ and treated or not with IFN-gamma or a stimulatory cytokine-LPS mix. Production of NO was quantified in supernatants, iNOS protein and mRNA expression were measured after immunoprecipitation and Western blotting and quantitative RT-PCT, respectively. Results Phagocytosis of HZ/sHZ by human monocytes did not increase iNOS protein and mRNA expression and NO production either after stimulation by IFN-gamma or the cytokine-LPS mix. By contrast, in HZ/sHZ-laden murine macrophages, identical treatment with IFN-gamma and the cytokine-LPS mix elicited significant increases in protein and mRNA expression of iNOS and NOS metabolites production, in agreement with literature data. Conclusion Results indicate that human monocytes fed or not with HZ/sHZ were constantly

  16. Ternary complex structures of human farnesyl pyrophosphate synthase bound with a novel inhibitor and secondary ligands provide insights into the molecular details of the enzyme’s active site closure

    Directory of Open Access Journals (Sweden)

    Park Jaeok

    2012-12-01

    Full Text Available Abstract Background Human farnesyl pyrophosphate synthase (FPPS controls intracellular levels of farnesyl pyrophosphate, which is essential for various biological processes. Bisphosphonate inhibitors of human FPPS are valuable therapeutics for the treatment of bone-resorption disorders and have also demonstrated efficacy in multiple tumor types. Inhibition of human FPPS by bisphosphonates in vivo is thought to involve closing of the enzyme’s C-terminal tail induced by the binding of the second substrate isopentenyl pyrophosphate (IPP. This conformational change, which occurs through a yet unclear mechanism, seals off the enzyme’s active site from the solvent environment and is essential for catalysis. The crystal structure of human FPPS in complex with a novel bisphosphonate YS0470 and in the absence of a second substrate showed partial ordering of the tail in the closed conformation. Results We have determined crystal structures of human FPPS in ternary complex with YS0470 and the secondary ligands inorganic phosphate (Pi, inorganic pyrophosphate (PPi, and IPP. Binding of PPi or IPP to the enzyme-inhibitor complex, but not that of Pi, resulted in full ordering of the C-terminal tail, which is most notably characterized by the anchoring of the R351 side chain to the main frame of the enzyme. Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering. Structure analysis identified a cascade of conformational changes required for the C-terminal tail rigidification involving Y349, F238, and Q242. The residues K57 and N59 upon PPi/IPP binding undergo subtler conformational changes, which may initiate this cascade. Conclusions In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the “off” position in the

  17. Mitochondrial HMG to CoA synthase (mHS): cDNA cloning in human, mouse and C. elegans, mapping to human chromosome 1p12-13 and partial human genomic cloning

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal, Quebec (Canada)]|[Kingston General Hospital, Ontario (Canada)] [and others

    1994-09-01

    mHS catalyzes the rate-limiting first step of ketogenesis in the liver. A cytoplasmic HS isozyme, encoded by another gene, catalyzes an early step in cholesterol synthesis. Starting from a rat mHS cDNA obtained by RT-PCR from the published rat cDNA sequence, we obtained and sequenced human and mouse cDNAs spanning the entire coding sequence of natural human and mouse mHS, as well as sequencing C. elegans HS-like cDNA. Consensus sequences for 3 mitochondrial and 4 cytoplasmic HSs were created and compared to invertebrate HS sequences. We found high conversation in the active site and at other regions presumably important for HS function. We mapped the mHS locus, HMGCS2 by in situ hybridization to chromosome 1P12-13, in contrast to the human cHS locus (HMGCS1) known to be on chromosome 5p13. Comparative mapping results suggest that these two chromosomal regions may be contiguous in other species, constant with a recent gene duplication event. Furthermore, we have characterized a human genomic mHS subclone containing 4 mHS exons, and found the position of all splice junctions to be identical to that of the hamster cHS gene except for one site in the 3{prime} nontranslated region. We calculate that the mHS and cHS genes were derived from a common ancestor 400-700 Myrs ago, implying that ketogenesis from fat may have become possible around the time of emergence of vertebrates ({approximately}500 Myr ago). Ketogenesis has evolved into an important pathway of energy metabolism, and we predict the mHS deficiency may prove to be responsible for some as yet explained cases of Reye-like syndromes in humans. This hypothesis can now be tested at the molecular level without the necessity of obtaining hepatic tissue.

  18. Oxidized low-density lipoprotein-induced periodontal inflammation is associated with the up-regulation of cyclooxygenase-2 and microsomal prostaglandin synthase 1 in human gingival epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagahama, Yu [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Obama, Takashi [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Usui, Michihiko [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Kanazawa, Yukari [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Iwamoto, Sanju [Department of Biochemistry, Showa University School of Medicine, Tokyo (Japan); Suzuki, Kazushige [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Miyazaki, Akira [Department of Biochemistry, Showa University School of Medicine, Tokyo (Japan); Yamaguchi, Tomohiro [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Yamamoto, Matsuo [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Itabe, Hiroyuki, E-mail: h-itabe@pharm.showa-u.ac.jp [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan)

    2011-10-07

    Highlights: {yields} OxLDL-induced responses in human gingival epithelial cells were studied. {yields} OxLDL enhanced the production of IL-8, IL-1{beta} and PGE{sub 2} in Ca9-22 cells. {yields} An NF-{kappa}B inhibitor suppressed the expression of COX-2 and mPGES1 induced by oxLDL. {yields} Unlike the case in macrophages, oxLDL did not increase the CD36 level. -- Abstract: Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E{sub 2} (PGE{sub 2}) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE{sub 2}-producing enzymes, cyclooxygenase-2 and microsomal PGE{sub 2} synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-{kappa}B) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-{kappa}B pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.

  19. Activin suppresses LPS-induced Toll-like receptor, cytokine and inducible nitric oxide synthase expression in normal human melanocytes by inhibiting NF-κB and MAPK pathway activation.

    Science.gov (United States)

    Kim, Young Il; Park, Seung-Won; Kang, In Jung; Shin, Min Kyung; Lee, Mu-Hyoung

    2015-10-01

    Activins are dimeric growth and differentiation factors that belong to the transforming growth factor (TGF)-β superfamily of structurally related signaling proteins. In the present study, we examined the mechanisms through which activin regulates the lipopolysaccharide (LPS)-induced transcription of Toll-like receptors (TLRs), cytokines and inducible nitric oxide synthase (iNOS) in human melanocytes, as well as the involvement of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling. Cell proliferation was analyzed by cell viability assay, mRNA expression was detected by RT-qPCR, and protein expression was measured by western blot analysis. LPS increased the mRNA expression of TLRs (TLR1-10) and cytokines [interleukin (IL)-1β, IL-6, IL-8 and TNF-α], as well as the mRNA and protein expression of iNOS. Activin decreased the LPS-induced TLR and cytokine mRNA expression, as well as the LPS-induced iNOS mRNA and protein expression. In addition, activin suppressed NF-κB p65 activation and blocked inhibitor of NF-κB (IκBα) degradation in LPS-stimulated melanocytes, and reduced LPS-induced p38 MAPK and MEK/ERK activation. On the whole, our results demonstrated that activin inhibited TLR and cytokine expression in LPS-activated normal human melanocytes and suppressed LPS-induced iNOS gene expression. Moreover, the anti-inflammatory effects of activin were shown to be mediated through the suppression of NF-κB and MAPK signaling, resulting in reduced TLR and iNOS expression, and in the inhibition of inflammatory cytokine expression.

  20. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yamane, Takumi; Kobayashi-Hattori, Kazuo [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan); Oishi, Yuichi, E-mail: y3oishi@nodai.ac.jp [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  1. Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

    Science.gov (United States)

    Lacroix Pépin, Nicolas; Chapdelaine, Pierre; Rodriguez, Yoima; Tremblay, Jacques-P; Fortier, Michel A

    2014-07-01

    Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

  2. Expression of cystathionine β-synthase and cystathionine γ-lyase in human pregnant myometrium and their roles in the control of uterine contractility.

    Directory of Open Access Journals (Sweden)

    Xing-Ji You

    Full Text Available BACKGROUND: Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H(2S has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H(2S produced locally in the control of uterine contractility during labour is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE and cystathionine-β-synthetase (CBS, the principle enzymes responsible for H(2S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H(2S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10(-7-10(-2 mol/L, a precursor of H(2S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10(-3 mol/L increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K(ATP channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips. CONCLUSION/SIGNIFICANCE: H(2S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H(2S synthetic enzymes is down-regulated during labour, suggesting that H(2S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.

  3. Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

    Science.gov (United States)

    Lopez-Rivera, Esther; Jayaraman, Padmini; Parikh, Falguni; Davies, Michael A.; Ekmekcioglu, Suhendan; Izadmehr, Sudeh; Milton, Denái R.; Chipuk, Jerry E.; Grimm, Elizabeth A.; Estrada, Yeriel; Aguirre-Ghiso, Julio; Sikora, Andrew G.

    2014-01-01

    Melanoma is one of the cancers of fastest-rising incidence in the world. iNOS is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K/AKT/mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p-P70S6K, p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of TSC2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Rheb, a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of mTOR pathway members. Exogenously-supplied NO was also sufficient to reverse mTOR pathway inhibition by the B-Raf inhibitor Vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers. PMID:24398473

  4. Biphenyl synthase, a novel type III polyketide synthase.

    Science.gov (United States)

    Liu, B; Raeth, T; Beuerle, T; Beerhues, L

    2007-05-01

    Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.

  5. Glycogen synthase kinase 3 (GSK3)-inhibitor SB216763 promotes the conversion of human umbilical cord mesenchymal stem cells into neural precursors in adherent culture.

    Science.gov (United States)

    Gao, Liyang; Zhao, Mingyan; Li, Peng; Kong, Junchao; Liu, Zhijun; Chen, Yonghua; Huang, Rui; Chu, Jiaqi; Quan, Juanhua; Zeng, Rong

    2017-01-01

    The ability to generate neural progenitor cells from human umbilical cord mesenchymal stem cells (hUC-MSCs) has provided an option to treat neurodegenerative diseases. To establish a method for this purpose, we characterized the early neural markers of hUC-MSCs-derived cells under different conditions. We found that neither the elimination of signals for alternative fate nor N2 supplement was sufficient to differentiate hUC-MSCs into neural precursor cells, but the GSK3 inhibitor SB216763 could promote an efficient neural commitment of hUC-MSCs. The results indicated that Wnt/β-catenin might play an important role during the early neural differentiation of hUC-MSCs. Here, we report a method for hUC-MSCs to commit efficiently into a neural fate within a short period of time. This protocol provides an efficient method for hUC-MSCs-based neural regeneration.

  6. Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

    Science.gov (United States)

    Fantappiè, Ornella; Sassoli, Chiara; Tani, Alessia; Nosi, Daniele; Marchetti, Serena; Formigli, Lucia; Mazzanti, Roberto

    2015-06-01

    Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  7. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions GM3 synthase deficiency GM3 synthase ...

  8. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    Science.gov (United States)

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  9. Expression,purification and identification of I278T-mutant human cystathionine βsynthase%人胱硫醚β合酶I278T突变体的表达、纯化及鉴定

    Institute of Scientific and Technical Information of China (English)

    牛卫宁; 许乐; 羊梦林; 曹珊珊

    2014-01-01

    Objective To investigate the expression and purification I278T-mutant human cystathionineβsynthase(CBS) in E . coli .Methods Site-directed mutagenesis by overlap extension using the polymerase chain reaction (PCR) was employed to construct mutant plasmids pGEX4T-1-CBS(I278T) ,which was induced and expressed in a medium containing 3% ethanol ,purified by affinity chromatography to obtain mutated CBS (I278T) protein .The activity ,UV-visible absorption spectroscopy ,protein particle size and Zeta potential of the purified protein were measured .Results Plasmid pGEX4T-1-CBS(I278T) was successfully constructed .The yield ,the specific activity and activity recovery of purified mutant CBS (I278T ) protein were 2 .3 mg/L ,21 .4 U/mg and 22 .6% .S-adenosylmethionine(AdoMet) with final concentration of 1 mmol/L showed no activation toward mutant CBS (I278T) protein .Ac-cording to UV-visible absorption spectroscopy analysis ,purified mutant CBS(I278T) had characteristic absorption peaks at 429 nm and 550 nm for heme-binding proteins .Protein average particle size was 7 .5 -10 .1 nm ,mainly in the form of tetramers ,and Zeta potential was - 16 .3 mV .Conclusion The methods of expression ,purification and identification of I278T-mutant human cystathionineβsynthase in E .coli were successfully established .%探讨在大肠埃希菌中重组表达和纯化人胱硫醚β合酶CBS(I278T )突变体。方法采用重叠延伸聚合酶链反应(PCR)定点突变技术构建突变质粒pGEX4T-1-CBS(I278T ),在含有3%乙醇的培养基中诱导表达,亲和层析纯化得到突变CBS (I278T)蛋白,测定纯化蛋白的活性、紫外可见吸收光谱、蛋白粒径及Zeta电位。结果成功构建了质粒pGEX4T-1-CBS(I278T)。CBS(I278T )蛋白产率、比活性及酶活性回收率分别为2.3 mg/L、21.4 U/mg、22.6%。终浓度为1 mmol/L的 S-腺苷甲硫氨酸(AdoMet)对突变CBS(I278T)蛋白没有激活作用。紫外可见吸

  10. Uric acid attenuates nitric oxide production by decreasing the interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: a mechanism for uric acid-induced cardiovascular disease development.

    Science.gov (United States)

    Park, Jung-Hyun; Jin, Yoon Mi; Hwang, Soojin; Cho, Du-Hyong; Kang, Duk-Hee; Jo, Inho

    2013-08-01

    The elevated level of uric acid in the body is associated with increased risk of cardiovascular diseases, which is mediated by endothelial dysfunction. However, its underlying mechanism is not fully understood, although dysregulation of endothelial nitric oxide (NO) production is likely to be involved. Using human umbilical vascular endothelial cells (HUVEC), we explored the molecular mechanism of uric acid on endothelial NO synthase (eNOS) activity and NO production. Although high dose of uric acid (12mg/dl for 24h treatment) significantly decreased eNOS activity and NO production, it did not alter eNOS expression and phosphorylations at eNOS-Ser(1177), eNOS-Thr(495) and eNOS-Ser(114). Under this condition, we also found no alterations in the dimerization and acetylation of eNOS, compared with the control. Furthermore, uric acid did not change the activity of arginase II, an enzyme degrading l-arginine, a substrate of eNOS, and intracellular level of calcium, a cofactor for eNOS activation. We also found that uric acid did not alter xanthine oxidase activity, suggesting no involvement of xanthine oxidase-derived O2(-) production in the observed inhibitory effects. In vitro and in cell coimmunoprecipitation studies, however, revealed that uric acid significantly decreased the interaction between eNOS and calmodulin (CaM), an eNOS activator, although it did not change the intracellular CaM level. Like in HUVEC, uric acid also decreased eNOS-CaM interaction in bovine aortic EC. Finally, uric acid attenuated ionomycin-induced increase in the interaction between eNOS and CaM. This study suggests firstly that uric acid decreased eNOS activity and NO production through reducing the binding between eNOS and CaM in EC. Our result may provide molecular mechanism by which uric acid induces endothelial dysfunction.

  11. Corticotropin-releasing hormone interacts with interleukin-1β to regulate prostaglandin H synthase-2 expression in human myometrium during pregnancy and labor.

    Science.gov (United States)

    Markovic, Danijela; Bari, Muhammad F; Lu, Buyu; Vatish, Manu; Grammatopoulos, Dimitris K

    2013-07-01

    The onset of labor appears to involve the activation of myometrial inflammatory pathways, and transcription factors such as nuclear factor-κB (NF-κB) control expression of the contraction-associated proteins required to induce a procontractile phenotype. These responses might involve CRH, which integrates immune and neuroendocrine systems. In human myometrium we investigated cyclooxygenase 2 (PGHS2) expression and regulation by CRH and the proinflammatory cytokine IL-1β before and after labor. Myometrial tissues obtained from pregnant women at term before (n = 12) or during labor (n = 10) and pathological cases of choriamnionitis-associated term labor (n = 5) were used to isolate primary myocytes and investigate in vitro, CRH effects on basal and IL-1β regulated p65 activation and PGHS2 expression. In nonlaboring myometrial cells, CRH was unable to induce NF-κB nuclear translocation; however, it altered the temporal dynamics of IL-1β-driven NF-κB nuclear entry by initially delaying entry and subsequently prolonging retention. These CRH-R1-driven effects were associated with a modest inhibitory action in the early phase (within 2 hours) of IL-1β stimulated PGHS2 mRNA expression, whereas prolonged stimulation for 6-18 hours augmented the IL-1β effects. The early-phase effect required intact protein kinase A activity and was diminished after the onset of labor. The presence of chorioamnionitis led to exaggerated PGHS2 mRNA responses to IL-1β but diminished effects of CRH. CRH is involved in the inflammatory regulation of PGHS2 expression before and during labor; these actions might be important in priming and preparing the myometrium for labor and cellular adaptive responses to inflammatory mediators.

  12. Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase[S

    Science.gov (United States)

    Ternes, Philipp; Brouwers, Jos F. H. M.; van den Dikkenberg, Joep; Holthuis, Joost C. M.

    2009-01-01

    Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually. PMID:19454763

  13. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    expression analysis and proteomics have pointed to abnormalities in mitochondrial oxidative phosphorylation and cellular stress in muscle of type 2 diabetic subjects, and recent work suggests that impaired mitochondrial activity is another early defect in the pathogenesis of type 2 diabetes. This review...... will discuss the latest advances in the understanding of the molecular mechanisms underlying insulin resistance in human skeletal muscle in type 2 diabetes with focus on possible links between impaired glycogen synthase activity and mitochondrial dysfunction....

  14. 冷却速率对砂型铸造Mg-10Gd-3Y-0.4Zr合金凝固行为的影响(英文)

    Institute of Scientific and Technical Information of China (English)

    庞松; 吴国华; 刘文才; 张亮; 张扬; Hans; CONRAD; 丁文江

    2014-01-01

    采用计算机辅助冷却曲线分析技术研究砂型铸造过程中冷却速率(1.4~3.5°C/s)对Mg-10Gd-3Y-0.4Zr合金凝固行为的影响。结果表明:随着冷却速率的提高,起始形核温度(Tα,N)由634.8°C升至636.3°C,再辉前最低温度(Tα,Min)由631.9°C降至630.7°C,形核过冷度(ΔTN)由2.9°C升至5.6°C,共晶反应起始温度(Teut,N)上升,固相线温度(T S)由546.0°C降至541.4°C,凝固温度区间(ΔTS)增加6.1°C。形核速率(N)的增加引起晶核数量的增加,从而降低α-Mg的平均晶粒粒径。相对于形核时间(ΔtN),形核过冷度(ΔTN)对α-Mg平均晶粒粒径的影响更大。温度增加值(T eut,N-T eut,G)与缩短的反应时间改变共晶反应进程,并且影响第二相的含量以及形貌。

  15. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  16. The key role of Cosmc and T-synthase in mucin-type O-glycan biosynthesis--implications in human diseases%Cosmc和T合酶在黏蛋白型O-聚糖合成中的重要作用及其与人类疾病的相关性

    Institute of Scientific and Technical Information of China (English)

    巨同忠; Richard D. Cummings; 靳嘉巍; 查锡良

    2011-01-01

    Mucin type O-glycans, primarily derived from the core 1 structure, play pivotal functions in many biological processes. The T-synthase (core 1 p3-galactosyltransferase) is the key enzyme responsible for synthesizing the core 1 O-glycan Galpl,3GalNAcal-Ser/Thr (T antigen) through addition of Gal to GalNAcal-Ser/ Thr (Tn antigen). Interestingly, formation of active T-synthase in human and other vertebrates requires a specific molecular chaperone, Cosmc. Dysfunction of Cosmc results in an inactive T-synthase leading to expression of the Tn antigen and its sialylated version, sialylTn (STn, Neu5Aca2,6GalNAcal-Ser/Thr). This review summarizes the current understanding of the T-synthase and Cosmc regarding their biochemistry and biology, as well as their roles in human diseases, such as Tn syndrome, igA nephropathy, and human tumors, which are associated with expression of abnormal O-glycans.%从核心1结构(Galβl,3GaINAcαl-O-Ser/Thr,core 1 structure,T antigen)中衍生出来的黏蛋白型0-聚糖在很多生理过程中发挥重要的生物学功能.T-合酶(core 1 β3-galactosyltransferase,Tsynthase)是合成核心1结构的唯一糖基转移酶,它主要的功能是将半乳糖(Galactose)添加到GaINAcαI-Ser/Thr (Tn抗原)糖链上.但是在人体和其他脊椎动物中有活性的T-合酶的形成需要一个重要的伴侣分子Cosmc ; Cosmc功能丧失将直接导致T-合酶失活,其结果是机体细胞只能合成Tn抗原以及唾液酰化Tn (sialylTn,STn,Neu5Aca2,6GaINAca1-O-Ser/Thr).综述目前对T-合酶和Cosmc的研究以及在人类疾病(如异常O-聚糖表达相关的Tn综合征、IgA肾病和肿瘤)发生发展中的作用.

  17. Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Viggiano, J M; Pérez Martínez, S; de Gimeno, M F

    1997-01-01

    In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitation in vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 microM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reaction in vitro and that nitric oxide induces this event.

  18. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  19. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    Science.gov (United States)

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  20. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian;

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS...... classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...

  1. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. (Univ. of Texas Medical School, Houston (United States))

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  2. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    OpenAIRE

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic ch...

  3. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    Science.gov (United States)

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  4. Terpene synthases from Cannabis sativa.

    Science.gov (United States)

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  5. Differential expression profiles of glycosphingolipids in human breast cancer stem cells vs. cancer non-stem cells.

    Science.gov (United States)

    Liang, Yuh-Jin; Ding, Yao; Levery, Steven B; Lobaton, Marlin; Handa, Kazuko; Hakomori, Sen-itiroh

    2013-03-26

    Previous studies demonstrated that certain glycosphingolipids (GSLs) are involved in various cell functions, such as cell growth and motility. Recent studies showed changes in GSL expression during differentiation of human embryonic stem cells; however, little is known about expression profiles of GSLs in cancer stem cells (CSCs). CSCs are a small subpopulation in cancer and are proposed as cancer-initiating cells, have been shown to be resistant to numerous chemotherapies, and may cause cancer recurrence. Here, we analyzed GSLs expressed in human breast CSCs by applying a CSC model induced through epithelial-mesenchymal transition, using mass spectrometry, TLC immunostaining, and cell staining. We found that (i) Fuc-(n)Lc4Cer and Gb3Cer were drastically reduced in CSCs, whereas GD2, GD3, GM2, and GD1a were greatly increased in CSCs; (ii) among various glycosyltransferases tested, mRNA levels for ST3GAL5, B4GALNT1, ST8SIA1, and ST3GAL2 were increased in CSCs, which could explain the increased expression of GD3, GD2, GM2, and GD1a in CSCs; (iii) the majority of GD2+ cells and GD3+ cells were detected in the CD44(hi)/CD24(lo) cell population; and (iv) knockdown of ST8SIA1 and B4GALNT1 significantly reduced the expression of GD2 and GD3 and caused a phenotype change from CSC to a non-CSC, which was detected by reduced mammosphere formation and cell motility. Our results provide insight into GSL profiles in human breast CSCs, indicate a functional role of GD2 and GD3 in CSCs, and suggest a possible novel approach in targeting human breast CSCs to interfere with cancer recurrence.

  6. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  7. Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes.

    Science.gov (United States)

    Mott, David M; Stone, Karen; Gessel, Mary C; Bunt, Joy C; Bogardus, Clifton

    2008-02-01

    Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.

  8. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...

  10. N-acetylglutamate synthase deficiency and the treatment of hyperammonemic encephalopathy.

    Science.gov (United States)

    Elpeleg, Orly; Shaag, Avraham; Ben-Shalom, Efrat; Schmid, Tal; Bachmann, Claude

    2002-12-01

    Carbamylphosphate synthase is the first enzymatic reaction of the urea cycle. Its activator, N-acetylglutamate, is synthesized from acetyl-CoA and glutamate in a reaction catalyzed by N-acetylglutamate synthase (NAGS). We have identified the putative human NAGS gene and report the first mutation in this gene in a family with carbamylglutamate responsive hyperammonemia and normal activity of the urea cycle enzymes. Mutation analysis has a higher diagnostic specificity than the enzymatic assay in NAGS deficiency. A therapeutic trial with carbamylglutamate is recommended whenever hyperammonemia without an organic aciduria, increased orotate excretion, or diagnostic amino acidemia/uria is detected.

  11. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  12. Autopsy and postmortem examination case study on genetic risk factors for cardiac death: Polymorphisms of endothelial nitric oxide synthase gene Glu298asp variant and T-786c mutation, human paraoxonas

    Directory of Open Access Journals (Sweden)

    Ameno Kiyoshi

    2006-01-01

    Full Text Available Background/Aim. The Glu298Asp variant in exon 7 and T-786C mutation in the 5'-flanking region of the endothelial nitric oxide synthase (eNOS gene, paraoxonase I gene (PON1, and α2β- adrenergic receptor gene (α2β-AR have been reported to be genetic risk factors for coronary heart disease (CHD. The aim of this study was to investigate the effects of these four genetic polymorphisms on the probability of death due to CHD, using data obtained from medico-legal autopsies. Methods. Blood samples from three groups: healthy controls, dead cases with CHD and without CHD (the latter as a control for dead cases were used. After DNA extraction, genotyping was performed by polymerase chain reaction − restriction fragment length polymorphism (PCR-RFLP test. Results. The frequency of the T allele in Glu298Asp variant in the dead cases with CHD was significantly higher than that in the healthy control (p < 0.001, OR = 4.47 and that in the dead cases without CHD (p < 0.001, OR = 7.62. The gene frequency of PON1 was significantly different (p = 0.007 between dead cases with and without CHD, and was also significantly different (p = 0.025 between the healthy control and dead cases without CHD. The gene frequency of PON1 was not significantly different (p = 0.401 between the healthy controls and dead cases with CHD. Hence this gene was not associated with death due to CHD. The other polymorphisms (T- 786C mutation, α2β-AR also showed no effect on death due to CHD. Conclusion. The polymorphism of Glu298Asp eNOS gene in dead cases may be useful for determining the cause of death in CHD cases in the Japanese population.

  13. Understanding microscopic binding of human microsomal prostaglandin E synthase-1 (mPGES-1) trimer with substrate PGH2 and cofactor GSH: insights from computational alanine scanning and site-directed mutagenesis.

    Science.gov (United States)

    Hamza, Adel; Tong, Min; AbdulHameed, Mohamed Diwan M; Liu, Junjun; Goren, Alan C; Tai, Hsin-Hsiung; Zhan, Chang-Guo

    2010-04-29

    Microsomal prostaglandin E synthase-1 (mPGES-1) is an essential enzyme involved in a variety of diseases and is the most promising target for the design of next-generation anti-inflammatory drugs. In order to establish a solid structural base, we recently developed a model of mPGES-1 trimer structure by using available crystal structures of both microsomal glutathione transferase-1 (MGST1) and ba3-cytochrome c oxidase as templates. The mPGES-1 trimer model has been used in the present study to examine the detailed binding of mPGES-1 trimer with substrate PGH(2) and cofactor GSH. Results obtained from the computational alanine scanning reveal the contribution of each residue at the protein-ligand interaction interface to the binding affinity, and the computational predictions are supported by the data obtained from the corresponding wet experimental tests. We have also compared our mPGES-1 trimer model with other available 3D models, including an alternative homology model and a low-resolution crystal structure, and found that our mPGES-1 trimer model based on the crystal structures of both MGST1 and ba3-cytochrome c oxidase is more reasonable than the other homology model of mPGES-1 trimer constructed by simply using a low-resolution crystal structure of MGST1 trimer alone as a template. The available low-resolution crystal structure of mPGES-1 trimer represents a closed conformation of the enzyme and thus is not suitable for studying mPGES-1 binding with ligands. Our mPGES-1 trimer model represents a reasonable open conformation of the enzyme and is therefore promising for studying mPGES-1 binding with ligands in future structure-based drug design targeting mPGES-1.

  14. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice.

    Science.gov (United States)

    Tsutsui, Masato; Tanimoto, Akihide; Tamura, Masahito; Mukae, Hiroshi; Yanagihara, Nobuyuki; Shimokawa, Hiroaki; Otsuji, Yutaka

    2015-01-01

    Nitric oxide (NO) is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs), all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling), lung abnormalities (accelerated pulmonary fibrosis), and bone abnormalities (increased bone mineral density and bone turnover). These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  15. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice

    Directory of Open Access Journals (Sweden)

    Masato Tsutsui

    2015-01-01

    Full Text Available Nitric oxide (NO is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs, all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling, lung abnormalities (accelerated pulmonary fibrosis, and bone abnormalities (increased bone mineral density and bone turnover. These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  16. The Remarkable Character of Porphobilinogen Synthase.

    Science.gov (United States)

    Jaffe, Eileen K

    2016-11-15

    Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn(2+); a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb(2+). PBGSs that do not require the soft metal ion Zn(2+) at the active site instead are suspected of using the hard metal Mg(2+). The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg(2+) as an allosteric activator. Mg(2+) functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do

  17. The tomato terpene synthase gene family

    NARCIS (Netherlands)

    Falara, V.; Akhtar, T.A.; Nguyen, T.T.H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R.C.; Pichersky, E.

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  18. Cloning of parsley flavone synthase I.

    Science.gov (United States)

    Martens, S; Forkmann, G; Matern, U; Lukacin, R

    2001-09-01

    A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.

  19. Inducible nitric oxide synthase in renal transplantation

    NARCIS (Netherlands)

    Joles, JA; Vos, IH; Grone, HJ; Rabelink, TJ

    2002-01-01

    The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the induc

  20. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    Science.gov (United States)

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  1. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  2. Differential expression profiles of glycosphingolipids in human breast cancer stem cells vs. cancer non-stem cells

    DEFF Research Database (Denmark)

    Liang, Yuh-Jin; Ding, Yao; Levery, Steven B;

    2013-01-01

    Previous studies demonstrated that certain glycosphingolipids (GSLs) are involved in various cell functions, such as cell growth and motility. Recent studies showed changes in GSL expression during differentiation of human embryonic stem cells; however, little is known about expression profiles...... of GSLs in cancer stem cells (CSCs). CSCs are a small subpopulation in cancer and are proposed as cancer-initiating cells, have been shown to be resistant to numerous chemotherapies, and may cause cancer recurrence. Here, we analyzed GSLs expressed in human breast CSCs by applying a CSC model induced...... significantly reduced the expression of GD2 and GD3 and caused a phenotype change from CSC to a non-CSC, which was detected by reduced mammosphere formation and cell motility. Our results provide insight into GSL profiles in human breast CSCs, indicate a functional role of GD2 and GD3 in CSCs, and suggest...

  3. Tracking sesamin synthase gene expression through seed maturity in wild and cultivated sesame species--a domestication footprint.

    Science.gov (United States)

    Pathak, N; Bhaduri, A; Bhat, K V; Rai, A K

    2015-09-01

    Sesamin and sesamolin are the major oil-soluble lignans present in sesame seed, having a wide range of biological functions beneficial to human health. Understanding sesame domestication history using sesamin synthase gene expression could enable delineation of the sesame putative progenitor. This report examined the functional expression of sesamin synthase (CYP81Q1) during capsule maturation (0-40 days after flowering) in three wild Sesamum species and four sesame cultivars. Among the cultivated accessions, only S. indicum (CO-1) exhibited transcript abundance of sesamin synthase along with high sesamin content similar to S. malabaricum, while the other cultivated sesame showed low expression. The sesamin synthase expression analysis, coupled with quantification of sesamin level, indicates that sesamin synthase was not positively favoured during domestication. The sesamin synthase expression pattern and lignan content, along with phylogenetic analysis suggested a close relationship of cultivated sesame and the wild species S. malabaricum. The high genetic identity between the two species S. indicum and S. malabaricum points towards the role of the putative progenitor S. malabaricum in sesame breeding programmes to broaden the genetic base of sesame cultivars. This study emphasises the need to investigate intraspecific and interspecific variation in the primary, secondary and tertiary gene pools to develop superior sesame genotypes. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

  4. Cellulose Synthases and Synthesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Anne Endler; Staffan Persson

    2011-01-01

    Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.

  5. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  6. Building-block selectivity of polyketide synthases.

    Science.gov (United States)

    Liou, Grace F; Khosla, Chaitan

    2003-04-01

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

  7. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    Science.gov (United States)

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  8. 离心作用下人牙周膜细胞内诱导型一氧化氮合酶和胱硫醚分解酶的表达%Expression of inducible nitric oxide synthase and cystathionine-γ-lyase in cultured human periodontal ligament cells following centrifugal force stimulation

    Institute of Scientific and Technical Information of China (English)

    廖崇珊; 华咏梅; 冯欣华

    2012-01-01

    目的 研究在离心力作用下人牙周膜细胞内的诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和硫化氢合酶胱硫醚分解酶(cystat hionine-γ-lyase,CSE)的表达变化.方法 采用组织块-酶消化联合法培养人牙周膜细胞,对细胞施加离心力(167 g),SABC法染色及胞浆透光度检测iNOS和CSE在10、30、60、90、120和240 min不同加力时间点的表达变化.结果 正常人牙周膜细胞内几乎无iNOS和CSE表达;加力10 min,iNOS和CSE有表达(P<0.01);之后两种酶表达逐渐加强,加力60 min,iNOS和CSE表达达到高峰(P<0.01);之后逐渐减弱,加力240min,iNOS和CSE恢复到基础水平.结论 离心力可诱导人牙周膜细胞内iNOS和CSE的表达短暂性升高,提示一氧化氮(N())和硫化氢(H2S)可能参与了牙周组织改建中的力学信号转导过程.%Objective This study was to examine the changes of inducible nitric oxide synthase (iNOS) and hydrogen sulfide (H2S) synthase,cystathionine-γ-lyase (CSE) in cultured Human periodontal ligament fibrohlasts (HPDLFs)in response to centrifugal force stimulation. Methods Cultured HPDLFs with the method of tissue-enzymatic digestion were stimulated mechanically by centrifugal force (167 g) for 10,30,60,90,120 and 240 min,respectively.The expression of iNOSand CSE were analyzed by Streptavidin-biotin complex (SABC) immunocytochemistry combined with cytoplasmic light transmission measurement.Results The basal levels of iNOS and CSE in the HPDLFs were very low.Application of centrifugal force for 10 min resulted in a rapid increase in the iNOS and CSE expression (P<0.01).The expression levels gradually increased to a peak 60 min after applying force (P<0.01),then declined to the basal level at 240 min of sustained force.Conclusions Centrifugal force induced the expression of HPDLFs iNOS and CSE in the same pattern within a narrow time frame,suggesting iNOS and CSE may play an essential role in periodontium remodeling.

  9. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  10. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  11. Chloroquine stimulates glucose uptake and glycogen synthase in muscle cells through activation of Akt.

    Science.gov (United States)

    Halaby, Marie-Jo; Kastein, Brandon K; Yang, Da-Qing

    2013-06-14

    Chloroquine is a pharmaceutical agent that has been widely used to treat patients with malaria. Chloroquine has also been reported to have hypoglycemic effects on humans and animal models of diabetes. Despite many previous studies, the mechanism responsible for its hypoglycemic effect is still unclear. Chloroquine was recently reported to be an activator of ATM, the protein deficient in the Ataxia-telagiectasia (A-T) disease. Since ATM is also known as an insulin responsive protein that mediates Akt activation, we tested the effect of chloroquine on the activity of Akt and its downstream targets. In L6 muscle cells treated with insulin and chloroquine, the phosphorylation of Akt and glucose uptake were dramatically increased compared to cells treated with insulin alone, suggesting that chloroquine is a potent activator of Akt and glucose uptake in these cells. We also found that the reduction of insulin-mediated Akt activity in muscle tissues of insulin resistant rats was partially reversed by chloroquine treatment. Moreover, insulin-mediated phosphorylation of glycogen synthase kinase-3β in L6 cells was greatly enhanced by chloroquine. A substantial decrease in phosphorylation of glycogen synthase was also observed in chloroquine-treated L6 cells, indicating enhanced activity of glycogen synthase. Taken together, our results not only show that chloroquine is a novel activator of Akt that stimulates glucose uptake and glycogen synthase, but also validate chloroquine as a potential therapeutic agent for patients with type 2 diabetes mellitus. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. β-Adrenergic-mediated vasodilation in young men and women: cyclooxygenase restrains nitric oxide synthase.

    Science.gov (United States)

    Limberg, Jacqueline K; Johansson, Rebecca E; Peltonen, Garrett L; Harrell, John W; Kellawan, J Mikhail; Eldridge, Marlowe W; Sebranek, Joshua J; Schrage, William G

    2016-03-15

    We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a greater contribution of nitric oxide synthase and cyclooxygenase to β-adrenergic-mediated vasodilation compared with men. Forearm blood flow (Doppler ultrasound) was measured in young men (n = 29, 26 ± 1 yr) and women (n = 33, 25 ± 1 yr) during intra-arterial infusion of isoproterenol (β-adrenergic agonist). In subset of subjects, isoproterenol responses were examined before and after local inhibition of nitric oxide synthase [N(G)-monomethyl-l-arginine (l-NMMA); 6 male/10 female] and/or cyclooxygenase (ketorolac; 5 male/5 female). Vascular conductance (blood flow ÷ mean arterial pressure) was calculated to assess vasodilation. Vascular conductance increased with isoproterenol infusion (P 0.99) or women (P = 0.21). In contrast, ketorolac infusion markedly increased isoproterenol-mediated responses in both men (P vasodilation is not different between men and women and sex differences in the independent contribution of nitric oxide synthase and cyclooxygenase to β-mediated vasodilation are not present. However, these data are the first to demonstrate β-adrenoceptor activation of cyclooxygenase suppresses nitric oxide synthase signaling in human forearm microcirculation and may have important implications for neurovascular control in both health and disease.

  13. Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates

    Science.gov (United States)

    Caldwell, Harlan D.; Wood, Heidi; Crane, Debbie; Bailey, Robin; Jones, Robert B.; Mabey, David; Maclean, Ian; Mohammed, Zeena; Peeling, Rosanna; Roshick, Christine; Schachter, Julius; Solomon, Anthony W.; Stamm, Walter E.; Suchland, Robert J.; Taylor, Lacey; West, Sheila K.; Quinn, Tom C.; Belland, Robert J.; McClarty, Grant

    2003-01-01

    We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogen’s tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates — specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-γ resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-γ–mediated eradication and thus establish persistent infection. PMID:12782678

  14. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    Science.gov (United States)

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  15. Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants.

    Science.gov (United States)

    Gray, Dennis W; Breneman, Steven R; Topper, Lauren A; Sharkey, Thomas D

    2011-06-10

    2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ~90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K(+), whereas isoprene production is inhibited by K(+) such that, at physiologically relevant [K(+)], little or no isoprene emission should be detected from MBO-emitting trees. The K(m) of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site.

  16. Calcium and calcium sensing receptor modulates the expression of thymidylate synthase, NAD(P)H:quinone oxidoreductase 1 and survivin in human colon carcinoma cells: promotion of cytotoxic response to mitomycin C and fluorouracil

    OpenAIRE

    Liu, Guangming; Hu, Xin; Varani, James; Chakrabarty, Subhas

    2009-01-01

    Ca2+ and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca2+ (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-re...

  17. CJ-15,183, a new inhibitor of squalene synthase produced by a fungus, Aspergillus aculeatus.

    Science.gov (United States)

    Watanabe, S; Hirai, H; Ishiguro, M; Kambara, T; Kojima, Y; Matsunaga, T; Nishida, H; Suzuki, Y; Sugiura, A; Harwood, H J; Huang, L H; Kojima, N

    2001-11-01

    A new squalene synthase (SSase) inhibitor, CJ-15,183 (I) was isolated from the fermentation broth of a fungus, Aspergillus aculeatus CL38916. The compound potently inhibited rat liver and Candida albicans microsomal SSases and also inhibited the human enzyme. It also showed antifungal activities against filamentous fungi and a yeast. The structure was determined to be an aliphatic tetracarboxylic acid compound consisting of an alkyl gamma-lactone, malic acid and isocitric acid moieties by spectroscopic studies.

  18. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  19. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives.

  20. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    OpenAIRE

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity ...

  1. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Science.gov (United States)

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

  2. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Berta Alquézar

    2017-08-01

    Full Text Available Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck genome sequence allowed us to characterize for the first time the terpene synthase (TPS family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  3. Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT

    Science.gov (United States)

    Vicidomini, R; Di Giovanni, A; Petrizzo, A; Iannucci, L F; Benvenuto, G; Nagel, A C; Preiss, A; Furia, M

    2015-01-01

    still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity. PMID:25811802

  4. Targeting of the Virulence Factor Acetohydroxyacid Synthase by Sulfonylureas Results in Inhibition of Intramacrophagic Multiplication of Brucella suis

    OpenAIRE

    Boigegrain, Rose-Anne; Liautard, Jean-Pierre; Köhler, Stephan

    2005-01-01

    The acetohydroxyacid synthase (AHAS) of Brucella suis can be effectively targeted by the sulfonylureas chlorimuron ethyl and metsulfuron methyl. Growth in minimal medium was inhibited, and multiplication in human macrophages was totally abolished with 100 μM of sulfonylureas. Metsulfuron methyl-resistant mutants showed reduced viability in macrophages and reduced AHAS activity.

  5. Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

    NARCIS (Netherlands)

    Gady, A.L.F.; Vriezen, W.; Wal, van de M.H.B.J.; Huang, P.; Bovy, A.G.; Visser, R.G.F.; Bachem, C.W.B.

    2012-01-01

    In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutatio

  6. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis

    Science.gov (United States)

    Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control caro...

  7. Evolution and function of phytochelatin synthases.

    Science.gov (United States)

    Clemens, Stephan

    2006-02-01

    Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

  8. Torque generation mechanism of ATP synthase

    Science.gov (United States)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  9. Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

    Directory of Open Access Journals (Sweden)

    Vinciane Régnier

    Full Text Available BACKGROUND: The cystathionine β-synthase (CBS gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA metabolism, a pathway important for several brain physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1 expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line. CONCLUSION/SIGNIFICANCE: We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

  10. Squalene Synthase As a Target for Chagas Disease Therapeutics

    Science.gov (United States)

    Chan, Hsiu-Chien; Li, Jikun; Zheng, Yingying; Huang, Chun-Hsiang; Ren, Feifei; Chen, Chun-Chi; Zhu, Zhen; Galizzi, Melina; Li, Zhu-Hong; Rodrigues-Poveda, Carlos A.; Gonzalez-Pacanowska, Dolores; Veiga-Santos, Phercyles; de Carvalho, Tecia Maria Ulisses; de Souza, Wanderley; Urbina, Julio A.; Wang, Andrew H.-J.; Docampo, Roberto; Li, Kai; Liu, Yi-Liang; Oldfield, Eric; Guo, Rey-Ting

    2014-01-01

    Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease. PMID:24789335

  11. Squalene synthase as a target for Chagas disease therapeutics.

    Directory of Open Access Journals (Sweden)

    Na Shang

    2014-05-01

    Full Text Available Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

  12. [Four cases of aldosterone synthase deficiency in childhood].

    Science.gov (United States)

    Collinet, E; Pelissier, P; Richard, O; Gay, C; Pugeat, M; Morel, Y; Stephan, J-L

    2012-11-01

    Neonatal salt-wasting syndromes are rare but potentially serious conditions. Isolated hypoaldosteronism is an autosomal recessive inherited disorder of terminal aldosterone synthesis, leading to selective aldosterone deficiency. Two different biochemical forms of this disease have been described, called aldosterone synthase deficiency or corticosterone methyl oxydase, types I and II. In type I, there is no aldosterone synthase activity and the 18 hydroxycorticosterone (18 OHB) level is low, whereas in type II, a residual activity of aldosterone synthase persists and 18 OHB is overproduced. We report on four patients with isolated hypoaldosteronism. In 2 of them, who were recently diagnosed with aldosterone synthase deficit, we discuss the symptoms and treatment. The 2 other patients are now adults. We discuss the long-term outcome, the quality of adult life, aldosterone synthase deficits, as well as the pathophysiology and molecular analysis.

  13. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  14. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  15. Expression of fatty acid synthase in nonalcoholic fatty liver disease.

    Science.gov (United States)

    Dorn, Christoph; Riener, Marc-Oliver; Kirovski, Georgi; Saugspier, Michael; Steib, Kathrin; Weiss, Thomas S; Gäbele, Erwin; Kristiansen, Glen; Hartmann, Arndt; Hellerbrand, Claus

    2010-03-25

    Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD.

  16. Complexing mechanism of the lanthanide cations Eu3+, Gd3+, and Tb3+ with 1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (dota)-characterization of three successive complexing phases: study of the thermodynamic and structural properties of the complexes by potentiometry, luminescence spectroscopy, and EXAFS.

    Science.gov (United States)

    Moreau, Juliette; Guillon, Emmanuel; Pierrard, Jean-Claude; Rimbault, Jean; Port, Marc; Aplincourt, Michel

    2004-10-11

    Complexation of the lanthanides Eu3+, Gd3+, and Tb3+ with 1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (dota) has been studied in solution by using potentiometry, luminescence spectrometry, and EXAFS. Three series of successive complexes were characterized by at least two of these methods: the immediate [LnHn(dota)](n-1)+** and intermediate [LnHn(dota)](n-1)+* complexes with 0 potentiometry. From the results, a complexation mechanism involving three steps has been proposed. In the [LnHn(dota)](n-1)+** complexes that are instantaneously formed, the lanthanide is bound to four oxygen atoms of the carboxylate groups and to five water molecules. These species evolve rapidly: the lanthanide moves into the macrocycle cavity, two new bonds are formed with two nitrogen atoms diametrically opposed in the tetraaza cycle and only three water molecules remain bound to the lanthanide in the [LnHn(dota)](n-1)+* (0

  17. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway.

    Science.gov (United States)

    Taura, Futoshi; Tanaka, Shinji; Taguchi, Chiho; Fukamizu, Tomohide; Tanaka, Hiroyuki; Shoyama, Yukihiro; Morimoto, Satoshi

    2009-06-18

    Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.

  18. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  19. The nitric oxide synthase of mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Goin, J C; Boquet, M; Canteros, M G; Franchi, A M; Perez Martinez, S; Polak, J M; Viggiano, J M; Gimeno, M A

    1997-07-01

    Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

  20. Endothelial nitric oxide synthase in the microcirculation.

    Science.gov (United States)

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

  1. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    Science.gov (United States)

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of BPS was rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. 3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor substrate for PPS. The aryl moiety of the triketide intermediate may be trapped in the new pocket by hydrogen bond formation with the backbone, thereby acting as an inhibitor. PPS is a promising biotechnological tool for manipulating benzoate-primed biosynthetic pathways to produce novel compounds. PMID:19710020

  2. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Science.gov (United States)

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  3. Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

    Science.gov (United States)

    Iskarpatyoti, Jason A; Morse, E Ashley; McClung, R Paul; Ikizler, Miné; Wetzel, J Denise; Contractor, Nikhat; Dermody, Terence S

    2012-11-25

    Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk.

  4. Role of cysteine residues in pseudouridine synthases of different families.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  5. Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins.

    Science.gov (United States)

    Vestergaard, Martin; Nøhr-Meldgaard, Katrine; Bojer, Martin Saxtorph; Krogsgård Nielsen, Christina; Meyer, Rikke Louise; Slavetinsky, Christoph; Peschel, Andreas; Ingmer, Hanne

    2017-09-05

    Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics.IMPORTANCE Bacterial pathogens that cause disease in humans remain a serious threat to public health, and antibiotics are still our primary weapon in treating bacterial diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available

  6. Subcellular localization of the homocitrate synthase in Penicillium chrysogenum.

    Science.gov (United States)

    Bañuelos, O; Casqueiro, J; Steidl, S; Gutiérrez, S; Brakhage, A; Martín, J F

    2002-01-01

    There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

  7. The Pseudouridine Synthases Proceed through a Glycal Intermediate.

    Science.gov (United States)

    Veerareddygari, Govardhan Reddy; Singh, Sanjay K; Mueller, Eugene G

    2016-06-29

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2'. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases.

  8. Peroxisomal and mitochondrial citrate synthase in CAM plants.

    Science.gov (United States)

    Zafra, M F; Segovia, J L; Alejandre, M J; García-Peregrín, E

    1981-12-01

    Citrate synthase wa studied for the first time in peroxisomes and mitochondria of crassulacean acid metabolism plants. Cellular organelles were isolated from Agave americana leaves by sucrose density gradient centrifugation and characterized by the use of catalase and cytochrome oxidase as marker enzymes, respectively. 48,000 X g centrifugation caused the breakdown of the cellular organelles. The presence of a glyoxylate cycle enzyme (citrate synthase) and a glycollate pathway enzyme (catalase) in the same organelles, besides the absence of another glyoxalate cycle enzyme (malate synthase) is reported for the first time, suggesting that peroxisomal and glyoxysomal proteins are synthesized at the same time and housed in he same organelle.

  9. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

    Science.gov (United States)

    Mühleip, Alexander W.; Dewar, Caroline E.; Schnaufer, Achim; Kühlbrandt, Werner; Davies, Karen M.

    2017-01-01

    We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis. PMID:28096380

  10. Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues.

    Science.gov (United States)

    Spedaliere, C J; Hamilton, C S; Mueller, E G

    2000-08-01

    On the basis of sequence alignments, the pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered [Koonin, E. V. (1996) Nucleic Acids. Res. 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762]. We have investigated the functional significance of these alignments by substituting the nearly invariant lysine and proline residues in Motif I of RluA and TruB, pseudouridine synthases belonging to different families. Contrary to our expectations, the altered enzymes display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate proline residue in Cbf5p, a yeast homologue of TruB [Zerbarjadian, Y., King, T., Fournier, M. J., Clarke, L., and Carbon, J. (1999) Mol. Cell. Biol. 19, 7461-7472]. Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated. By extrapolation, our findings also support the assignment of pseudouridine synthase function to certain physiologically important eukaryotic proteins that contain Motif I, including the human protein dyskerin, alteration of which leads to the disease dyskeratosis congenita.

  11. Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

    Science.gov (United States)

    De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

    2014-01-01

    Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

  12. Loss of ceramide synthase 3 causes lethal skin barrier disruption.

    Science.gov (United States)

    Jennemann, Richard; Rabionet, Mariona; Gorgas, Karin; Epstein, Sharon; Dalpke, Alexander; Rothermel, Ulrike; Bayerle, Aline; van der Hoeven, Franciscus; Imgrund, Silke; Kirsch, Joachim; Nickel, Walter; Willecke, Klaus; Riezman, Howard; Gröne, Hermann-Josef; Sandhoff, Roger

    2012-02-01

    The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1-6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.

  13. N-acetylglutamate synthase: structure, function and defects.

    Science.gov (United States)

    Caldovic, Ljubica; Ah Mew, Nicholas; Shi, Dashuang; Morizono, Hiroki; Yudkoff, Marc; Tuchman, Mendel

    2010-01-01

    N-acetylglutamate (NAG) is a unique enzyme cofactor, essential for liver ureagenesis in mammals while it is the first committed substrate for de novo arginine biosynthesis in microorganisms and plants. The enzyme that produces NAG from glutamate and CoA, NAG synthase (NAGS), is allosterically inhibited by arginine in microorganisms and plants and activated in mammals. This transition of the allosteric effect occurred when tetrapods moved from sea to land. The first mammalian NAGS gene (from mouse) was cloned in 2002 and revealed significant differences from the NAGS ortholog in microorganisms. Almost all NAGS genes possess a C-terminus transferase domain in which the catalytic activity resides and an N-terminus kinase domain where arginine binds. The three-dimensional structure of NAGS shows two distinctly folded domains. The kinase domain binds arginine while the acetyltransferase domain contains the catalytic site. NAGS deficiency in humans leads to hyperammonemia and can be primary, due to mutations in the NAGS gene or secondary due to other mitochondrial aberrations that interfere with the normal function of the same enzyme. For either condition, N-carbamylglutamate (NCG), a stable functional analog of NAG, was found to either restore or improve the deficient urea-cycle function.

  14. Helicobacter pylori does not use spermidine synthase to produce spermidine.

    Science.gov (United States)

    Zhang, Huawei; Au, Shannon Wing Ngor

    2017-08-26

    Helicobacter pylori is the primary pathogen associated to gastritis and gastric cancer. Growth of H. pylori depends on the availability of spermidine in vivo. Interestingly, the genome of H. pylori contains an incomplete set of genes for the classical pathway of spermidine biosynthesis. It is thus not clear whether some other genes remained in the pathway would have any functions in spermidine biosynthesis. Here, we study spermidine synthase, which is responsible for the final catalytic process in the classical route. Protein sequence alignment reveals that H. pylori SpeE (HpSpeE) lacks key residues for substrate binding. By using isothermal titration calorimetry, we show that purified recombinant HpSpeE does not interact with the putative substrates putrescine and decarboxylated S-adenosylmethionine, and the product spermidine. High performance liquid chromatography analysis further demonstrates that HpSpeE has no detectable in vitro enzymatic activity. Additionally, intracellular spermidine level in speE-null mutant strain is comparable to that in the wild type strain. Collectively, our results suggest that HpSpeE is functionally distinct from spermidine production. H. pylori may instead employ the alternative pathway for spermidine synthesis which is dominantly exploited by other human gut microbes. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Gelatinization temperature of rice explained by polymorphisms in starch synthase.

    Science.gov (United States)

    Waters, Daniel L E; Henry, Robert J; Reinke, Russell F; Fitzgerald, Melissa A

    2006-01-01

    The cooking quality of rice is associated with the starch gelatinization temperature (GT). Rice genotypes with low GT have probably been selected for their cooking quality by humans during domestication. We now report polymorphisms in starch synthase IIa (SSIIa) that explain the variation in rice starch GT. Sequence analysis of the eight exons of SSIIa identified significant polymorphism in only exon 8. These single nucleotide polymorphisms (SNPs) were determined in 70 diverse genotypes of rice. Two SNPs could classify all 70 genotypes into either high GT or low GT types which differed in GT by 8 degrees C. 'A' rather than 'G' at base 2412 determined whether a methionine or valine was present at the corresponding amino acid residue in SSIIa, whilst two adjacent SNPs at bases 2543 and 2544 coded for either leucine (GC) or phenylalanine (TT). Rice varieties with high GT starch had a combination of valine and leucine at these residues. In contrast, rice varieties with low GT starch had a combination of either methionine and leucine or valine and phenylalanine at these same residues. At least two distinct polymorphisms have apparently been selected for their desirable cooking qualities in the domestication of rice.

  16. Nitric oxide synthase: non-canonical expression patterns

    Directory of Open Access Journals (Sweden)

    Mattila eJoshua

    2014-10-01

    Full Text Available Science can move ahead by questioning established or canonical views and, so it may be with the enzymes, nitric oxide synthases (NOS. Nitric oxide (NO is generated by NOS isoforms that are often described by their tissue-specific expression patterns. NOS1 (nNOS is abundant in neural tissue, NOS2 is upregulated in activated macrophages and known as inducible NOS (iNOS, and NOS3 (eNOS is abundant in endothelium where it regulates vascular tone. These isoforms are described as constitutive or inducible, but in this Perspective we question the broad application of these labels. Are there instances where ‘constitutive’ NOS (NOS1 and NOS3 are inducibly expressed; conversely, are there instances where NOS2 is constitutively expressed? NOS1 and NOS3 inducibility may be linked to post-translational regulation, making their actual patterns activity much more difficult to detect. Constitutive NOS2 expression has been observed several tissues, especially the human pulmonary epithelium where it may regulate airway tone. These data suggest expression of the three NOS enzymes may include non-established patterns. Such information should be useful in designing strategies to modulate these important enzymes in different disease states.

  17. Increased resting intracellular calcium modulates NF-κB-dependent inducible nitric-oxide synthase gene expression in dystrophic mdx skeletal myotubes.

    Science.gov (United States)

    Altamirano, Francisco; López, Jose R; Henríquez, Carlos; Molinski, Tadeusz; Allen, Paul D; Jaimovich, Enrique

    2012-06-15

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca(2+)](rest)) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca(2+)](rest) was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca(2+) entry (low Ca(2+) solution, Ca(2+)-free solution, and Gd(3+)) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca(2+)](rest). Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca(2+)](rest) was reduced. Levels of mRNA for TNFα, IL-1β, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca(2+)](rest) using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca(2+)](rest), is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells.

  18. ATP synthase ecto-α-subunit: a novel therapeutic target for breast cancer

    Directory of Open Access Journals (Sweden)

    Pan Jian

    2011-12-01

    Full Text Available Abstract Background Treatment failure for breast cancer is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in breast cancer cells in vitro and in vivo. Identification of new targets will facilitate the developmental pace of new techniques in screening and early diagnosis. Improved abilities to predict progression and metastasis, therapeutic response and toxicity will help to increase survival of breast cancer patients. Methods Differential protein expression in two breast cancer cell lines, one with high and the other with low metastatic potential, was analyzed using two-dimensional liquid phase chromatographic fractionation (Proteome Lab PF 2D system followed by matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI-TOF/MS. Results Up regulation of α-subunit of ATP synthase was identified in high metastatic cells compared with low metastatic cells. Immunohistochemical analysis of 168 human breast cancer specimens on tissue microarrays revealed a high frequency of ATP synthase α-subunit expression in breast cancer (94.6% compared to normal (21.2% and atypical hyperplasia (23% breast tissues. Levels of ATP synthase expression levels strongly correlated with large tumor size, poor tumor differentiation and advanced tumor stages (P Conclusions Over-expression of ATP synthase α-subunit may be involved in the progression and metastasis of breast cancer, perhaps representing a potential biomarker for diagnosis, prognosis and a therapeutic target for breast cancer. This finding of this study will help us to better understand the molecular mechanism of tumor metastasis and to improve the screening, diagnosis, as well as prognosis and/or prediction of responses to therapy for breast cancer.

  19. Glutamine analogs promote cytoophidium assembly in human and Drosophila cells

    Institute of Scientific and Technical Information of China (English)

    Kangni Chen; Jing Zhang; (O)mür Yilmaz Tastan; Zillah Anne Deussen; Mayte Yu-Yin Siswick; Ji-Long Liu

    2011-01-01

    CTP synthase is compartmentalized within a subcellular structure,termed the cytoophidium,in a range of organisms including bacteria,yeast,fruit fly and rat.Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells.Surprisingly,the occurrence of cyloophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-L-norleucine (DON),an inhibitor of glutaminedependent enzymes including CTP synthase.Experiments in flies confirmned that DON globally promotes cytoophidium assembly.Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia.Moreover,DON facilitates cytoophidium assembly even when CTP synthase level is low.A second glutamine analog azaserine also promotes cytoophidum formation.Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia.

  20. Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

    NARCIS (Netherlands)

    Banzhaf, M.; van den Berg van Saparoea, B.; Terrak, M.; Fraipont, C.; Egan, A.; Philippe, J.; Zapun, A.; Breukink, E.; Nguyen-Distèche, M.; den Blaauwen, T.; Vollmer, W.

    2012-01-01

    Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan

  1. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Directory of Open Access Journals (Sweden)

    Zulfiqar Ahmad

    Full Text Available We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  2. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Science.gov (United States)

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  3. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... 1Department of Environmental Biotechnology, Bharathidasan University, ... script and UA cloning vector (QIAGEN PCR Cloning Kit) was used to clone ..... Expression of a Arabidopsis sucrose synthase gene indicates a role.

  4. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function

    OpenAIRE

    Haines, Ricci J.; Corbin, Karen D.; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C.

    2012-01-01

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regu...

  5. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences.

    Directory of Open Access Journals (Sweden)

    Andrew eCarroll

    2011-03-01

    Full Text Available The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized subfamilies and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair cellulose synthase function, and compare these sites to those observed in the closest cellulose synthase-like (Csl families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  6. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  7. Nitric Oxide Synthases in Heart Failure

    Science.gov (United States)

    Carnicer, Ricardo; Crabtree, Mark J.; Sivakumaran, Vidhya

    2013-01-01

    Abstract Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal. 18, 1078–1099. PMID:22871241

  8. Effect of a selective inducible nitric oxide synthase inhibitor on cell growth in human colorectal cancer Lovo cell line%选择性诱生型一氧化氮合酶抑制剂对人结直肠癌Lovo细胞生长的影响

    Institute of Scientific and Technical Information of China (English)

    魏波; 卫洪波; 亓翠玲; 韩晓燕; 王天宝

    2008-01-01

    Objective To investigate the effect of a selective inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), on the proliferation and apoptosis of human colorectal cancer (CRC) Lovo cell line, and explore its possible mechanism. Methods MTT assay was used to detect the inhibition of Lovo cell growth by aminoguanidine. Apoptosis and cell cycle of Lovo cells were examined by flow cytometry (FCM). Morphologic change of Lovo cell treated by AG was observed with AO/EB staining. Results There were significant differences in 0.5 mmol/L and 1.0 mmol/L AG groups as compared to the control group (P<0.05). The absorbance (A) values of Lovo cells in each time point were significantly different (P<0.05). Growth of Lovo cells was inhibited by aminoguanidine in a dose-and time-dependent manner. FCM analysis showed that the cell ratio of G0/G1 phase increased with the increasing of the concentration of aminoguanidine, but the cell ratio of S-and G2/M phase decreased correspondingly (P<0.05). S phase fraction and proliferation index (PI)decreased remarkabely, and the apoptotic rate of Lovo cells increased. After AG treatment, AO/EB staining revealed some apoptotic morphological features such as cell shrinkage, nuclear condensation,DNA fragmentation, and formation of apoptosis bodies. Conclusions Aminoguanidine inhibits the proliferation and facilitates the apoptosis of human CRC Lovo cells. One of the mechanisms may be explained as blocking the progress of cell cycle of CRC Lovo cells by aminoguanidine.%目的 研究选择性诱生型一氧化氮合酶(iNOS)抑制剂氨基胍(AG)对人结直肠癌细胞株Lovo增殖与凋亡的影响,并对其作用机制进行初步探讨.方法 应用四唑盐(MTT)比色法检测AG对Lovo细胞增殖的抑制作用,流式细胞术检测分析不同浓度AG作用后Lovo细胞的凋亡率和细胞周期分布变化.并用丫啶橙结合溴化乙锭染色荧光显微镜观察凋亡细胞形态学改变.结果 AG 0.5 mmol

  9. RNA diversity has profound effects on the translation of neuronal nitric oxide synthase

    OpenAIRE

    Wang, Yang; Newton, Derek C.; Robb, G. Brett; Kau, Cheng-Lin; Miller, Tricia L.; Cheung, Anthony H.; Hall,Anne V.; VanDamme, Suzannah; Wilcox, Josiah N.; Marsden, Philip A.

    1999-01-01

    A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) mRNA species revealed NOS1 to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5′-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts wi...

  10. Structure-Based Inhibitors Exhibit Differential Activities against Helicobacter pylori and Escherichia coli Undecaprenyl Pyrophosphate Synthases

    Directory of Open Access Journals (Sweden)

    Chih-Jung Kuo

    2008-01-01

    Full Text Available Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS, which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.

  11. ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

    Directory of Open Access Journals (Sweden)

    Kexiu Song

    2014-01-01

    Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

  12. Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

    Science.gov (United States)

    Lahuta, Lesław B; Pluskota, Wioletta E; Stelmaszewska, Joanna; Szablińska, Joanna

    2014-09-01

    The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

  13. Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii

    Directory of Open Access Journals (Sweden)

    Huan Zhao

    2017-03-01

    Full Text Available Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS and cycloartenol synthase (CAS are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR approach. The SgSQS cDNA has a 1254 bp open reading frame (ORF encoding 417 amino acids, and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression.

  14. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  15. Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    Nassar Marwa

    2006-10-01

    Full Text Available Abstract Background Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. Results We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098 after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7, suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human

  16. Cystathionine beta-synthase deficiency causes fat loss in mice.

    Directory of Open Access Journals (Sweden)

    Sapna Gupta

    Full Text Available Cystathionine beta synthase (CBS is the rate-limiting enzyme responsible for the de novo synthesis of cysteine. Patients with CBS deficiency have greatly elevated plasma total homocysteine (tHcy, decreased levels of plasma total cysteine (tCys, and often a marfanoid appearance characterized by thinness and low body-mass index (BMI. Here, we characterize the growth and body mass characteristics of CBS deficient TgI278T Cbs(-/- mice and show that these animals have significantly decreased fat mass and tCys compared to heterozygous sibling mice. The decrease in fat mass is accompanied by a 34% decrease in liver glutathione (GSH along with a significant decrease in liver mRNA and protein for the critical fat biosynthesizing enzyme Stearoyl CoA desaturase-1 (Scd-1. Because plasma tCys has been positively associated with fat mass in humans, we tested the hypothesis that decreased tCys in TgI278T Cbs(-/- mice was the cause of the lean phenotype by placing the animals on water supplemented with N-acetyl cysteine (NAC from birth to 240 days of age. Although NAC treatment in TgI278T Cbs(-/- mice caused significant increase in serum tCys and liver GSH, there was no increase in body fat content or in liver Scd-1 levels. Our results show that lack of CBS activity causes loss of fat mass, and that this effect appears to be independent of low serum tCys.

  17. Characterization of the phytochelatin synthase of Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Debalina Ray

    2011-05-01

    Full Text Available Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS. Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS. The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism.

  18. Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration

    DEFF Research Database (Denmark)

    Guterbaum, Thomas Jeremy; Braunstein, Thomas Hartig; Fossum, A;

    2013-01-01

    Hydrogen peroxide (H₂O₂) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein end...... endothelial cells (HUVECs) exposed to H₂O₂....

  19. Common 894G>T single nucleotide polymorphism in the gene coding for endothelial nitric oxide synthase (eNOS) and risk of congenital heart defects

    NARCIS (Netherlands)

    Beynum, van I.M.; Mooij, C.; Kapusta, L.; Heil, S.G.; Heijer, den M.

    2008-01-01

    BACKGROUND: Endothelial nitric oxide synthase (eNOS) produces nitric oxide, which plays a role in vasodilatation and in the regulation of cell growth and apoptosis. eNOS-deficient mice have impaired cardiac development resulting in congenital heart defects (CHDs). In humans, a single nucleotide

  20. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  1. Properties of peroxisomal and mitochondrial citrate synthase from Agave americana.

    Science.gov (United States)

    Segovia, J L; Zafra, M F; Alejandre, M J; García-Peregrín, E

    1982-09-01

    Adenine nucleotides were tested as effectors of peroxisomal and mitochondrial citrate synthase from Agave americana leaves in the presence of different concentrations of acetyl-CoA and oxalacetate substrates. ATP inhibited both enzyme activities but with a different inhibition profile. 1.0-7.5 mM ADP did not inhibit the peroxisomal citrate synthase in the presence of high substrate concentrations, while the mitochondrial enzyme was strongly inhibited by 1.0 mM ADP in the same conditions. Likewise, a different pattern was obtained with AMP on both peroxisomal and mitochondrial activities. The rate of citrate formation as function of acetyl-CoA and oxalacetate concentration was also studied in both fractions. Maximal velocity was highest in the peroxisomal fraction, whether acetyl-CoA or oxalacetate were the variable substrates. These differences indicate that peroxisomal and mitochondrial citrate synthases seem to be two different isoenzymes.

  2. 重组腺病毒气管途径反复转染大鼠肺组织人类eNOS基因的转导效果%Efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase gene into lung tissue by repeated intratracheal transfection in rats

    Institute of Scientific and Technical Information of China (English)

    周锦; 曹惠鹃; 张铁铮; 金强; 王俊科

    2012-01-01

    Objective To investigate the efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase (eNOS) into lung tissue by repeated intratracheal transfection in rats.Methods Sixty 3-4 month old male Wistar rats weighing 220-280 g were randomly divided into 2 groups:control group (group C,n =10) and eNOS gene transduction group (group T,n =50).The animals were anesthetized with intraperitoneal 10% chloral hydrate 35 mg/kg,tracheally intubated and mechanically ventilated (VT 2.5 ml,RR 60 bpm,FiO2 1.0).Recombinant adenovirus carrying human eNOS gene was given as gift by Professor Gerard from Texas University,Southwest Medical Center.In group T 50 μl of the recombinant adenovirus in concentration of 5 × 109 PFU/ml was instilled into trachea every 5 minutes for 12 times,while in group C equal volume of vector conservation solution was instilled instead.Pulmonary arterial blood samples were obtained at 2,5,7,14 and 21 d after intratracheal transfection (n =10 at each time point) for determination of serum NO concentration.The animals were immediately sacrificed after blood sample collection for determination of expression of eNOS protein in the lung tissue and RNA.The eNOS expression in the trachea,bronchus,lung,liver,spleen and kidney was detected by immuno-histochemistry.Results The serum NO concentrations were significantly higher at all time points in group T than in group C.The eNOS expression was detected in the epithelial cells of trachea and bronchi,and endothelial cells of alveoli and pulmonary blood vessels in group T but not in group C.eNOS expression was not detected in liver,spleen and kidney at 7 d after intratracheal transfection in group T.Conclusion Human eNOS gene mediated by recombinant adenovirus was transducted into rat lung tissue with normal enzyme activity by repeated intratracheal administration without being detected in distant organs.%目的 重组腺病毒气管途径反复转染大鼠肺组织人类内

  3. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    Science.gov (United States)

    Robinson, M L; Carman, G M

    1982-01-01

    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  4. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

    Directory of Open Access Journals (Sweden)

    Yan Ping Lim

    2016-06-01

    Full Text Available Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

  5. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

    OpenAIRE

    2010-01-01

    Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphib...

  6. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    OpenAIRE

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control...

  7. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...... PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  8. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  9. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

    Science.gov (United States)

    Belkheir, Asma K; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

  10. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    Science.gov (United States)

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  11. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    Science.gov (United States)

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-26

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

  12. Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p.

    Science.gov (United States)

    Maceluch, J; Kmieciak, M; Szweykowska-Kulińska, Z; Jarmołowski, A

    2001-01-01

    Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

  13. Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses1[OPEN

    Science.gov (United States)

    Schwerdt, Julian G.; Wright, Frank; Oehme, Daniel; Wagner, John M.; Shirley, Neil J.; Burton, Rachel A.; Schreiber, Miriam; Zimmer, Jochen; Marshall, David F.; Waugh, Robbie; Fincher, Geoffrey B.

    2015-01-01

    Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-β-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-β-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels. PMID:25999407

  14. Trichothecenes induce accumulation of glucosylceramide in neural cells by interfering with lactosylceramide synthase activity.

    Science.gov (United States)

    Kralj, Ana; Gurgui, Mihaela; König, Gabriele M; van Echten-Deckert, Gerhild

    2007-11-15

    Trichothecenes are sesquiterpenoid metabolites produced by several fungal strains that impair human and animal health. Since sphingolipids were connected with fungal toxicity the aim of the present study was to test the influence of fungal metabolites on sphingolipid metabolism in neural cells. The crude extract of fungal strain Spicellum roseum induced accumulation of glucosylceramide (GlcCer), and simultaneous reduction of the formation of lactosylceramide (LacCer) and complex gangliosides in primary cultured neurons. Following a bioassay-guided fractionation of the respective fungal extract we could demonstrate that the two isolated trichothecene derivatives, 8-deoxy-trichothecin (8-dT) and trichodermol (Td-ol) were responsible for this effect. Thus, incubation of primary cultured neurons as well as of neuroblastoma B104 cells for 24 h with 30 microM of either of the two fungal metabolites resulted in uncoupling of sphingolipid biosynthesis at the level of LacCer. For the observed reduction of LacCer synthase activity by about 90% cell integrity was crucial in both cell types. In neuroblastoma cells the amount of LacCer synthase mRNA was reduced in the presence of trichothecenes, whereas in primary cultured neurons this was not the case, suggesting a post-transcriptional mechanism of action in the latter cell type. The data also show that the compounds did not interfere with the translocation of GlcCer in neuroblastoma cells. Collectively, our results demonstrate that trichodermol and 8-deoxy-trichothecin inhibit LacCer synthase activity in a cell-type-specific manner.

  15. Two polyketide-synthase-associated acyltransferases are required for sulfolipid biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Bhatt, Kiranmai; Gurcha, Sudagar S; Bhatt, Apoorva; Besra, Gurdyal S; Jacobs, William R

    2007-02-01

    The methyl-branched fatty acyl components of sulfolipid-I (SL-I), a major glycolipid of the human pathogen Mycobacterium tuberculosis, are synthesized by the polyketide synthase Pks2. Rv3824c (papA1), located downstream of pks2, encodes a protein that belongs to a subfamily of acyltransferases associated with mycobacterial polyketide synthases [polyketide synthase-associated proteins (PAPs)]. The presence of a conserved acyltransferase motif (HX(3)DX(14)Y) suggested a role for PapA1 in acylation of sulfated trehalose to form SL-I. Targeted deletion of the H37Rv papA1 resulted in loss of SL-I, demonstrating its role in mycobacterial sulfolipid biosynthesis. Furthermore, SL-I synthesis was restored in the mutant strain following complementation with papA1, but not with mutant alleles of papA1 containing alterations of key residues in the acyltransferase motif, confirming that PapA1 was an acyltransferase. While other M. tuberculosis pks clusters are associated with a single PAP-encoding gene, it was demonstrated that another open reading frame, Rv3820c (papA2), located 5.8 kb downstream of papA1 is also an acyltransferase gene involved in SL-I biosynthesis: deletion of papA2 abolished SL-I production. The absence of any partially acylated intermediates in either null mutant indicated that both PapA1 and PapA2 were required for all acylation steps of SL-I assembly.

  16. Expression, Purification and Activity Assay of the Full-length and Truncated Human Cystathionine β-Synthase%人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

    Institute of Scientific and Technical Information of China (English)

    牛卫宁; 羊梦林; 曹珊珊; 许乐; 钦传光

    2011-01-01

    将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta( DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS).重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基.将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1% CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1 mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735 U/mgo同时构建了表达CBS1413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E coli Rosetta (pETDuet-1-CBS1413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965 U/rng;还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法.%The human cystathionine β-synthase (CBS) gene was ligated into vector pGEX-4T-l. The recombinant pGEX-4T-1 -CBS was transformed into E. Coli Rosetta (DE3) , and the recombinant E. Coli Rosetta (pGEX4T-1-CBS) strain which highly express CBS gene was constructed. After the recombinant E. Coli was grown at 37℃ to an A600 of 0.4~0. 6, induced with IPTG at a final concentration of 0. 1mmol/L for 16h at 30℃. The productivity of the soluble CBS reached 28mg/L. The supernatant of the disrupted cells by sonication was directly loaded on GSTrap Fast Flow, and the GST-CBS fusion protein was absorbed on the column. The GST- tagged CBS fusion protein bound to the column was treated with thrombin(10 unit of thrombin/mg protein) at 22℃ for 12-16h in the presence of 3% glycerol and 0. 1% CHAPS. An easy one-step protocol to purify recombinant human CBS and in 54.8% overall yield had been

  17. Computer Simulation of Gd(III) Speciation in Human Interstitial Fluid

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The speciation and distribution of Gd(III) in human interstitial fluid was studied by computer simulation. Meantime artificial neural network was applied to the estimation of log β values of complexes. The results show that the precipitate species, GdPO4 and Gd2(CO3)3, are the predominant species. Among soluble species, the free Gd(III), [Gd(HSA)] , [Gd(Ox)] and then the ternary complexes of Gd(III) with citrate are main species and [Gd3(OH)4] becomes the predominant species at the Gd(III) total concentration of 2.2×10-2mol/L.

  18. Relationship between single nucleotide polymorphism of glycogen synthase gene of Pacific oyster Crassostrea gigas and its glycogen content

    Science.gov (United States)

    Liu, Siwei; Li, Qi; Yu, Hong; Kong, Lingfeng

    2017-02-01

    Glycogen is important not only for the energy supplementary of oysters, but also for human consumption. High glycogen content can improve the stress survival of oyster. A key enzyme in glycogenesis is glycogen synthase that is encoded by glycogen synthase gene GYS. In this study, the relationship between single nucleotide polymorphisms (SNPs) in coding regions of Crassostrea gigas GYS (Cg-GYS) and individual glycogen content was investigated with 321 individuals from five full-sib families. Single-strand conformation polymorphism (SSCP) procedure was combined with sequencing to confirm individual SNP genotypes of Cg-GYS. Least-square analysis of variance was performed to assess the relationship of variation in glycogen content of C. gigas with single SNP genotype and SNP haplotype. As a consequence, six SNPs were found in coding regions to be significantly associated with glycogen content ( P polymorphism on the glycogen content and provided molecular biological information for the selective breeding of good quality traits of C. gigas.

  19. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  20. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B;

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  1. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  2. Biosynthesis of polyketides by trans-AT polyketide synthases.

    Science.gov (United States)

    Piel, Jörn

    2010-07-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that have recently been recognized as one of the major group of proteins involved in the production of bioactive polyketides. 436 references are cited.

  3. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    Directory of Open Access Journals (Sweden)

    David O. Hooks

    2014-06-01

    Full Text Available Polyhydroxyalkanoate (PHA is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC. Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

  4. Functional Characterization of Sesquiterpene Synthase from Polygonum minus

    Directory of Open Access Journals (Sweden)

    Su-Fang Ee

    2014-01-01

    Full Text Available Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS has a complete open reading frame (ORF of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

  5. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    Science.gov (United States)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data. PMID:27721652

  6. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highl

  7. Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

    Science.gov (United States)

    Le Gal, Solène; Robert-Gangneux, Florence; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Damiani, Céline; Totet, Anne; Gangneux, Jean-Pierre; Nevez, Gilles

    2013-05-01

    Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

  8. Cloning and expression pattern of chitin synthase (CHS) gene in ...

    African Journals Online (AJOL)

    USER

    2010-08-16

    Aug 16, 2010 ... African Journal of Biotechnology Vol. 9(33), pp. 5297-5308, 16 ... Chitin synthase (CHS) plays an important role in biosynthesis of chitin .... strand cDNA Synthesis kit, 5'/3' RACE kit and pMD18-T vector were purchased from ...

  9. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  10. Phytochelatin synthase: of a protease a peptide polymerase made.

    Science.gov (United States)

    Rea, Philip A

    2012-05-01

    Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.

  11. 二甲双胍对人肝癌细胞 HepG2增殖及脂肪酸合酶的影响%Effects of metformin on cell proliferation and fatty acid synthase in human hepatocellular carcinoma cell line HepG2

    Institute of Scientific and Technical Information of China (English)

    彭晓韧; 刘燕; 邹大进; 李娟

    2015-01-01

    Objective The cancer risk of patients with diabetes mellitus who are treated by metformin declines remarkably in comparison to patients receiving other drug therapies.The article was to investigate the relationship between antineopastic activity and fatty acid synthase (FASN) of metformin in human hepatocellular carcinoma cell(HCC) line HepG2. Methods HepG2 cells were treated with various concentrations of metformin( 0, 1, 5, 10, 15 mmol/L) for 24, 48 and 72 h respectively and cell growth was assessed by CCK-8 assay.Positive control(paclitaxel 10μg/mL) and negative control(metformin 0mmol/L) were set up simultaneously.After being treated with doses of metformin(0, 5, 10,15mmol/L) for 72h, protein expression levels of AMPKα、P-AMPKα、FASN、P-mTOR and P-Akt were measured by western blotting analysis and FASN mRNA expression levels were measured by RT-PCR. Results Being treated with vari-ous doses of metformin(1, 5, 10, 15 mmol/L) for 24, 48 and 72 h, the growth of HepG2 cells were inhibited by metformin in dose-dependent and time-dependent manner( P0.05) .FASN mRNA expression levels decreased significantly in all metformin-treated groups( P<0.05) . Conclusion Met-formin actitiviates AMPK, inhibits mTOR and downregulates FASN, which are implicated in its antineopastic activity on HCC.Although metformin inhibits mTOR activation, it is not involved in Akt upregulation through a negative loop.%目的:二甲双胍治疗的糖尿病患者癌症发生风险较其他药物治疗者显著降低。探讨二甲双胍在人肝癌细胞HepG2中的抗肿瘤活性与脂肪酸合酶的关系。方法选取不同浓度(1、5、10、15 mmol/L)二甲双胍处理HepG2细胞24、48、72 h,用CCK-8法检测其对细胞增殖的影响。同时设阳性对照(紫杉醇10μg/mL),阴性对照(二甲双胍0 mmol/L)。设0、5、10、15 mmol/L二甲双胍处理72 h,用Western blot检测腺苷酸活化蛋白激酶( adenosine monophosphate activated protein

  12. Allosteric Activation of Trypanosomatid Deoxyhypusine Synthase by a Catalytically Dead Paralog*♦

    Science.gov (United States)

    Nguyen, Suong; Jones, Deuan C.; Wyllie, Susan; Fairlamb, Alan H.; Phillips, Margaret A.

    2013-01-01

    Polyamine biosynthesis is a key drug target in African trypanosomes. The “resurrection drug” eflornithine (difluoromethylornithine), which is used clinically to treat human African trypanosomiasis, inhibits the first step in polyamine (spermidine) biosynthesis, a highly regulated pathway in most eukaryotic cells. Previously, we showed that activity of a key trypanosomatid spermidine biosynthetic enzyme, S-adenosylmethionine decarboxylase, is regulated by heterodimer formation with a catalytically dead paralog (a prozyme). Here, we describe an expansion of this prozyme paradigm to the enzyme deoxyhypusine synthase, which is required for spermidine-dependent hypusine modification of a lysine residue in the essential translation factor eIF5A. Trypanosoma brucei encodes two deoxyhypusine synthase paralogs, one that is catalytically functional but grossly impaired, and the other is inactive. Co-expression in Escherichia coli results in heterotetramer formation with a 3000-fold increase in enzyme activity