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Sample records for beta-phorbol 12-myristate 13-acetate

  1. Induction of CD3 delta epsilon omega by phorbol 12-myristate 13-acetate

    DEFF Research Database (Denmark)

    Vangsted, A; Neisig, A; Wallin, H

    1993-01-01

    The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3 omega chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Ti alpha, CD......3 gamma and CG3 zeta chains two- to threefold and the synthesis of Ti beta and CD3 delta epsilon omega complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount...... of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3 omega in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3 delta epsilon omega complexes was immunoprecipitated as compared...

  2. Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment.

    Science.gov (United States)

    Zhou, Zhuo Long; Ma, Jing; Tong, Ming-Hui; Chan, Barbara Pui; Wong, Alice Sze Tsai; Ngan, Alfonso Hing Wan

    The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell-protein or cell-cell contact was also demonstrated.

  3. Galangin and kaempferol suppress phorbol-12-myristate-13-acetate-induced matrix metalloproteinase-9 expression in human fibrosarcoma HT-1080 cells.

    Science.gov (United States)

    Choi, Yu Jung; Lee, Young Hun; Lee, Seung-Taek

    2015-01-01

    Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.

  4. Effect of Dark Chocolate Extracts on Phorbol 12-Myristate 13-Acetate-Induced Oxidative Burst in Leukocytes Isolated by Normo-Weight and Overweight/Obese Subjects.

    Science.gov (United States)

    Ioannone, Francesca; Sacchetti, Giampiero; Serafini, Mauro

    2017-01-01

    Oxidative and inflammatory stress represents a major risk factor for cardiovascular disease (CVD) in overweight and obese subjects. Between the different plant foods, chocolate has been shown to decrease CVD risk due to its antioxidant and anti-inflammatory properties. However, as we recently showed in epidemiological studies, meta-analyses, and human trials, dietary antioxidants resulted more effective in subjects characterized by an ongoing oxidative stress, than in healthy people. Aim of this work was to investigate the effect of different concentrations of chocolate phenolic extract (CPE) on in vitro free radical production, stimulated by phorbol 12-myristate 13-acetate (PMA), in leukocytes extracted from blood of normo-weight and overweight/obese subjects. Neutrophils from overweight/obese group had a significantly higher free radical production compared to the normo-weight group. In neutrophils, the lowest CPE concentration significantly reduced free radical production in overweight/obese group only, and higher CPE concentrations were effective in both groups. In monocytes, the CPE concentration that was significantly effective in reducing free radical production was lower in overweight/obese subjects than in normo-weight subjects. Chocolate polyphenol extracts inhibit oxidative burst in human neutrophils and monocytes with a higher efficiency in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese. Results of this study provide further evidence about a differential role of dietary antioxidant strictly related to the "stress" condition of the subjects.

  5. Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-BL6 melanoma variants.

    Science.gov (United States)

    Huijzer, J C; McFarland, M; Niles, R M; Meadows, G G

    1996-03-01

    The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential.

  6. Effect of Dark Chocolate Extracts on Phorbol 12-Myristate 13-Acetate-Induced Oxidative Burst in Leukocytes Isolated by Normo-Weight and Overweight/Obese Subjects

    Directory of Open Access Journals (Sweden)

    Francesca Ioannone

    2017-06-01

    Full Text Available Oxidative and inflammatory stress represents a major risk factor for cardiovascular disease (CVD in overweight and obese subjects. Between the different plant foods, chocolate has been shown to decrease CVD risk due to its antioxidant and anti-inflammatory properties. However, as we recently showed in epidemiological studies, meta-analyses, and human trials, dietary antioxidants resulted more effective in subjects characterized by an ongoing oxidative stress, than in healthy people. Aim of this work was to investigate the effect of different concentrations of chocolate phenolic extract (CPE on in vitro free radical production, stimulated by phorbol 12-myristate 13-acetate (PMA, in leukocytes extracted from blood of normo-weight and overweight/obese subjects. Neutrophils from overweight/obese group had a significantly higher free radical production compared to the normo-weight group. In neutrophils, the lowest CPE concentration significantly reduced free radical production in overweight/obese group only, and higher CPE concentrations were effective in both groups. In monocytes, the CPE concentration that was significantly effective in reducing free radical production was lower in overweight/obese subjects than in normo-weight subjects. Chocolate polyphenol extracts inhibit oxidative burst in human neutrophils and monocytes with a higher efficiency in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese. Results of this study provide further evidence about a differential role of dietary antioxidant strictly related to the “stress” condition of the subjects.

  7. Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

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    Tada T

    2016-09-01

    Full Text Available Tomoki Tada,1 Kensaku Aki,2 Wataru Oboshi,1,3 Kazuyoshi Kawazoe,4 Toshiyuki Yasui,5 Eiji Hosoi2 1Subdivision of Biomedical Laboratory Sciences, Graduate School of Health Sciences, Tokushima University, 2Department of Cells and Immunity Analytics, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, 3Department of Medical Technology, Kagawa Prefectural University of Health Sciences, Kagawa, 4Department of Clinical Pharmacy Practice Pedagogy, Institute of Biomedical Sciences, 5Department of Reproductive and Menopausal Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan Background: The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. Objective: The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. Materials and methods: Human erythroid leukemia (HEL cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. Results: There was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6–560 µM and cilostazol (5–10 µM significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8–200 µM and sodium valproate (50–1,000 µg/mL also significantly inhibited

  8. Involvement of phorbol-12-myristate-13-acetate-induced protein 1 in goniothalamin-induced TP53-dependent and -independent apoptosis in hepatocellular carcinoma-derived cells

    International Nuclear Information System (INIS)

    Kuo, Kung-Kai; Chen, Yi-Ling; Chen, Lih-Ren; Li, Chien-Feng; Lan, Yu-Hsuan; Chang, Fang-Rong; Wu, Yang-Chang; Shiue, Yow-Ling

    2011-01-01

    The objective was to investigate the upstream apoptotic mechanisms that were triggered by a styrylpyrone derivative, goniothalamin (GTN), in tumor protein p53 (TP53)-positive and -negative hepatocellular carcinoma (HCC)-derived cells. Effects of GTN were evaluated by the flow cytometry, alkaline comet assay, immunocytochemistry, small-hairpin RNA interference, mitochondria/cytosol fractionation, quantitative reverse transcription-polymerase chain reaction, immunoblotting analysis and caspase 3 activity assays in two HCC-derived cell lines. Results indicated that GTN triggered phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA)-mediated apoptosis via TP53-dependent and -independent pathways. In TP53-positive SK-Hep1 cells, GTN furthermore induced TP53 transcription-dependent and -independent apoptosis. After GTN treatment, accumulation of reactive oxygen species, formation of DNA double-strand breaks, transactivation of TP53 and/or PMAIP1 gene, translocation of TP53 and/or PMAIP1 proteins to mitochondria, release of cytochrome c from mitochondria, cleavage of caspases and induction of apoptosis in both cell lines were sustained. GTN might represent a novel class of anticancer drug that induces apoptosis in HCC-derived cells through PMAIP1 transactivation regardless of the status of TP53 gene. - Highlights: → Goniothalamin (GTN) induced apoptosis in hepatocellular carcinomas-derived cells. → The apoptosis induced by GTN is PMAIP1-dependent, regardless of TP53 status. → The apoptosis induced by GTN might be TP53 transcription-dependent or -independent. → GTN-induced apoptosis is mitochondria- and caspases-mediated.

  9. "Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

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    Lei Zhu

    Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

  10. NRF2 Signaling Negatively Regulates Phorbol-12-Myristate-13-Acetate (PMA-Induced Differentiation of Human Monocytic U937 Cells into Pro-Inflammatory Macrophages.

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    Min-Gu Song

    Full Text Available Blood monocytes are recruited to injured tissue sites and differentiate into macrophages, which protect against pathogens and repair damaged tissues. Reactive oxygen species (ROS are known to be an important contributor to monocytes' differentiation and macrophages' function. NF-E2-related factor 2 (NRF2, a transcription factor regulating cellular redox homeostasis, is known to be a critical modulator of inflammatory responses. We herein investigated the role of NRF2 in macrophage differentiation using the human monocytic U937 cell line and phorbol-12-myristate-13-acetate (PMA. In U937 cells with NRF2 silencing, PMA-stimulated cell adherence was significantly facilitated when compared to control U937 cells. Both transcript and protein levels for pro-inflammatory cytokines, including interleukine-1β (IL-1β, IL-6, and tumor necrosis factor-α (TNFα were highly elevated in PMA-stimulated NRF2-silenced U937 compared to the control. In addition, PMA-inducible secretion of monocyte chemotactic protein 1 (MCP-1 was significantly high in NRF2-silenced U937. As an underlying mechanism, we showed that NRF2-knockdown U937 retained high levels of cellular ROS and endoplasmic reticulum (ER stress markers expression; and subsequently, PMA-stimulated levels of Ca2+ and PKCα were greater in NRF2-knockdown U937 cells, which caused enhanced nuclear accumulation of nuclear factor-ҡB (NFҡB p50 and extracellular signal-regulated kinase (ERK-1/2 phosphorylation. Whereas the treatment of NRF2-silenced U937 cells with pharmacological inhibitors of NFҡB or ERK1/2 largely blocked PMA-induced IL-1β and IL-6 expression, indicating that these pathways are associated with cell differentiation. Taken together, our results suggest that the NRF2 system functions to suppress PMA-stimulated U937 cell differentiation into pro-inflammatory macrophages and provide evidence that the ROS-PKCα-ERK-NFҡB axis is involved in PMA-facilitated differentiation of NRF2-silenced U937

  11. Differential effects of phorbol 12-myristate 13-acetate and diacylglycerols on thromboxane A2-independent phospholipase A2 activation in collage-stimulated human platelets.

    Science.gov (United States)

    Reddy, S; Rao, G H; Murthy, M

    1994-04-01

    We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC8 and OAG, and 1,3-DiC8 (a poor activator of PKC) on thromboxane A2 (TxA2)-independent phospholipase A2 (PLA2) activation in human platelets using collagen and A23187 as agonists. We measured PLA2 activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA2 synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC8, OAG, and 1,3-DiC8 increased TxA2-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC8, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC8) in priming TxA2-independent PLA2 activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA2-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligatory for priming PLA2 activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC8, OAG, and 1,3-DiC8 likely enhanced PLA2 activation via intracellular Ca2+ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA1, or nonspecific PLA2. Since both 1,2-DiC8 and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA1 may not

  12. Hepatic stellate cells lack AP-1 responsiveness to electrophiles and phorbol 12-myristate-13-acetate

    International Nuclear Information System (INIS)

    Reichard, John F.; Petersen, Dennis R.

    2004-01-01

    Stellate cell profibrotic gene induction and transdifferentiation are central events in liver fibrosis. Oxidative stress has been implicated as an activator of the transcription factors Nrf2 and AP-1 through shared kinase signaling pathways that also purportedly contribute to stellate cell activation. The present study examined the role of oxidative stress in ARE- and TRE-regulated gene induction in isolated hepatic stellate cells. Using a portion of the human Nqo1 promoter consisting of an ARE imbedded TRE, it was demonstrated that while the ARE was responsible for mediating inducible gene expression in response to the electrophiles 4-HNE and tBHQ, the TRE was refractory to induction by either electrophiles or PMA. It was demonstrated that stellate cells possess nuclear TRE-binding proteins that were identified as JunB, JunD, Fra1, and Fra2, which were unaffected by either electrophiles or PMA treatment. This report demonstrates that, in contrast to the ARE, the TRE and its binding cognate AP-1 did not mediate independent gene induction in hepatic stellate cells. This observation is significant given the presumed importance attributed to AP-1 in mediating profibrogenic gene expression

  13. Antioxidant and Antiradical Activities of Manihot esculenta Crantz (Euphorbiaceae Leaves and Other Selected Tropical Green Vegetables Investigated on Lipoperoxidation and Phorbol-12-myristate-13-acetate (PMA Activated Monocytes

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    Ange Mouithys-Mickalad

    2011-09-01

    Full Text Available Abelmoschus esculentus (Malvaceae, Hibiscus acetosella (Malvaceae, Manihot esculenta Crantz (Euphorbiaceae and Pteridium aquilinum (Dennstaedtiaceae leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N′-dimethyl-p-phenylene-diamine (DMPD. The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS in “inflammation like” conditions was studied by fluorescence technique using 2′,7′-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration.

  14. α-Mangostin suppresses 12-o-tetradecanoylphorbol-13- acetate ...

    African Journals Online (AJOL)

    α-Mangostin, Matrix metalloproteinase, Osteosarcoma, Cell migration, 12-O-. Tetradecanoylphorbol-13-acetate ... International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African. Index Medicus, JournalSeek .... The results were analyzed using Student's t-test, and differences were.

  15. Antipain and 12-O-tetradecanoyl-phorbol-13-acetate

    International Nuclear Information System (INIS)

    Fujiwara, Y.; Tatsumi, M.

    1980-01-01

    Antipain (AP) and 12-O-tetradecanoyl-phorbooe-13-acetate (TPA) were tested in V79 Chinese hamster cells for cytotoxicity and effects on survival and 6-thioguanine-resistant (6TGsup(r)) mutation after UV-irradiation. AP and/or TPA were relatively non-cytotoxic and had no significant effects on UV survival. Despite their non-mutagenicity, the recovery of UV-induced 6TGsup(r) colonies was significantly enhanced by the pretreatment with either AP (0,5-2 mM) or TPA (0.1-1 μg/ml) only during the expression period before the 6TG selection at a low density of cells in the absence of AP or TPA. Such enhancing effects were maximal when AP or TPA was present during the late expression period after the mutation fixation and extensive dilution of DNA lesions. Reconstruction experiments revelaed the antgonisitc actions that TPA and AP tended to eliminate and increase, respectively, the metabolic co-operation. In the TPA-plus-AP treatment, AP abolished the TPA-enhanced recovery of induced mutants. Thus, it seems that TPA increases the mutant recovery langely through decreased metabolic co-operation and AP could modulate the mutation expression. Further, an error-prone inducible repair may not exist or, if it exists, AP may not inhibit it in V79 Chinese hamster cells. (orig.)

  16. Local anesthetics inhibit induction of ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

    OpenAIRE

    Yuspa, S H; Lichti, U; Ben, T

    1980-01-01

    The induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in mouse epidermal cells in vivo and in vitro occurs rapidly after exposure to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). This induction has characteristics of a cell surface receptor-mediated process. Local anesthetics modify a variety of cellular responses mediated by membrane receptors. When cultured mouse epidermal cells were exposed to the local anesthetics lidocaine, tetracaine...

  17. Interferon-gamma up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1993-01-01

    AMP (Bt2cAMP), dibutyryl cGMP (Bt2cGMP)] and compounds known to affect keratinocytes [4 beta-phorbol 12-myristate 13-acetate (PMA), retinoic acid, Ca2+, dexamethasone, lipopolysaccharides, foetal calf serum]. Protein IGUP I-5111 was selected for further studies as its level is affected by simian-virus-40......, which migrated with the AMA variant of keratinocyte protein IEF SSP 5111, is novel although it exhibits weak similarity to cytoskeletal proteins. IGUP I-5111 contains the RGD sequence found in many extracellular glycoprotein ligands of the integrin receptor family and it is found at least partially...... in the culture supernatant. Considering the presence of IFN-gamma in psoriatic plaques as well as its putative involvement in the pathophysiology of the disease it was of interest to determine whether the set of proteins was upregulated in these cells. Two-dimensional gel analysis of the protein phenotype of non-cultured...

  18. Effects of glucocorticoid hormones on radiation induced and 12-O-tetradecanoylphorbol-13-acetate enhanced radiation transformation in vitro

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Umans, R.S.

    1988-01-01

    We have studied the interactions of glucocorticoid hormones with radiation in the induction of transformation in vitro in C3AH10T1/2 cells. We have observed that cortisone has its primary enhancing effect on radiation transformation when present after the radiation exposure during the ''expression period'', or the time after carcinogen exposure during which promoting agents have been shown to enhance radiation transformation in vitro, and that two different glucocorticoid hormones, dexamethasone and cortisone, have a suppressive effect on the 12-O-tetradecanoylphorbol-13-acetate (TPA) enhancement of radiation transformation in vitro

  19. Differential retinoic acid inhibition of ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate and by germicidal ultraviolet light

    International Nuclear Information System (INIS)

    Lichti, U.; Patterson, E.; Hennings, H.; Yuspa, S.H.

    1981-01-01

    Several retinoids including retinoic acid effectively inhibit phorbol ester-mediated tumor promotion and ornithine decarboxylase (ODC) induction in mouse epidermis. To understand better the possible cellular site of action of retinoids, the inhibitory action of retinoic acid on the induction of ODC was compared for two distinctly different inducers, namely, 12-O-tetradecanoylphorbol-13-acetate (TPA) and germicidal ultraviolet light (uv), in primary mouse epidermal cell cultures. It was found that the induction of ODC by TPA is almost completely prevented by retinoic acid while the induction by uv is only moderately inhibited. The differential inhibition of enzyme induction cannot be accounted for by selective retinoid inhibition of DNA, RNA, or protein synthesis either alone or in concert with TPA or uv. These agents possibly act at transcription or translation, both of which are required for ODC induction by TPA or uv

  20. 12-O-Tetradecanoyl-phorbol-13-acetate and its relationship to SCE induction in Syrian and Chinese hamster cells

    International Nuclear Information System (INIS)

    Popescu, N.C.; Amsbaugh, S.C.; Larramendy, M.L.; DiPaolo

    1982-01-01

    12-O-Tetradecanoyl-phorbol-13-acetate (TPA) in conditions that produce enhancement of ultraviolet light (UV) and x-irradiation Syrian hamster embryo cell (HEC) transformation did not cause further increase in the sister chromatid exchange (SCE) frequency induced by UV and x-irradiation, two physical carcinogens that differ in their mode of DNA interaction and efficiency of SCE induction. Several factors which might influence SCE induction by TPA were studied on HEC and Chinese hamster V79-4 cells. Heat-inactivated serum was used because of the possibility that a serum component may interfere with TPA ability to cause SCE. TPA effect on SCE was determined at the first and second division post treatment on cells exposed to different 5-bromodeoxyuridine (BrdUrd) concentrations. Independent of BrdUrd concentration (1-10μg/ml medium) and the number of cells divisions post treatment, TPA (0.01-2μg/ml medium) was ineffective in inducing SCE in exponentially and stationary HEC cultures cultivated in medium supplemented with heat-inactivated serum. Also, TPA did not increase the SCE frequency in V79-4 Chinese hamster cells cultured in heat-activated or noninactivated serum. Although SCE induction, a cellular response to carcinogen-induced DNA damage, may be important for the induction of transformation by environmental agents, the enhancement of transformation frequency caused by TPA occurs without further DNA alterations involved in SCE formation

  1. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) accelerates expression of differentiation markers in cultures of rat palatal epithelial cells

    DEFF Research Database (Denmark)

    Arenholt, D; Dabelsteen, Erik

    1987-01-01

    Cultures of rat palatal epithelium grown on collagen rafts were treated with different doses of the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Sections from biopsies taken 1, 6, 24, and 48 hr after the addition of TPA were examined for the localization of staining by blood ...

  2. Effect of 12-O-tetradecanoylphorbol-13-acetate-induced psoriasis-like skin lesions on systemic inflammation and atherosclerosis in hypercholesterolaemic apolipoprotein E deficient mice

    DEFF Research Database (Denmark)

    Madsen, Marie; Hansen, Peter Riis; Nielsen, Lars Bo

    2016-01-01

    BACKGROUND: Risk of cardiovascular disease is increased in patients with psoriasis, but molecular mechanisms linking the two conditions have not been clearly established. Lack of appropriate animal models has hampered generation of new knowledge in this area of research and we therefore sought...... to develop an animal model with combined atherosclerosis and psoriasis-like skin inflammation. METHODS: Topical 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to the ears twice per week for 8 weeks in atherosclerosis-prone apolipoprotein E deficient (ApoE(-/-)) mice. RESULTS: TPA led to localized...

  3. Transduced PEP-1-ribosomal protein S3 (rpS3) ameliorates 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice

    International Nuclear Information System (INIS)

    Ahn, Eun Hee; Kim, Dae Won; Kang, Hye Won; Shin, Min Jae; Won, Moo Ho; Kim, Joon; Kim, Dong Joon; Kwon, Oh-Shin; Kang, Tae-Cheon; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2010-01-01

    This study investigated the preventive effect of ribosomal protein S3 (rpS3) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. A cell permeable expression vector PEP-1-rpS3 was constructed. Topical application of the vector markedly inhibited TPA-induced expression levels of cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines. Application of PEP-1-rpS3 also resulted in a significant reduction in the activation of nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) in TPA-treated ears. These results indicate that PEP-1-rpS3 inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK, prompting the suggestion that PEP-1-rpS3 can be used as a therapeutic agent against skin inflammation.

  4. Flaccidoxide-13-Acetate Extracted from the Soft Coral Cladiella kashmani Reduces Human Bladder Cancer Cell Migration and Invasion through Reducing Activation of the FAK/PI3K/AKT/mTOR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Choo-Aun Neoh

    2017-12-01

    Full Text Available Metastasis of cancer is the cause of the majority of cancer deaths. Active compound flaccidoxide-13-acetate, isolated from the soft coral Cladiella kashmani, has been found to exhibit anti-tumor activity. In this study, Boyden chamber analysis, Western blotting and gelatin zymography assays indicated that flaccidoxide-13-acetate exerted inhibitory effects on the migration and invasion of RT4 and T24 human bladder cancer cells. The results demonstrated that flaccidoxide-13-acetate, in a concentration-dependent manner, reduced the levels of matrix metalloproteinase-2 (MMP-2, MMP-9, urokinase-type plasminogen activator receptor (uPAR, focal adhesion kinase (FAK, phosphatidylinositide-3 kinases (PI3K, p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR, p-mTOR, Ras homolog gene family, member A (Rho A, Ras, mitogen-activated protein kinase kinase 7 (MKK7 and mitogen-activated protein kinase kinase kinase 3 (MEKK3, and increased the expressions of tissue inhibitor of metalloproteinase-1 (TIMP-1 and TIMP-2 in RT4 and T24 cells. This study revealed that flaccidoxide-13-acetate suppressed cell migration and invasion by reducing the expressions of MMP-2 and MMP-9, regulated by the FAK/PI3K/AKT/mTOR pathway. In conclusion, our study was the first to demonstrate that flaccidoxide-13-acetate could be a potent medical agent for use in controlling the migration and invasion of bladder cancer.

  5. Altered sensitivity to ellagic acid in neuroblastoma cells undergoing differentiation with 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid.

    Science.gov (United States)

    Alfredsson, Christina Fjæraa; Rendel, Filip; Liang, Qui-Li; Sundström, Birgitta E; Nånberg, Eewa

    2015-12-01

    Ellagic acid has previously been reported to induce reduced proliferation and activation of apoptosis in several tumor cell lines including our own previous data from non-differentiated human neuroblastoma SH-SY5Y cells. The aim of this study was now to investigate if in vitro differentiation with the phorbol ester 12-O- tetradecanoylphorbol-13-acetate or the vitamin A derivative all-trans retinoic acid altered the sensitivity to ellagic acid in SH-SY5Y cells. The methods used were cell counting and LDH-assay for evaluation of cell number and cell death, flow cytometric analysis of SubG1- and TUNEL-analysis for apoptosis and western blot for expression of apoptosis-associated proteins. In vitro differentiation was shown to reduce the sensitivity to ellagic acid with respect to cell detachment, loss of viability and activation of apoptosis. The protective effect was phenotype-specific and most prominent in all-trans retinoic acid-differentiated cultures. Differentiation-dependent up-regulation of Bcl-2 and integrin expression is introduced as possible protective mechanisms. The presented data also point to a positive correlation between proliferative activity and sensitivity to ellagic-acid-induced cell detachment. In conclusion, the presented data emphasize the need to consider degree of neuronal differentiation and phenotype of neuroblastoma cells when discussing a potential pharmaceutical application of ellagic acid in tumor treatment. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  6. Modifiers of free radicals inhibit in vitro the oncogenic actions of x-rays, bleomycin, and the tumor promoter 12-O-tetradecanoylphorbol 13-acetate

    International Nuclear Information System (INIS)

    Borek, C.; Troll, W.

    1983-01-01

    Using short-term cultures of hamster embryo cells, we have examined the effects of the free-radical scavenger superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and the enzyme catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) on x-ray-and bleomycin-induced transformation and on the enhancement of radiogenic transformation by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). We find that superoxide dismutase inhibits (i) transformation induced by x-ray and bleomycin and (ii) promotional action of TPA in vitro. The results suggest that the oncogenic action of x-rays and bleomycin and the enhancement of oncogenic transformation by TPA are mediated in part by free radicals. The findings also suggest that superoxide dismutase can serve as an inhibitor of oncogenesis and that its actions, as seen in this in vitro system, are most predominantly on inhibiting late events in the progression of cellular transformation--those associated with promotion

  7. Effects of 12-O-tetradecanoyl-phorbol-13-acetate [corrected] and sodium lauryl sulfate on the production and expression of cytokines and proto-oncogenes in photoaged and intrinsically aged human keratinocytes.

    Science.gov (United States)

    Suh, D H; Youn, J I; Eun, H C

    2001-11-01

    Skin aging may be divided into photoaging and intrinsic aging. The purpose of this study was to investigate the effects of 12-O-tetradecanoyl-phorbol-13-acetate and sodium lauryl sulfate on the production and expression of cytokines and proto-oncogenes in photoaged and intrinsically aged skin, compared with young skin. Keratinocytes were taken from newborns, young adults in their twenties, and from the forearm and thigh of volunteers in their fifties and seventies. Interleukin-1alpha and -6, and interleukin-1 receptor antagonist, c-fos and c-myc were measured after cultured keratinocytes had been treated with 12-O-tetradecanoyl-phorbol-13-acetate and sodium lauryl sulfate. There has been no report concerning the dependence of cytokine production by sodium lauryl sulfate upon photoaging and intrinsic aging. This study also involves the first investigation of the effects of aging on c-myc expression by 12-O-tetradecanoyl-phorbol-13-acetate treatment. Cytokine production decreased markedly with age. These results suggest the progressive decline of cellular function with age. The ratio of cytokine production in the irritant-treated group compared with that in the control group showed a different pattern in photoaging and intrinsic aging. With the significant difference between photoaging and intrinsic aging, T/C ratio decreased in interleukin-1alpha and interleukin-1 receptor antagonist upon aging, whereas it increased in interleukin-6. S/C ratio was uniquely elevated on photoaged skin in the 50 y age group. It is suggested that photoaged skin shows an exaggerated reaction to surfactant. Compared with the control, c-fos expression in 12-O-tetradecanoyl-phorbol-13-acetate-treated keratinocytes decreased with age in the thigh, but increased in the photoaged skin of forearm. The increased c-fos expression in 12-O-tetradecanoyl-phorbol-13-acetate-treated keratinocytes could be relevant for the predisposition of photoaged keratinocytes to malignant transformation.

  8. Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae Hee; Lee, Chang Ki [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Oral Cancer Research Institute, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Hwang, Young Sun [Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Park, Kwang-Kyun [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Chung, Won-Yoon [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of)], E-mail: wychung@yuhs.ac

    2008-07-03

    Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H{sub 2}O{sub 2} formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-{kappa}B) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-{kappa}B activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-{kappa}B signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.

  9. Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

    International Nuclear Information System (INIS)

    Park, Jae Hee; Lee, Chang Ki; Hwang, Young Sun; Park, Kwang-Kyun; Chung, Won-Yoon

    2008-01-01

    Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H 2 O 2 formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-κB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-κB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent

  10. Structure-dependent inhibitory effects of synthetic cannabinoids against 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and skin tumour promotion in mice.

    Science.gov (United States)

    Nakajima, Jun'ichi; Nakae, Dai; Yasukawa, Ken

    2013-08-01

    Whether and how synthetic cannabinoids affect inflammation and carcinogenesis has not been well studied. The present study was thus conducted to assess effects of synthetic cannabinoids on inflammation and carcinogenesis in vivo in mice. Twenty-three analogues of synthetic cannabinoids were isolated from, and identified as adulterants in, illegal drugs distributed in the Tokyo metropolitan area, and were examined for their inhibitory effects on the induction of oedema in mouse ears by 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, selected cannabinoids, JWH-018, -122 and -210, were studied for their effects on carcinogenesis induced in mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by TPA. Among cannabinoids, naphthoylindoles mostly exhibited superior inhibitory effects against TPA-induced ear oedema and, especially, JWH-018, -122 and -210 showed potent activity with 50% inhibitory dose (ID50) values of 168, 346 and 542 nm, respectively (an activity corresponding to that of indometacin (ID50 = 908 nm)). Furthermore these three compounds also markedly suppressed the tumour-promoting activity of TPA. This is the first report indicating the structure-activity relationships for the anti-inflammatory activity of synthetic cannabinoids on TPA-induced inflammation in mice. Naphthoylindoles, JWH-018, -122 and -210, had the most potent anti-inflammatory activity and also markedly inhibited tumour promotion by TPA in the two-stage mouse skin carcinogenesis model. The present results suggest that synthetic cannabinoids, such as JWH-018, -122 and -210, may be used as cancer chemopreventive agents in the future. © 2013 Royal Pharmaceutical Society.

  11. Tumor-promoting phorbol ester transiently down-modulates the p53 level and blocks the cell cycle

    DEFF Research Database (Denmark)

    Skouv, J.; Jensen, P O; Forchhammer, J

    1994-01-01

    Activation of the protein kinase C signaling pathway by tumor-promoting phorbol esters, such as 4 beta-phorbol 12-myristate 13-acetate (PMA), induced a decrease in the level of p53 mRNA in several serum-starved human cell lines. Also, the tumor-promoting phosphatase inhibitor okadaic acid induced...... a decrease in the p53 mRNA level in the cell lines. Normal diploid as well as various tumor cell lines were tested. Two tumor cell lines, HeLa and A549, both containing the wild-type p53 gene, but very different levels of p53 protein, were studied in detail. In both cell lines, the level of p53 m......RNA was minimal after 9 h of exposure to PMA. After approximately 120 h, the p53 mRNA level was similar to the pretreatment level. PMA induced a similar transient decrease in the level of p53 protein in the A549 cell line. The decrease in the p53 mRNA level could not be explained by changes in the transcriptional...

  12. Structural and functional analysis of an enhancer GPEI having a phorbol 12-O-tetradecanoate 13-acetate responsive element-like sequence found in the rat glutathione transferase P gene.

    Science.gov (United States)

    Okuda, A; Imagawa, M; Maeda, Y; Sakai, M; Muramatsu, M

    1989-10-05

    We have recently identified a typical enhancer, termed GPEI, located about 2.5 kilobases upstream from the transcription initiation site of the rat glutathione transferase P gene. Analyses of 5' and 3' deletion mutants revealed that the cis-acting sequence of GPEI contained the phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE)-like sequence in it. For the maximal activity, however, GPEI required an adjacent upstream sequence of about 19 base pairs in addition to the TRE-like sequence. With the DNA binding gel-shift assay, we could detect protein(s) that specifically binds to the TRE-like sequence of GPEI fragment, which was possibly c-jun.c-fos complex or a similar protein complex. The sequence immediately upstream of the TRE-like sequence did not have any activity by itself, but augmented the latter activity by about 5-fold.

  13. Chemical profiling with cytokine stimulating investigations of Sutherlandia frutescens L.R. (Br.) (Fabaceae)

    CSIR Research Space (South Africa)

    Faleschini, MT

    2013-03-01

    Full Text Available gave the best performance in recruiting various inflammatory cytokines to the site of infection upon stimulation with phorbol 12-myristate 13-acetate, where essentially the non-polar compounds present in the ethanol extract contributed to most...

  14. GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

    International Nuclear Information System (INIS)

    Moore, K.L.; Varki, A.; McEver, R.P.

    1991-01-01

    GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

  15. Effects of 12-O-tetradecanoylphorbol-13-acetate in combination with gemcitabine on Panc-1 pancreatic cancer cells cultured in vitro or Panc-1 tumors grown in immunodeficient mice.

    Science.gov (United States)

    Zheng, Xi; Cui, Xiao-Xing; Gao, Zhi; Verano, Michael; Huang, Mou-Tuan; Liu, Yue; Rabson, Arnold B; Conney, Allan H

    2012-12-01

    In the present study, the effects of 12-O-tetra-decanoylphorbol-13-acetate (TPA) alone or in combination with gemcitabine on the growth of Panc-1 pancreatic cancer cells cultured in vitro or grown in NCr immunodeficient nude mice were investigated. Combinations of TPA and gemcitabine synergi-stically inhibited the growth and induced apoptosis in Panc-1 cells. The combination of TPA (0.16 nM) and gemcitabine (0.5 µM) induced a marked increase in phosphorylated c-Jun NH2-terminal kinase (JNK) in the Panc-1 cells. In animal experiments, NCr nude mice with established Panc-1 tumors received daily intraperitoneal (i.p.) injections of TPA (50 ng/g body weight/day) or gemcitabine (0.5 µg/g body weight/day) alone or in combination for 26 days. Treatment with daily i.p. injections of low doses of TPA or gemcitabine alone had a modest inhibitory effect on the growth of the tumors. However, the combination of low doses of TPA and gemcitabine more potently inhibited the growth of Panc-1 tumors than either agent used individually. Treatment with TPA or gemcitabine alone or in combination did not affect the body weight of the animals. Clinical trials with TPA alone or in combination with gemcitabine on patients with pancreatic cancer are warranted in order to confirm our results.

  16. CpG Oligonucleotide and Interleukin 2 stimulation enables higher cytogenetic abnormality detection rates than 12-o-tetradecanolyphorbol-13-acetate in Asian patients with B-cell chronic lymphocytic leukemia (B-CLL).

    Science.gov (United States)

    Liaw, Fiona Pui San; Lau, Lai Ching; Lim, Alvin Soon Tiong; Lim, Tse Hui; Lee, Geok Yee; Tien, Sim Leng

    2014-12-01

    The present study was designed to compare abnormality detection rates using DSP30 + IL2 and 12-O-Tetradecanoylphorbol-13-acetate (TPA) in Asian patients with B-CLL. Hematological specimens from 47 patients (29 newly diagnosed, 18 relapsed) were established as 72 h-DSP30 + IL2 and TPA cultures. Standard methods were employed to identify clonal aberrations by conventional cytogenetics (CC). The B-CLL fluorescence in situ hybridization (FISH) panel comprised ATM, CEP12, D13S25, and TP53 probes. DSP30 + IL2 cultures had a higher chromosomal abnormality detection rate (67 %) compared to TPA (44 %, p 0.05). Thirteen cases with abnormalities were found exclusively in DSP30 + IL2 cultures compared to one found solely in TPA cultures. DSP30 + IL2 cultures were comparable to the FISH panel in detecting 11q-, +12 and 17p- but not 13q-. It also has a predilection for 11q- bearing leukemic cells compared to TPA. FISH had a higher abnormality detection rate (84.1 %) compared to CC (66.0 %) with borderline significance (p = 0.051), albeit limited by its coverage. In conclusion, DSP30 + IL2 showed a higher abnormality detection rate. However, FISH is indispensable to circumvent low mitotic indices and detect subtle abnormalities.

  17. Geraniol attenuates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammation in mouse skin: possible role of p38 MAP Kinase and NF-κB.

    Science.gov (United States)

    Khan, Abdul Quaiyoom; Khan, Rehan; Qamar, Wajhul; Lateef, Abdul; Rehman, Muneeb U; Tahir, Mir; Ali, Farrah; Hamiza, Oday O; Hasan, Syed Kazim; Sultana, Sarwat

    2013-06-01

    Abnormal production of reactive oxygen species (ROS) and proinflammatory cytokines often act as trigger for development of most of the chronic human diseases including cancer via up-regulation of transcription factors and activation of MAP kinases. We investigated the protective effects of geraniol (GOH) against 12-O-tetradecanoyl phorbol-13-acetate (TPA) induced oxidative and inflammatory responses, expression of p38MAPK, NF-κB and COX-2 in mouse skin. Animals were divided into four groups I-IV (n=6). Group II and III received topical application of TPA at the dose of 10 nmol/0.2 ml of acetone/animal/day, for two days. Group III was pre-treated with GOH (250 μg) topically 30 min prior to each TPA administration. While group I and IV were given acetone (0.2 ml) and GOH respectively. Our results show that GOH significantly inhibited TPA induced lipid peroxidation (LPO), inflammatory responses, proinflammatory cytokine release, up regulates reduced glutathione (GSH) content and the activity of different antioxidant enzymes. Interestingly, GOH also inhibited TPA induced altered activity of p38MAPK. Further, TPA induced altered expression of NF-κB (p65) and COX-2 was also attenuated by GOH. Thus, our results suggest that GOH attenuates early tumor promotional changes, and it may serve as one of the various ways to prevent carcinogenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Epidermal changes following application of 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate to human skin transplanted to nude mice studied with histological species markers

    International Nuclear Information System (INIS)

    Graem, N.

    1986-01-01

    Effects of the tumor initiator 7,12-dimethylbenz(a)anthracene (DMBA) and of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on epidermis of human fetal and adult skin were studied in the nude mouse/human skin model. Human skin grafts on NC nude mice were exposed to two topical applications of 1 mg of DMBA in 50 microliter of acetone with an interval of 3 days and/or to applications of 10 micrograms of TPA in 50 microliter of acetone twice weekly. In some animals, it was attempted to augment the susceptibility of the grafts to the tumor-initiating effect of DMBA by pretreatment with TPA or ultraviolet light. The mice were sacrificed 8-32 wk after the initial treatment. Tumors did not appear in the central portions of any of the grafts, but epidermal tumors were seen at the graft border in 34.9% of the DMBA-treated animals. To identify human epidermis on the grafts and to determine the species origin of the induced tumors, two independently working histological marker methods were applied. (a) The first is detection of a human Blood Group B-like antigen present in mouse epidermis and in chemically induced murine epidermal tumors. This antigen cannot be demonstrated in human epidermis and in epidermal tumors of human patients. (b) The second is histological staining with the DNA-specific fluorochrome, bisbenzimide, displaying a characteristic pattern of 5-10 intranuclear fluorescent bodies in murine nonneoplastic epidermal cells and in murine epidermal tumor cells. Such a pattern is not seen in human epidermis and in epidermal tumors of human patients. The studies showed that TPA treatment resulted in epidermal hyperplasia in both the human epidermis and the adjacent mouse epidermis and that the induced tumors were derived from murine tissue

  19. Effects of 12-O-tetradecanoylphorbol-13-acetate on the incorporation of labelled precursors into RNA, DNA and protein in epidermis, dermis and subcutis from precancerous mouse skin with reference to enhanced tumorigenesis

    International Nuclear Information System (INIS)

    Bhisey, R.A.; Ramchandani, A.G.; Sirsat, S.M.

    1984-01-01

    The effects of a single application of 1.8 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) on precursor incorporation into RNA, DNA and protein in the epidermis, dermis and subcutis from 3-methylcholanthrene (MCA) injected precancerous mouse skin were studied at various time points between 3 and 96 h. In the precancerous tissues, the rates of incorporation of [ 3 H]uridine into RNA did not alter appreciably from those in the control tissues; while the rates of [ 3 H]methylthymidine incorporation into DNA were elevated with peaks appearing between 6 and 12 h, at 24 h and at 72 h in epidermis, dermis and subcutis. The rate of incorporation of [ 14 C]leucine into protein was markedly elevated in all the three tissues which showed 3-4 sharp peaks. The maximum stimulation ranged between 14 and 20 times that of the control. A single application of TPA to the precancerous mouse skin induced early stimulation of precursor incorporation into all the three macromolecules in epidermis, dermis and subcutis. The increased stimulation was maintained for 36-72 h. The patterns of incorporation of [ 3 H]methylthymidine into DNA gave rise to 2-3 peaks of elevated uptake in each tissue up to 36-48 h. A lowered rate of DNA synthesis between 48 and 60 h was followed by a peak at 72 h. In each group, epidermal mitotic activity correlated well with spurts of precursor incorporation into cellular DNA. The observations indicate that TPA recruits more cells into the DNA synthetic phase and accelerates selective growth of preneoplastic cells during tumor progression

  20. Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane

    NARCIS (Netherlands)

    Bosch, Remko R.; Bazuine, Merlijn; Wake, Michelle M.; Span, Paul N.; Olthaar, André J.; Schürmann, Annette; Maassen, J. Antonie; Hermus, Ad R. M. M.; Willems, Peter H. G. M.; Sweep, C. G. J.

    2003-01-01

    The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the

  1. Immunomodulating effects of food compounds : a study using the THP-1 cell line

    NARCIS (Netherlands)

    Chanput, W.

    2012-01-01

    THP-1 is a human leukaemia monocytic cell line from the peripheral blood of a 1 year old human male. After exposure to phorbol-12-myristate-13-acetate (PMA), THP-1 cells in monocyte state start to adhere to culture plates and alter their morphology with an indication for differentiation into

  2. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    International Nuclear Information System (INIS)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-01-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis

  3. Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro

    NARCIS (Netherlands)

    Arts, J.; Herr, I.; Lansink, M.; Angel, P.; Kooistra, T.

    1997-01-01

    Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a

  4. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Ploug, M

    1990-01-01

    The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u...

  5. Biological responsiveness to the phorbol esters and specific binding of [3H]phorbol 12,13-dibutyrate in the nematode Caenorhabditis elegans, a manipulable genetic system

    International Nuclear Information System (INIS)

    Lew, K.K.; Chritton, S.; Blumberg, P.M.

    1982-01-01

    Because of its suitability for genetic studies, the nematode Caenorhabditis elegans was examined for its responsiveness to the phorbol esters. Phorbol 12-myristate 13-acetate had three effects. It inhibited the increase in animal size during growth; it decreased the yield of progeny; and it caused uncoordinated movement of the adult. The effects on nematode size, progeny yield, and movement were quantitated. Concentrations of phorbol 12-myristate 13-acetate yielding half-maximal responses were 440, 460, and 170 nM, respectively. As was expected from the biological responsiveness of the nematodes, specific, saturable binding of phorbol ester to nematode extracts was found. [ 3 H]phorbol 12,13-dibutyrate bound with a dissociation constant of 26.8 +/- 3.9 nM. At saturation, 5.7 +/- 1.4 pmole/mg protein was bound

  6. Differential Expression of FAK and Pyk2 in Metastatic and Non-metastatic EL4 Lymphoma Cell Lines

    OpenAIRE

    Zhang, Zhihong; Knoepp, Stewart M.; Ku, Hsun; Sansbury, Heather M.; Xie, Yuhuan; Chahal, Manpreet S.; Tomlinson, Stephen; Meier, Kathryn E.

    2011-01-01

    The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases...

  7. Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Rasmussen, Søren G F; Jørgensen, Trine Nygaard

    2009-01-01

    (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than approximately 30%). DAT was constitutively internalized into vesicular...... and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular...

  8. Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain?

    OpenAIRE

    Nisimoto, Yukio; Jackson, Heather M.; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J. David

    2010-01-01

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47 phox and p67 phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K m for NADPH of 55 ? 10 ?M. The concentration of Nox4 in cell lysates was esti...

  9. Regulation of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of MMP, and progesterone secretion in luteinized granulosa cells from normally ovulating women with polycystic ovary disease.

    Science.gov (United States)

    Ben-Shlomo, Izhar; Goldman, Shlomit; Shalev, Eliezer

    2003-03-01

    To investigate the regulation of MMP-9, TIMP-1, and progesterone via three signal transduction pathways in luteinized granulosa cells from normal ovulatory and PCOD women. In vitro study. Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, Ha'Emek Hospital, Afula, Israel. Ten normal ovulatory and 10 women with polycystic ovary disease (PCOD) treated in an assisted reproduction program. Cultured cells were exposed to phorbol 12-myristate 13-acetate (TPA), acting via protein kinase C (PKC), to epidermal growth factor (EGF), acting via protein tyrosine kinase (PTK), and to forskolin, acting via protein kinase A (PKA). Secretion of MMP-9, TIMP-1, and progesterone. Phorbol 12-myristate 13-acetate elicited an increase in MMP-9 and TIMP-1 secretion in both groups and apparently did not affect progesterone secretion. Epidermal growth factor did not change significantly neither MMP-9 nor TIMP-1 secretion but dose dependently decreased MMP-9-TIMP-1 ratio and increased progesterone secretion in the PCOD group. Forskolin inhibited MMP-9 activity and increased TIMP-1 and progesterone secretion in both groups. Progesterone production was inversely related to the ratio of MMP-9-TIMP-1 regardless of cell origin. In this preliminary study, similar and divergent patterns have emerged in the regulation of MMP-9 and TIMP-1 in human luteinized granulosa cells. Repressing MMP-9-TIMP-1 ratio may have an important modulatory effect on progesterone secretion.

  10. Coordinate viral induction of tumor necrosis factor α and interferon β in human B cells and monocytes

    International Nuclear Information System (INIS)

    Goldfeld, A.E.; Maniatis, T.

    1989-01-01

    Human tumor necrosis factor α (TNF-α) gene expression can be induced primarily in cells of the monocyte/macrophage lineage by a variety of inducers, including lipopolysaccharide, phorbol esters such as phorbol 12-myristate 13-acetate, and virus or synthetic double-stranded RNA [poly(I)·poly(C)]. In this paper the authors show that the TNF-α gene also responds to virus and phorbol 12-myristate 13-acetate in B lymphocytes and that virus is the most potent inducer of TNF-α mRNA in both monocyte and B-cell lines. In addition, they show that viral infection coinduces the expression of TNF-α and interferon β mRNA and that viral induction of both genes is blocked by the kinase inhibitor 2-aminopurine. Inhibition of protein synthesis with cycloheximide had no effect on mRNA expression of the genes in one of three cell lines tested (U937) but blocked the viral induction of both genes in another (Namalwa). Thus, the regulatory factors required for mRNA induction of both genes are present prior to the addition of virus in U937 but not in Namalwa cells. However, in a third cell line (JY), cycloheximide blocked viral induction of the interferon β gene but not the TNF-α gene. Taken together, these observations suggest that viral induction of TNF-α and interferon β gene expression may involve overlapping pathways with both common and distinct regulatory factors

  11. Isolation and culture of melanocytes from the arctic fox (Alopex lagopus

    Directory of Open Access Journals (Sweden)

    Jiarong Bao

    2015-09-01

    Full Text Available Coat colour is a phenotypic marker of fur animal species, which was determined by the pigment generated from melanocytes. In this study, we developed and validated a method for isolation, purification and passage culture of melanocytes from the arctic fox (Alopex lagopus. Skin biopsies were harvested from the dorsal region of adult foxes and enzyme digestion by Dispase II. The primary culture of melanocytes from arctic fox skin was obtained by using keratinocyte serum-free medium supplemented with epidermal growth factor and bovine pituitary extract with/without phorbol- 12-myristate-13-acetate, and by carrying out a medium change strategy. After serial passages, it yielded pure population of melanocytes, which become efficient tools for investigating the function of colour genes and unraveling the process of melanin synthesis.

  12. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J

    2001-01-01

    The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  13. Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

    Energy Technology Data Exchange (ETDEWEB)

    Summers, S.; Florio, T.; Cronin, M.

    1986-05-01

    Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitory hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.

  14. Involvement of PKCα in PMA-induced facilitation of exocytosis and vesicle fusion in PC12 cells

    International Nuclear Information System (INIS)

    Xue Renhao; Zhao Yanying; Chen Peng

    2009-01-01

    Phorbol-12-myristate-13-acetate, a stable analog of the important signaling membrane lipid diacylglycerol (DAG), is known to potentiate exocytosis and modulate vesicle fusion kinetics in neurons and endocrine cells. The exact mechanisms underlying the actions of PMA, however, is often not clear, largely because of the diversity of the DAG/PMA receptors involved in the exocytotic process, which include, most notably, various isoforms of protein kinase C (PKC). In this study, the roles of PKCα in PMA-mediated regulation of exocytosis were investigated by over-expressing wild-type PKCα (wt-PKCα) or dominant negative PKCα (dn-PKCα). Amperometric measurements based on carbon fiber microelectrodes demonstrated that PKCα has a key role in the PMA-mediated facilitation of exocytosis and vesicle fusion in neuroendocrine PC12 cells.

  15. REGULATION OF TLR/RLR GENE ACTIVITY AND SYNTHESIS OF CYTOKINES DURING PHORBOL MYRISTATE ACETATE (PMA-INDUCED DIFFERENTIATION OF THP-1 MONOCYTES INTO MACROPHAGE-LIKE CELLS

    Directory of Open Access Journals (Sweden)

    T. M. Sokolova

    2017-01-01

    Full Text Available The levels of TLR/RLR gene expression and production of some cytokines were studied in monocytic THP-1 cell line during its differentiation to mature macrophage-like forms induced by phorbol 12-myristate 13-acetate (PMA treatment for 1 and 5 days in vitro. For the first time, we have shown high induction levels for the genes that encode signaling immune receptors and transcription factors in response to PMA, as well as inhibitory effects of TLR3, TLR7/TLR8, TLR9-agonists in mature macrophages. The PMAactivated THP-1 macrophage-like cells secreted large quantitities of inflammatory IL-1β and TNFα cytokines into culture medium.

  16. Design, synthesis and biological evaluation of novel desmuramyldipeptide analogs.

    Science.gov (United States)

    Jakopin, Žiga; Corsini, Emanuela; Gobec, Martina; Mlinarič-Raščan, Irena; Dolenc, Marija Sollner

    2011-09-01

    A series of novel desmuramyldipeptides have been designed and synthesized as part of our search for therapeutically useful muramyldipeptide (MDP) analogs. Their immunomodulatory properties were initially assessed in vitro, evaluating their effect on lipopolysaccharide (LPS)-induced cytokine release in THP-1 cells. Following the initial screening, selected compounds were further investigated for immunomodulatory properties using LPS and phorbol 12-myristate 13-acetate (PMA)/ionomycin-stimulated human peripheral blood mononuclear cells. The results confirmed the immunomodulatory properties of some of the synthesized desmuramyldipeptide analogs. Taken together, presented data confirmed the immunostimulatory effect of compound 44, MDP derivative incorporating a pyrido-fused [1,2]-benzisothiazole moiety, while for compounds 32 and 39, indole scaffold-based derivatives of MDP, an immunosuppressive effect was observed. Further studies will be necessary to address their potential therapeutic use as immunomodulatory drugs, both as immunostimulants or anti-inflammatory agents. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  17. Reduced response of splenocytes after mitogen-stimulation in the prion protein (PrP) gene-deficient mouse: PrPLP/Doppel production and cerebral degeneration

    International Nuclear Information System (INIS)

    Kim, Chi-Kyeong; Hirose, Yuko; Sakudo, Akikazu; Takeyama, Natsumi; Kang, Chung-Boo; Taniuchi, Yojiro; Matsumoto, Yoshitsugu; Itohara, Shigeyoshi; Sakaguchi, Suehiro; Onodera, Takashi

    2007-01-01

    Splenocytes of wild-type (Prnp +/+ ) and prion protein gene-deficient (Prnp -/- ) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA) + ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrP C ) expression was enhanced following ConA stimulation, but not PMA + Io or LPS in Prnp +/+ splenocytes. Rikn Prnp -/- splenocytes elicited lower cell proliferations than Prnp +/+ or Zrch I Prnp -/- splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrP C and PrPLP/Doppel

  18. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    , inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...... phosphorylation of the MAP kinases extracellular signal-regulated kinases ERK1 and ERK2. The MAP kinase activation was sustained, because ERK1 and ERK2 were phosphorylated in PC12-E2 cells and primary hippocampal neurons even after 24 hr of cultivation on NCAM-expressing fibroblasts. Based on these results, we...

  19. EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells.

    Science.gov (United States)

    Braundmeier, A G; Dayger, C A; Mehrotra, P; Belton, R J; Nowak, R A

    2012-12-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

  20. Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis

    DEFF Research Database (Denmark)

    Roepstorff, Kirstine; Rasmussen, Izabela Zorawska; Sawada, Makoto

    2008-01-01

    dominant-positive mutants enhanced, whereas dominant-negative mutants inhibited, NADPH oxidase-mediated superoxide generation following formyl-methionyl-leucylphenylalanine or phorbol 12-myristate 13-acetate stimulation. Both Rac1 and the GTP exchange factor VAV1 were required as upstream signaling......The p21-activated kinase-1 (PAK1) is best known for its role in the regulation of cytoskeletal and transcriptional signaling pathways. We show here in the microglia cell line Ra2 that PAK1 regulates NADPH oxidase (NOX-2) activity in a stimulus-specific manner. Thus, conditional expression of PAK1...... proteins in the formyl-methionyl-leucyl-phenylalanine-induced activation of endogenous PAK1. In contrast, PAK1 mutants had no effect on superoxide generation downstream of FcgammaR signaling during phagocytosis of IgG-immune complexes. We further present evidence that the effect of PAK1 on the respiratory...

  1. Strenuous exercise decreases the percentage of type 1 T cells in the circulation

    DEFF Research Database (Denmark)

    Steensberg, A; Toft, A D; Bruunsgaard, H

    2001-01-01

    -gamma and interleukin (IL)-2, and type 2 (Th2 and Tc2) cells, which produce IL-4. The question addressed in the present study was whether exercise affected the relative balance between the circulating levels of these cytokine-producing T cells. Nine male runners performed treadmill running for 2.5 h at 75% of maximal...... oxygen consumption. The intracellular expression of cytokines was detected following stimulation with ionomycin and phorbol 12-myristate 13-acetate in blood obtained before, during, and after exercise. The percentage of type 1 T cells in the circulation was suppressed at the end of exercise and 2 h after......Prolonged strenuous exercise is followed by a temporary functional immune impairment. Low numbers of CD4+ T helper (Th) and CD8+ T cytotoxic (Tc) cells are found in the circulation. These cells can be divided according to their cytokine profile into type 1 (Th1 and Tc1), which produce interferon...

  2. Effect of gamma-ray irradiated natural herbal extracts on NF-kB activation in HMC-1 cells

    International Nuclear Information System (INIS)

    Kim, Yong Soo; Lim, Youn Mook; Gwon, Hui Jeong; Choi, Bo Ram; Nho, Young Chang

    2009-01-01

    Recently, studies have documented various health benefits of some natural herbal extracts (NHE) such as Houttuynia cordata (H), Centella asiatica (C), Plantago asiatica (P), Morus alba L. (M), and Ulmus davidiana (U). The aim of the present study was to demonstrate the radiation effect on NF-kB activation of the NHE in the human mast cell line (HMC-1). The HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187. Both non-and irradiated NHE also significantly inhibited the PMA plus A23187-induced nuclear factor NF-kB activation and also suppressed the expression of activation of NF-kB. These results indicated that the NHE exerted a regulatory effect on inflammatory reactions mediated by mast cells

  3. Effect of gamma-ray irradiated natural herbal extracts on NF-kB activation in HMC-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yong Soo; Lim, Youn Mook; Gwon, Hui Jeong; Choi, Bo Ram; Nho, Young Chang [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-12-15

    Recently, studies have documented various health benefits of some natural herbal extracts (NHE) such as Houttuynia cordata (H), Centella asiatica (C), Plantago asiatica (P), Morus alba L. (M), and Ulmus davidiana (U). The aim of the present study was to demonstrate the radiation effect on NF-kB activation of the NHE in the human mast cell line (HMC-1). The HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187. Both non-and irradiated NHE also significantly inhibited the PMA plus A23187-induced nuclear factor NF-kB activation and also suppressed the expression of activation of NF-kB. These results indicated that the NHE exerted a regulatory effect on inflammatory reactions mediated by mast cells.

  4. Immune-suppressive activity of punicalagin via inhibition of NFAT activation

    International Nuclear Information System (INIS)

    Lee, Sang-Ik; Kim, Byoung-Soo; Kim, Kyoung-Shin; Lee, Samkeun; Shin, Kwang-Soo; Lim, Jong-Soon

    2008-01-01

    Since T cell activation is central to the development of autoimmune diseases, we screened a natural product library comprising 1400 samples of medicinal herbal extracts, to identify compounds that suppress T cell activity. Punicalagin (PCG) isolated from the fruit of Punica granatum was identified as a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated T cells (NFAT). PCG downregulated the mRNA and soluble protein expression of interleukin-2 from anti-CD3/anti-CD28-stimulated murine splenic CD4+ T cells and suppressed mixed leukocytes reaction (MLR) without exhibiting cytotoxicity to the cells. In vivo, the PCG treatment inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice and decreased CD3+ T cell infiltration of the inflamed tissue. These results suggest that PCG could be a potential candidate for the therapeutics of various immune pathologies

  5. The Small Colony Variant of Listeria monocytogenes Is More Tolerant to Antibiotics and Has Altered Survival in RAW 264.7 Murine Macrophages

    DEFF Research Database (Denmark)

    Curtis, Thomas; Gram, Lone; Knudsen, Gitte Maegaard

    2016-01-01

    Small Colony Variant (SCV) cells of bacteria are a slow-growing phenotype that result from specific defects in the electron transport chain. They form pinpoint colonies on agar plates and have a variety of phenotypic characteristics, such as altered carbon metabolism, decreased toxin and lytic...... monocytogenes (strain SCV E18), similar to the high persister mutant phenotype, survived significantly better than the wild type when exposed over a 48-h period to concentrations above Minimal Inhibitory Concentration for most tested antibiotics. SCV E18 survived more poorly than the wildtype in unactivated RAW......264.7 macrophage cells, presumably because of its reduced listeriolysin O expression, however, it survived better in reactive oxygen species producing, phorbol 12-myristate 13-acetate-activated macrophages. Although SCV E18 was sensitive to oxygen as it entered the stationary phase...

  6. High circulating levels of tumor necrosis factor-alpha in centenarians are not associated with increased production in T lymphocytes

    DEFF Research Database (Denmark)

    Sandmand, Marie; Bruunsgaard, Helle; Kemp, Kåre

    2003-01-01

    BACKGROUND: Aging is characterized by increased inflammatory activity reflected by increased plasma levels of proinflammatory cytokines, concomitant with an altered cytokine profile of T lymphocytes. High plasma levels of tumor necrosis factor (TNF)-alpha are strongly associated with morbidity...... and mortality in elderly humans. However, the cellular source and mechanisms for the increased circulating TNF-alpha levels are unknown. OBJECTIVE: The aim of the present study was to investigate if high plasma levels of TNF-alpha are associated with increased production of TNF-alpha by T lymphocytes in elderly...... humans. METHODS: TNF-alpha production by CD4+ and CD8+ T lymphocytes was measured by flow cytometry following stimulation with phorbol 12-myristate 13-acetate and ionomycin in 28 young controls, 14, 81-year-olds and 25 centenarians. RESULTS: Plasma levels of TNF-alpha increased with increasing age...

  7. Regulation of ATP-sensitive K+ channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    International Nuclear Information System (INIS)

    De Weille, J.R.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M.

    1989-01-01

    The actions of somatostatin and of the phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and 86 Rb + flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K + channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive 86 Rb + efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K + channels are discussed

  8. The stimulation of EL-4 cells to produce interleukin-2 and its potential use in immunocytotoxicity testing

    International Nuclear Information System (INIS)

    Lasek, W.; Steer, S.; Clothier, R.; Balls, M.

    1989-01-01

    The ability of EL-4 thymoma cells to produce interleukin-2 (IL-2) following exposure to phorbol-12-myristate 13-acetate (PMA) and Concanavalin A (Con A) has been studied in vitro using medium containing either 10% or 1% fetal calf serum (FCS). The potent stimulatory effect of PMA on IL-2 production by EL-4 cells has been confirmed by measuring 3H-thymidine incorporation by the IL-2-dependent T cell line, CTLL-2, in the presence of conditioned medium (CM) from stimulated cultures. EL-4 cells produced several times more IL-2 when cultured in medium containing 10% FCS than when only 1% FCS was present. Added together, PMA and Con A acted synergistically in some EL-4 cell cultures. The ability of E:-4 cells to produce IL-2 was maintained after further incubation without stimulants. CM with IL-2 activity from stimulated EL-4 cells could prove useful in immunotoxicity testing

  9. [ABIN1 is not involved in imatinib upregulating A20 to inhibit the activation of NF-κB pathway in Jurkat T cells].

    Science.gov (United States)

    Chen, Qian; Wang, Senlin; Lin, Chen; Chen, Shaohua; Zhao, Xiaoling; Li, Yangqiu

    2017-05-01

    Objective To investigate the effect of imatinib (IM) on the expressions of A20-binding inhibitor of NF-κB1 (ABIN1) and A20 in Jurkat T cells. Methods Jurkat T cells were treated with 25, 50 and 100 nmol/L IM for 24 hours. The mRNA and protein levels of ABIN1, A20 and NF-κB were detected by real-time quantitative PCR and Western blotting. Results IM significantly inhibited both mRNA and protein levels of ABIN1 and NF-κB, but raised the mRNA and protein levels of A20; while phorbol 12-myristate 13-acetate/ionomycin increased the expression levels of ABIN1 and A20 mRNA and protein. Conclusion IM could upregulate A20 protein to inhibit the activation of NF-κB pathway in Jurkat T cells, which was independent of the ABIN1 protein.

  10. The effect of medium composition on interleukin-2 production by murine EL-4 thymoma cells

    Directory of Open Access Journals (Sweden)

    Galesi A. L. L.

    2004-01-01

    Full Text Available Due to the role of interleukin-2 (IL-2 in the mediation of immune response, this cytokine has been used in the treatment of some types of cancer and infectious diseases. However, relatively high levels of this cytokine are required to achieve significant activity. The aim of this work was to study a culture medium composition designed to increase the production of IL-2 by suspended murine EL-4 cells. The cultivations were carried out aiming at producing IL-2 in stirred bioreactors. The effects of concentration of glutamine, phorbol-12-myristate-13-acetate (PMA, concanavalin A (Con A, Pluronic F68, and fetal calf serum (FCS on cell viability and IL-2 production were evaluated. PMA alone was more efficient in IL-2 production than it was in association with Con A. The maximum IL-2 production was around 162 ng/mL with 856 ng/mL PMA and 1.45% (v/v FCS.

  11. Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway.

    Science.gov (United States)

    Sancho, Rocío; Márquez, Nieves; Gómez-Gonzalo, Marta; Calzado, Marco A; Bettoni, Giorgio; Coiras, Maria Teresa; Alcamí, José; López-Cabrera, Manuel; Appendino, Giovanni; Muñoz, Eduardo

    2004-09-03

    Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.

  12. Lymphocyte adhesion to endothelial cell (EC) is stimulated by phorbol esters

    International Nuclear Information System (INIS)

    Haskard, D.; Cavender, D.; Ziff, M.

    1986-01-01

    The effect of phorbol esters on T cell adhesion to EC has been studied. The phorbol esters 12-0-tetradecanoyl-phorbol-13-acetate and 4-beta-phorbol-12-13-dibutyrate, but not the biologically inert 4-0-methyl-phorbol-12-13-didecanoate strongly increased the binding of 51 Cr-labeled T cells to human umbilical vein EC monolayers in microtiter wells. Increase in binding was observed at 0.3 ng/ml with maximal enhancement at 50 ng/ml. Both unstimulated and phorbol ester activated T cells displayed a substantially greater binding affinity for EC than for fibroblasts or plastic. Binding enhancement occurred within one minute, with maximal increase after 15 min. Preincubation studies showed that binding enhancement was entirely attributable to an effect on T cells, with no action on EC. Additive binding enhancement was seen when phorbol esters and reagents that increase adhesion by actions on EC (LPS, IL-1 and IFN-γ) were used together. Increase in adhesion of activated T lymphocytes to EC may explain the greater emigration of activated T cells than small resting T cells into inflammatory foci in vivo. The rapid onset of the phorbol effect suggests that this may be an important mechanism for immediate localization of circulating T cells in the cellular immune response, activated, perhaps, at the endothelial blood-tissue interface

  13. Protein kinase C and α 2-adrenoceptor-mediated inhibition of noradrenaline release from the rat tail artery

    International Nuclear Information System (INIS)

    Bucher, B.; Neuburger, J.; Illes, P.

    1991-01-01

    In isolated rat tail arteries preincubated with [3H]noradrenaline, electrical field stimulation evoked the overflow of tritium. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activating phorbol ester, time-dependently increased the overflow at 1 mumol/L but not at 0.1 mumol/L. In contrast, the overflow was not altered by phorbol 13-acetate (PA, 1 mumol/L), which does not influence the activity of PKC. Polymyxin B (70 mumol/L), an inhibitor of PKC, depressed the overflow when given alone and, in addition, attenuated the effect of PMA, 1 mumol/L. The selective alpha 2-adrenoceptor agonist B-HT 933 depressed the overflow; PMA, 1 mumol/L, did not interfere with the effect of B-HT 933, 10 mumol/L. The results provide evidence for the participation of prejunctionally located PKC in the release of noradrenaline. However, PKC does not seem to be involved in the alpha 2-adrenoceptor-agonist-mediated inhibition of noradrenaline release

  14. Anti-Tumour Promoting Activity and Antioxidant Properties of Girinimbine Isolated from the Stem Bark of Murraya koenigii S.

    Directory of Open Access Journals (Sweden)

    Yih Yih Kok

    2012-04-01

    Full Text Available Girinimbine, a carbazole alkaloid isolated from the stem bark of Murraya koenigii was tested for the in vitro anti-tumour promoting and antioxidant activities. Anti-tumour promoting activity was determined by assaying the capability of this compound to inhibit the expression of early antigen of Epstein-Barr virus (EA-EBV in Raji cells that was induced by the tumour promoter, phorbol 12-myristate 13-acetate. The concentration of this compound that gave an inhibition rate at fifty percent was 6.0 µg/mL and was not cytotoxic to the cells. Immunoblotting analysis of the expression of EA-EBV showed that girinimbine was able to suppress restricted early antigen (EA-R. However, diffused early antigen (EA-D was partially suppressed when used at 32.0 µg/mL. Girinimbine exhibited a very strong antioxidant activity as compared to a-tocopherol and was able to inhibit superoxide generation in the 12-O-tetradecanoylphorbol-13-acetate (TPA-induced differentiated premyelocytic HL-60 cells more than 95%, when treated with the compound at 5.3 and 26.3 µg/mL, respectively. However girinimbine failed to scavenge the stable diphenyl picryl hydrazyl (DPPH-free radical.

  15. Palytoxin: exploiting a novel skin tumor promoter to explore signal transduction and carcinogenesis.

    Science.gov (United States)

    Wattenberg, Elizabeth V

    2007-01-01

    Palytoxin is a novel skin tumor promoter, which has been used to help probe the role of different types of signaling mechanisms in carcinogenesis. The multistage mouse skin model indicates that tumor promotion is an early, prolonged, and reversible phase of carcinogenesis. Understanding the molecular mechanisms underlying tumor promotion is therefore important for developing strategies to prevent and treat cancer. Naturally occurring tumor promoters that bind to specific cellular receptors have proven to be useful tools for investigating important biochemical events in multistage carcinogenesis. For example, the identification of protein kinase C as the receptor for the prototypical skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (also called phorbol 12-myristate 13-acetate, PMA) provided key evidence that tumor promotion involves the aberrant modulation of signaling cascades that govern cell fate and function. The subsequent discovery that palytoxin, a marine toxin isolated from zoanthids (genus Palythoa), is a potent skin tumor promoter yet does not activate protein kinase C indicated that investigating palytoxin action could help reveal new aspects of tumor promotion. Interestingly, the putative receptor for palytoxin is the Na(+),K(+)-ATPase. This review focuses on palytoxin-stimulated signaling and how palytoxin has been used to investigate alternate biochemical mechanisms by which important targets in carcinogenesis can be modulated.

  16. Induction of rat hepatic zinc thionein by phorbol ester-mediated protein kinase C pathway

    Energy Technology Data Exchange (ETDEWEB)

    Garrett, S.H.; Funk, A.E.; Brady, F.O.

    1986-05-01

    Metallothionein (MT) exists in rat liver mainly as a zinc protein. The levels of this protein fluctuate in response to a variety of internal and external stimuli. Among these inducers of MT are metals, glucocorticoids, catecholamines, and polypeptide hormones. Metals and glucocorticoids are primary inducers of MT, while the others operate either via adenylate cyclase/cAMP/cAMP-dependent protein kinase, or via phospholipase C/inositol 1,4,5-triphosphate, diacylglycerol/Ca/sup 2 +/-dependent protein kinase, protein kinase C. The authors have examined the role of the protein kinase C pathway in the induction of MT by using a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), to activate it. In vivo TPA is a good inducer of Zn/sub 7/-MT with an ED/sub 0.5/ of 26.5 nmoles/kg b.w. Maximal levels reached were about 7..mu..g Zn in MT/g liver, an induction increase of 8 to 10-fold. An inactive compound, 4..beta..-phorbol, and the vehicle (DMSO) did not stimulate the synthesis of Zn/sub 7/-MT. This induction by TPA requires de novo protein synthesis, as demonstrated by a cycloheximide/(/sup 35/S)-cysteine experiment. TPA stimulated Zn incorporation by 8.6-fold and (/sup 35/S)-cysteine incorporation by 4.8-fold during an 11h induction. These increases were blocked 100% by treatment with cycloheximide at -1 and +5h. These experiments have been repeated in cultured hepatocytes, using (/sup 35/S)-cysteine incorporation, slab SDS-PAGE, and autoradiography to quantitate MT levels.

  17. Isolation and characterization of human glycophorin A cDNAs using a synthetic oligonucleotide approach: nucleotide sequence, mRNA structure and regulation by 12-O-tetradecanoylphorbol 13-acetate (TPA)

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1986-01-01

    The authors have previously shown that treatment of human erythroleukemic K562 cells with the tumor-promoting phorbol ester, TPA, results in a diminished expression of glycophorin A at the level of protein biosynthesis and in vitro mRNA translation activity. To further examine the structure, relationships and expression of human glycophorins they have successfully isolated and sequenced several glycophorin A specific cDNA clones derived from K562 cells, by making extensive use of mixed and exact synthetic oligonucleotides as primers and radioactively labeled probes. The nucleotide sequence obtained from the largest glycophorin A cDNA suggests the presence of a hydrophobic leader-like peptide of at least 19 amino acids. Northern gel analysis using both whole cDNA-plasmid and synthetic oligonucleotide probes revealed the existence of multiple mRNAs, three of which they believe to be glycophorin A-specific, whereas a fourth and smaller mRNA appears to be glycophorin B-specific. Furthermore, the abundance of all four glycophorin mRNAs were found to be extensively reduced following treatment of K562 cells with TPA suggesting coordinate regulation, possibly at the level of gene transcription

  18. Ribosomal protein S6 phosphorylation and morphological changes in response to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate in primary human tumour cells, established and transformed cell lines

    DEFF Research Database (Denmark)

    Rance, A J; Thönnes, M; Issinger, O G

    1985-01-01

    lifespan (fibroblasts, primary human tumour cells) can be mimicked by unknown steps also associated with immortalization (establishment function) and the transformed state of the tumour cells. Another interesting observation were morphological changes of the established and SV40-transformed cells which...

  19. Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor

    International Nuclear Information System (INIS)

    Morrison, W.J.; Dhar, A.; Shukla, S.D.

    1989-01-01

    The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF stimulated incorporation of 32 P into proteins and caused [ 3 H]InsP 3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [ 3 H]InsP 3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [ 3 H]InsP 3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF

  20. Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type II cells

    International Nuclear Information System (INIS)

    Dobbs, L.G.; Wright, J.R.; Hawgood, S.; Gonzalez, R.; Venstrom, K.; Nellenbogen, J.

    1987-01-01

    Pulmonary surfactant is synthesized and secreted by alveolar type II cells. Radioactive phosphatidylcholine has been used as a marker for surfactant secretion. The authors report findings that suggest that surfactant inhibits secretion of 3 H-labeled phosphatidylcholine by cultured rat type II cells. The lipid components and the surfactant protein group of M/sub r/ 26,000-36,000 (SP 26-36) inhibit secretion to different extents. Surfactant lipids do not completely inhibit release; in concentrations of 100 μg/ml, lipids inhibit stimulated secretion by 40%. SP 26-36 inhibits release with an EC 50 of 0.1 μg/ml. At concentrations of 1.0 μg/ml, SP 26-36 inhibits basal secretion and reduces to basal levels secretion stimulated by terbutaline, phorbol 12-myristate 13-acetate, and the ionophore A23187. The inhibitory effect of SP 26-36 can be blocked by washing type II cells after adding SP 26-36, by heating the proteins to 100 0 C for 10 min, by adding antiserum specific to SP 26-36, or by incubating cells in the presence of 0.2 mM EGTA. SP 26-36 isolated from canine and human sources also inhibits phosphatidylcholine release from rat type II cells. Neither type I collagen nor serum apolipoprotein A-1 inhibits secretion. These findings are compatible with the hypothesis that surfactant secretion is under feedback regulatory control

  1. Extracellular ultrathin fibers sensitive to intracellular reactive oxygen species: Formation of intercellular membrane bridges

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Se-Hui; Park, Jin-Young; Joo, Jung-Hoon; Kim, Young-Myeong; Ha, Kwon-Soo, E-mail: ksha@kangwon.ac.kr

    2011-07-15

    Membrane bridges are key cellular structures involved in intercellular communication; however, dynamics for their formation are not well understood. We demonstrated the formation and regulation of novel extracellular ultrathin fibers in NIH3T3 cells using confocal and atomic force microscopy. At adjacent regions of neighboring cells, phorbol 12-myristate 13-acetate (PMA) and glucose oxidase induced ultrathin fiber formation, which was prevented by Trolox, a reactive oxygen species (ROS) scavenger. The height of ROS-sensitive ultrathin fibers ranged from 2 to 4 nm. PMA-induced formation of ultrathin fibers was inhibited by cytochalasin D, but not by Taxol or colchicine, indicating that ultrathin fibers mainly comprise microfilaments. PMA-induced ultrathin fibers underwent dynamic structural changes, resulting in formation of intercellular membrane bridges. Thus, these fibers are formed by a mechanism(s) involving ROS and involved in formation of intercellular membrane bridges. Furthermore, ultrastructural imaging of ultrathin fibers may contribute to understanding the diverse mechanisms of cell-to-cell communication and the intercellular transfer of biomolecules, including proteins and cell organelles.

  2. Extract of corn silk (stigma of Zea mays) inhibits the tumour necrosis factor-alpha- and bacterial lipopolysaccharide-induced cell adhesion and ICAM-1 expression.

    Science.gov (United States)

    Habtemariam, S

    1998-05-01

    Treatment of human endothelial cells with cytokines such as tumour necrosis factor-alpha (TNF) or E. coli lipopolysaccharide (LPS) induces the expression of several adhesion molecules and enhances leukocyte adhesion to endothelial cell surface. Interfering with this leukocyte adhesion or adhesion molecules upregulation is an important therapeutic target for the treatment of bacterial sepsis and various inflammatory diseases. In the course of screening marketed European anti-inflammatory herbal drugs for TNF antagonistic activity, a crude ethanolic extract of corn silk (stigma of Zea mays) exhibited significant activity. The extract at concentrations of 9-250 micrograms/ml effectively inhibited the TNF- and LPS-induced adhesiveness of EAhy 926 endothelial cells to monocytic U937 cells. Similar concentration ranges of corn silk extract did also block the TNF and LPS but not the phorbol 12-myristate 13-acetate-induced ICAM-1 expression on EAhy 926 endothelial cell surface. The extract did not alter the production of TNF by LPS-activated macrophages and failed to inhibit the cytotoxic activity of TNF. It is concluded that corn silk possesses important therapeutic potential for TNF- and LPS-mediated leukocyte adhesion and trafficking.

  3. Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1987-01-01

    The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH 2 -terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

  4. Cloning the human lysozyme cDNA: Inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells

    International Nuclear Information System (INIS)

    Chung, L.P.; Keshav, S.; Gordon, S.

    1988-01-01

    Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has in 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a >1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression

  5. Coenzyme Q10 partially restores pathological alterations in a macrophage model of Gaucher disease.

    Science.gov (United States)

    de la Mata, Mario; Cotán, David; Oropesa-Ávila, Manuel; Villanueva-Paz, Marina; de Lavera, Isabel; Álvarez-Córdoba, Mónica; Luzón-Hidalgo, Raquel; Suárez-Rivero, Juan M; Tiscornia, Gustavo; Sánchez-Alcázar, José A

    2017-02-06

    Gaucher disease (GD) is caused by mutations in the GBA1 gene which encodes lysosomal β-glucocerebrosidase (GCase). In GD, partial or complete loss of GCase activity causes the accumulation of the glycolipids glucosylceramide (GlcCer) and glucosylsphingosine in the lysosomes of macrophages. In this manuscript, we investigated the effects of glycolipids accumulation on lysosomal and mitochondrial function, inflammasome activation and efferocytosis capacity in a THP-1 macrophage model of Gaucher disease. In addition, the beneficial effects of coenzyme Q 10 (CoQ) supplementation on cellular alterations were evaluated. Chemically-induced Gaucher macrophages were developed by differentiateing THP-1 monocytes to macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) and then inhibiting intracellular GCase with conduritol B-epoxide (CBE), a specific irreversible inhibitor of GCase activity, and supplementing the medium with exogenous GlcCer. This cell model accumulated up to 16-fold more GlcCer compared with control THP-1 cells. Chemically-induced Gaucher macrophages showed impaired autophagy flux associated with mitochondrial dysfunction and increased oxidative stress, inflammasome activation and impaired efferocytosis. All abnormalities were partially restored by supplementation with CoQ. These data suggest that targeting mitochondria function and oxidative stress by CoQ can ameliorate the pathological phenotype of Gaucher cells. Chemically-induced Gaucher macrophages provide cellular models that can be used to investigate disease pathogenesis and explore new therapeutics for GD.

  6. Interleukin-1α Induction in Human Keratinocytes (HaCaT: An In Vitro Model for Chemoprevention in Skin

    Directory of Open Access Journals (Sweden)

    T. Magcwebeba

    2012-01-01

    Full Text Available Long-term exposure to UV irradiation and toxic chemicals is associated with chronic inflammation that contributes to skin cancer development with interleukin-1 alpha (IL-1α, constitutively produced by keratinocytes, playing a pivotal role in skin inflammation. The aim of this study was to investigate the modulation of IL-1α production in the HaCaT keratinocyte cell line. Phorbol 12-myristate 13-acetate failed to induce IL-1α in HaCaT cells, and this might be associated with the specific deficiency known to affect downstream signalling of the MEK/ERK pathway in these cells. The calcium ionophore, ionomycin, slightly enhanced the production of intracellular (icIL-1α, but this resulted in a necrotic release at higher concentrations. UV-B exposure significantly increased the production of icIL-1α in a dose-dependent manner with a maximal induction exhibited at 24 h with minimal necrotic and apoptotic effects. Validation of the HaCaT cell model indicated that the nonsteroidal anti-inflammatory drug (NSAID, ibuprofen, and the glucocorticoid, dexamethasone, inhibited icIL-1α production, and this was associated with a slight inhibition of cell viability. The UV-B-induced keratinocyte cell model provides an in vitro system that could, apart from phorbol ester-like compounds, be utilised as a screening assay in identifying skin irritants and/or therapeutic topical agents via the modulation of IL-1α production.

  7. KCl stimulation increases norepinephrine transporter function in PC12 cells.

    Science.gov (United States)

    Mandela, Prashant; Ordway, Gregory A

    2006-09-01

    The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

  8. Protein phosphorylation in pancreatic islets induced by 3-phosphoglycerate and 2-phosphoglycerate

    International Nuclear Information System (INIS)

    Pek, S.B.; Usami, Masaru; Bilir, N.; Fischer-Bovenkerk, C.; Ueda, Tetsufumi

    1990-01-01

    The authors have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, they considered the possibility that 3-phosphoglycerate may act as a coupling factor, and they searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [γ- 32 P]ATP and appropriate test substances and were subjected to NaDodSO 4 /PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion

  9. Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study.

    Science.gov (United States)

    Westera, Liset; van Viegen, Tanja; Jeyarajah, Jenny; Azad, Azar; Bilsborough, Janine; van den Brink, Gijs R; Cremer, Jonathan; Danese, Silvio; D'Haens, Geert; Eckmann, Lars; Faubion, William; Filice, Melissa; Korf, Hannelie; McGovern, Dermot; Panes, Julian; Salas, Azucena; Sandborn, William J; Silverberg, Mark S; Smith, Michelle I; Vermeire, Severine; Vetrano, Stefania; Shackelton, Lisa M; Stitt, Larry; Jairath, Vipul; Levesque, Barrett G; Spencer, David M; Feagan, Brian G; Vande Casteele, Niels

    2017-11-02

    Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies. Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor. Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG. Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

  10. Role and mechanism of PKC on radiosensitization in pancreatic carcinoma cell line Panc-1

    International Nuclear Information System (INIS)

    Qiao Qiao; Zhang Shuo; Chen Yanzhi; Li Guang

    2008-01-01

    Objective: To explore the effect of PKC on radiosensitization in pancreatic carcinoma cell line Panc-1, and its mediating mechanism. Methods: Panc-1 cells were treated with the specific activator of PKC (phorbol 12-myristate 13-acetate, PMA) and the specific inhibitor of PKC (chelerythrine, CH) to observe the SF2 changes. Cell survival was determined by clonogenic assay. The apoptosis rates of the cells were analyzed by flow cytometry with Annexin V/PI staining. The expression of apoptosis related protein Bcl-2 and Bax after the treatment of CH and/or irradiation was determined by immunocytochemistry. Results: The SF 2 values of radiation group, PMA group and CH group were 0.78 ± 0.02, 0.92 ± 0.11 and 0.19 ± 0.20, respectively. CH can significantly increase the sensitivity of Panc-1 to irradiation. SERs of Panc-1 cells were 1.05, 1.24 and 1.77 after the treatment of 0.5, 2 and 8 μmol/L of CH, respectively. The result of flow cytometry analysis showed that PMA decreased the apoptosis index with irradiation, while CH significantly increased the apoptosis index. Expression of Bax protein was increased significantly (P<0.05) while that of Bcl-2 was not influenced; however, the ratio of Bax/Bcl-2 was increased. Conclusions: PKC regulates the radiosensitivity of Panc-1 by mediating the apoptosis of tumor cells. (authors)

  11. Acetylshikonin Inhibits Human Pancreatic PANC-1 Cancer Cell Proliferation by Suppressing the NF-κB Activity.

    Science.gov (United States)

    Cho, Seok-Cheol; Choi, Bu Young

    2015-09-01

    Acetylshikonin, a natural naphthoquinone derivative compound, has been used for treatment of inflammation and cancer. In the present study, we have investigated whether acetylshikonin could regulate the NF-κB signaling pathway, thereby leading to suppression of tumorigenesis. We observed that acetylshikonin significantly reduced proliferation of several cancer cell lines, including human pancreatic PANC-1 cancer cells. In addition, acetylshikonin inhibited phorbol 12-myristate 13-acetate (PMA) or tumor necrosis-α (TNF-α)-induced NF-κB reporter activity. Proteome cytokine array and real-time RT-PCR results illustrated that acetylshikonin inhibition of PMA-induced production of cytokines was mediated at the transcriptional level and it was associated with suppression of NF-κB activity and matrix metalloprotenases. Finally, we observed that an exposure of acetylshikonin significantly inhibited the anchorage-independent growth of PANC-1 cells. Together, our results indicate that acetylshikonin could serve as a promising therapeutic agent for future treatment of pancreatic cancer.

  12. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Hsin-Yu Chou

    2016-06-01

    Full Text Available Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA, and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR, we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

  13. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

    Science.gov (United States)

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

  14. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

    Directory of Open Access Journals (Sweden)

    Omar Rafael Alemán

    2016-01-01

    Full Text Available Neutrophils (PMN are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

  15. PMA Induces SnoN Proteolysis and CD61 Expression through an Autocrine Mechanism

    Science.gov (United States)

    Li, Chonghua; Peart, Natoya; Xuan, Zhenyu; Lewis, Dorothy E; Xia, Yang; Jin, Jianping

    2014-01-01

    Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-β family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APCCdh1 ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation. PMID:24637302

  16. [Protective effect of indirect activator of calcium pump on noise-induced hearing loss].

    Science.gov (United States)

    Liu, Jun; Yu, Ning; Han, Dongyi; Yang, Weiyan; Li, Xingqi

    2002-12-01

    To investigate the possible protective effect of phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC) and indirect activator of Ca2+ pump, on noise-induced hearing loss (NIHL). Twenty guinea pigs were divided randomly into two groups, and then perfused with artificial perilymph solutions in one group and with artificial perilymph solutions containing 3 mumol/L PMA in the other one, respectively. All animals were exposed with 100 dB SPL white noise for 2 hours. Cochlear microphonics (CM) and compound action potential (CAP) were recorded from the round window (RW) before noise exposure and 2 hours after noise exposure. There was no significant difference in CAP threshold and CM amplitude between two groups before noise exposure. A significant difference was observed in CAP threshold and CM amplitude between two groups after noise exposure. The amplitude of CM decreased and the threshold of CAP increased in both group after noise exposure, but in the PMA group the decrease of the amplitude of CM was higher while the increase of threshold of CAP lower than that in control (P < 0.05). PMA might have partly protective effect on NIHL. These findings indirectly proved that intracellular Ca2+ overload might involve in the mechanism of NIHL.

  17. Demonstration of interleukin-1 beta transcripts in acute myeloblastic leukemic cells by in situ hybridization.

    Science.gov (United States)

    Nakamura, M; Kanakura, Y; Furukawa, Y; Ernst, T J; Griffin, J D

    1990-07-01

    The cells from some patients with acute myeloblastic leukemia will secrete autostimulatory cytokines in tissue culture without the addition of stimulators such as phorbol 12-myristate 13-acetate. Production of interleukin-1 beta (IL-1 beta), for example, has been observed in up to 50% of cases. In order to investigate the nature of the cell secreting IL-1 beta in AML, we used an antisense RNA probe to detect specific IL-1 beta transcripts in individual leukemic cells by in situ hybridization. In fresh, uncultured cells, IL-1 beta transcripts were observed in 1-40% of undifferentiated leukemic blast cells in 17 of 19 cases. In situ hybridization was at least as sensitive as Northern blot analysis in detecting IL-1 beta transcripts. No correlation of IL-1 beta transcript expression with FAB classification was observed. Normal blood and bone marrow mononuclear cells did not contain cells expressing IL-1 beta transcripts. These results support the concept that the regulation of cytokine genes in AML cells is aberrant.

  18. [Phenotypic and functional features of NK and NKT cells in chronic hepatitis B].

    Science.gov (United States)

    Wu, Shaofei; Li, Man; Sun, Xuehua; Zhou, Zhenhua; Zhu, Xiaojun; Zhang, Xin; Gao, Yueqiu

    2015-06-01

    To detect the ratio of natural killer (NK)/natural killer T (NKT) cells in peripheral blood, the levels of NKG2D/NKG2A, interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) in patients with chronic hepatitis B (CHB). Peripheral blood mononuclear cells (PBMCs) were harvested from CHB patients. The ratio of NK/NKT cells in PBMCs and the levels of NKG2D and NKG2A were detected by flow cytometry. The expressions of intracellular IFN-γ and TNF-α were analyzed by flow cytometry after the treatment with phorbol 12-myristate 13-acetate (PMA), brefeldin A (BFA) or ionomycin in vitro. The comparison between two groups was performed by independent sample t-test. The relationship of each index to hepatitis B virus load and serum alanine aminotransferase was analyzed by Pearson correlation analysis. Compared with healthy controls, CHB patients presented with significantly decreased peripheral blood NK/NKT cell ratio and significantly elevated proportions of NKG2A+ NK and NKG2A+NKT cells, and after the treatment with PMA/BFA/ionomycin, IFN-γ+ NK and IFN-γ+ NKT cells were significantly reduced in CHB patients. NK and NKT cells showed a reduced ratio, disordered receptor expressions and decreased cytokine secretion capacity in CHB patients.

  19. Changes in NK and NKT cells in mesenteric lymph nodes after a Schistosoma japonicum infection.

    Science.gov (United States)

    Luo, Xueping; Xie, Hongyan; Chen, Dianhui; Yu, Xiuxue; Wu, Fan; Li, Lu; Wu, Changyou; Huang, Jun

    2014-03-01

    The mesenteric lymph node (MLN) is the main draining lymph node in mouse enterocoelia, which contains many types of immune cells. Among these cells, natural killer (NK) and natural killer T (NKT) cells belong to innate lymphoid cells (ILCs), which have potent activities for controlling a variety of pathogenic infections. In this study, C57BL/6 mice were infected with Schistosoma japonicum for 5-7 weeks. Lymphocytes were isolated from the MLN to detect changes in the phenotype and function of NK and NKT cells using a fluorescence activating cell sorter (FACS). These results demonstrated that a S. japonicum infection could significantly increase the percentage of NK cells in the mouse MLN, (P cell number of both NK and NKT cells. In addition, we found that NK and NKT cells from infected mice expressed higher levels of CD69 compared to normal mice (P NKT cell activation. Moreover, we found that the expression of CD4 was increased in infected MLN NK cells (P NKT cells of infected mice after phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation (P NKT cells might play roles in modulating the classical T cell response. Finally, our results indicated that the expression of CD94 was decreased in NK cells, suggesting that the downregulation of CD94 expression might served as a mechanism in NK cell activation.

  20. Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

    Science.gov (United States)

    Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

    2016-06-01

    Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

  1. Phorbol Esters Isolated from Jatropha Meal Induced Apoptosis-Mediated Inhibition in Proliferation of Chang and Vero Cell Lines

    Directory of Open Access Journals (Sweden)

    Syahida Ahmad

    2012-10-01

    Full Text Available The direct feeding of Jatropha meal containing phorbol esters (PEs indicated mild to severe toxicity symptoms in various organs of different animals. However, limited information is available on cellular and molecular mechanism of toxicity caused by PEs present in Jatropha meal. Thus, the present study was conducted to determine the cytotoxic and mode of action of PEs isolated from Jatropha meal using human hepatocyte (Chang and African green monkey kidney (Vero cell lines. The results showed that isolated PEs inhibited cell proliferation in a dose-dependent manner in both cell lines with the CC50 of 125.9 and 110.3 μg/mL, respectively. These values were compatible to that of phorbol 12-myristate 13-acetate (PMA values as positive control i.e., 124.5 and 106.3 μg/mL respectively. Microscopic examination, flow cytometry and DNA fragmentation results confirmed cell death due to apoptosis upon treatment with PEs and PMA at CC50 concentration for 24 h in both cell lines. The Western blot analysis revealed the overexpression of PKC-δ and activation of caspase-3 proteins which could be involved in the mechanism of action of PEs and PMA. Consequently, the PEs isolated form Jatropha meal caused toxicity and induced apoptosis-mediated proliferation inhibition toward Chang and Vero cell lines involving over-expression of PKC-δ and caspase-3 as their mode of actions.

  2. Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis.

    Science.gov (United States)

    Roum, J H; Borok, Z; McElvaney, N G; Grimes, G J; Bokser, A D; Buhl, R; Crystal, R G

    1999-07-01

    Cystic fibrosis (CF) is characterized by accumulation of activated neutrophils and macrophages on the respiratory epithelial surface (RES); these cells release toxic oxidants, which contribute to the marked epithelial derangements seen in CF. These deleterious consequences are magnified, since reduced glutathione (GSH), an antioxidant present in high concentrations in normal respiratory epithelial lining fluid (ELF), is deficient in CF ELF. To evaluate the feasibility of increasing ELF GSH levels and enhancing RES antioxidant protection, GSH aerosol was delivered (600 mg twice daily for 3 days) to seven patients with CF. ELF total, reduced, and oxidized GSH increased (P < 0.05, all compared with before GSH therapy), suggesting adequate RES delivery and utilization of GSH. Phorbol 12-myristate 13-acetate-stimulated superoxide anion (O2-.) release by ELF inflammatory cells decreased after GSH therapy (P < 0.002). This paralleled observations that GSH added in vitro to CF ELF inflammatory cells suppressed O2-. release (P < 0.001). No adverse effects were noted during treatment. Together, these observations demonstrate the feasibility of using GSH aerosol to restore RES oxidant-antioxidant balance in CF and support the rationale for further clinical evaluation.

  3. Inhibition of CD147 expression promotes chemosensitivity in HNSCC cells by deactivating MAPK/ERK signaling pathway.

    Science.gov (United States)

    Ma, Chao; Wang, Jianqi; Fan, Longkun; Guo, Yanjun

    2017-02-01

    Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world. CD147, a transmembrane glycoprotein, has been reported to be correlated with cancer progression, metastasis, and chemoresistance in various cancers. In this study, we aimed to investigate the mechanism of CD147 in regulating drug resistance in HNSCC cells. qRT-PCR were used to evaluated the expression of CD147 in 57 HNSCC tumorous tissues and 2 cell lines. Increased expression of CD147 was found in most HNSCC samples, and the expression level of CD147 was correlated with multidrug resistance. CD147 RNA silencing decreased the chemoresistance of HNSCC cells by deactivating MAPK/ERK signaling pathway. Further investigation revealed that either rescue expression of CD147 or treatment of MAPK/ERK activator phorbol 12-myristate 13-acetate (PMA) in CD147 knockdown CRC cell line attenuated the decreased chemoresistance in CD147 knockdown cells. Taken together, our results suggest that CD147 promotes chemoresistance by activating MAPK/ERK signaling pathway in HNSCC. Copyright © 2017. Published by Elsevier Inc.

  4. Comparison of the phosphorylation events in membranes prepared from proliferating versus quiescent endothelial cells

    International Nuclear Information System (INIS)

    Kazlauskas, A.; DiColeto, P.E.

    1986-01-01

    Little is known of the intracellular events which regulate the proliferation of endothelial cells (EC). Triton-solubilized membranes from proliferating (sparse) and quiescent (confluent) EC were incubated at pH 6.5 in the presence of divalent cations and [ 32 P]ATP. Membrane proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The overall kinase activity per mg protein was slightly greater in membranes prepared from proliferating versus quiescent cells. They found four proteins labeled in sparse cells to a dramatically greater extent having the following approximate molecular masses: 180, 100, 97 and 55 kilodalton (kd). The first two phosphoproteins were phosphorylated on serine residues exclusively; the 97 kd phosphoprotein contained 39% phosphoserine (p-ser) and 61% phosphothreonine (p-thr); and the 55 kd phosphoprotein contained 62% p-ser, 16% p-thr, and 22% phosphotyrosine (p-tyr). The kinases acting on all four phosphoproteins were independent of Ca 2+ , cAMP, cGMP, or phorbol 12-myristate 13-acetate. The observed differences in phosphorylation events between sparse and confluent membranes occurred in membranes from two EC lines - pig aortic and bovine aortic - but were not apparent in membranes prepared from human foreskin fibroblasts or 3T3 cells. Sparse endothelial cells made quiescent by serum deprivation were found to resemble confluent cells in the kinase activity; therefore, the enhanced kinase activity in sparse membranes may be growth dependent

  5. Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

    Science.gov (United States)

    Taupin, J L; Anderson, P

    1994-12-01

    The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

  6. Anti-Inflammatory Activity of Haskap Cultivars is Polyphenols-Dependent

    Directory of Open Access Journals (Sweden)

    H. P. Vasantha Rupasinghe

    2015-06-01

    Full Text Available Haskap (Lonicera caerulea L. berries have long been used for their health promoting properties against chronic conditions. The current study investigated the effect of Canadian haskap berry extracts on pro-inflammatory cytokines using a human monocytic cell line THP-1 derived macrophages stimulated by lipopolysaccharide. Methanol extracts of haskap from different growing locations in Canada were prepared and characterized for their total phenolic profile using colorimetric assays and liquid chromatography—Mass spectrometry (UPLC-MS/MS. Human THP-1 monocytes were seeded in 24-well plates (5 × 105/well and treated with phorbol 12-myristate 13-acetate (PMA, 0.1 μg/mL for 48 h to induce macrophage differentiation. After 48 h, the differentiated macrophages were washed with Hank’s buffer and treated with various concentrations of test compounds for 4 h, followed by the lipopolysaccharide (LPS-stimulation (18 h. Borealis cultivar showed the highest phenolic content, flavonoid content and anthocyanin content (p < 0.05. A negative correlation existed between the polyphenol concentration of the extracts and pro-inflammatory cytokines: Interleukin-6 (IL-6, tumour necrosis factor-alpha (TNF-α, prostaglandin (PGE2, and cyclooxygenase-2 (COX-2 enzyme. Borealis exhibited comparable anti-inflammatory effects to COX inhibitory drug, diclofenac. The results showed that haskap berry polyphenols has the potential to act as an effective inflammation inhibitor.

  7. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    International Nuclear Information System (INIS)

    Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

    1986-01-01

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [ 32 P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

  8. Fenspiride and membrane transduction signals in rat alveolar macrophages.

    Science.gov (United States)

    Féray, J C; Mohammadi, K; Taouil, K; Brunet, J; Garay, R P; Hannaert, P

    1997-07-15

    Fenspiride inhibits the calcium signal evoked by the inflammatory peptide formyl-Met-Leu-Phe (fMLP) in peritoneal macrophages, but at concentrations (approximately 1 mM) far above the therapeutic range (approximately 1 microM). Here, in rat alveolar macrophages, high fenspiride concentrations (1 mM) were required to inhibit the calcium signals evoked by the calcium agonist Bay K8644 or by ionomycin. Moreover, fenspiride (1 mM) was a poor inhibitor of the cell membrane depolarization induced by gramicidine D. By contrast, fenspiride blocked Na+-H+ antiport activation by (i) fMLP with an IC50 = 3.1 +/- 1.9 nM and (ii) PMA (phorbol 12-myristate 13-acetate) with an IC50 = 9.2 +/- 3.1 nM. Finally, protein kinase C (PKC) activity of macrophage homogenate was not significantly modified by 10 or 100 microM fenspiride (at 100 microM: 2.57 +/- 1.60 vs. 2.80 +/- 1.71 pmol/10(6) cells/min). In conclusion, fenspiride inhibits fMLP- and PMA-induced pH signals in rat alveolar macrophages, probably by acting distally on the PKC transduction signal. This pH antagonistic action may be relevant for the antiinflammatory mechanism of fenspiride and requires further investigation.

  9. Hydroxychloroquine decreases Th17-related cytokines in systemic lupus erythematosus and rheumatoid arthritis patients

    Directory of Open Access Journals (Sweden)

    Juliana Cruz da Silva

    2013-06-01

    Full Text Available OBJECTIVES: Hydroxychloroquine is an antimalarial agent that has been used in systemic lupus erythematosus and rheumatoid arthritis treatment for many years. Recently, novel mechanisms of action have been proposed, thereby broadening the therapeutic perspective of this medication. The purpose of this study was to evaluate the immunomodulatory activity of hydroxychloroquine in T helper 17 (Th17 cytokines in healthy individuals and patients. METHODS: Eighteen female patients with systemic lupus erythematosus (mean age 39.0±12.9 years and 13 female patients with rheumatoid arthritis (mean age 51.5±7.7 years were recruited from Universidade Federal de Pernambuco-Brazil. The patients were included after fulfilling four classification criteria for systemic lupus erythematosus or rheumatoid arthritis from the American College of Rheumatology. After being stimulated with phorbol 12-myristate 13-acetate and ionomycin in the absence or presence of different concentrations of hydroxychloroquine, the interleukin 6, 17 and 22 levels were quantified with an enzyme-linked immunosorbent assay in culture supernatants of peripheral blood mononuclear cells from healthy individuals and patients. RESULTS: We demonstrated that in peripheral blood mononuclear cells from healthy volunteers and in systemic lupus erythematosus and rheumatoid arthritis patients, there was a significant reduction in the IL-6, IL-17 and IL-22 supernatant levels after adding hydroxychloroquine. CONCLUSIONS Our in vitro results demonstrated that hydroxychloroquine inhibits IL-6, IL-17 and IL-22 production and contributes to a better understanding of the mechanism of action of this medication.

  10. Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

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    Alessia Arcaro

    2015-01-01

    Full Text Available Heat shock 60 kDa protein 1 (HSP60 is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM of 4-hydroxy-2-nonenal (HNE yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL or by copper-catalyzed oxidation (oxLDL, but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

  11. A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

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    Snehal Shabrish

    2016-01-01

    Full Text Available Natural killer (NK cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA and calcium ionophore (Ca2+-ionophore instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p<0.0001. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells.

  12. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    Science.gov (United States)

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  13. Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

    Science.gov (United States)

    Kim, Min Sun; Hwang, Yoon Jung; Yoon, Ki Joon; Zenke, Kosuke; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Ki Hong

    2009-11-01

    Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

  14. Anti-inflammatory properties of clovamide and Theobroma cacao phenolic extracts in human monocytes: evaluation of respiratory burst, cytokine release, NF-κB activation, and PPARγ modulation.

    Science.gov (United States)

    Zeng, Huawu; Locatelli, Monica; Bardelli, Claudio; Amoruso, Angela; Coisson, Jean Daniel; Travaglia, Fabiano; Arlorio, Marco; Brunelleschi, Sandra

    2011-05-25

    There is a great interest in the potential health benefits of biologically active phenolic compounds in cocoa (Theobroma cacao) and dark chocolate. We investigated the anti-inflammatory potential of clovamide (a N-phenylpropenoyl-L-amino acid amide present in cocoa beans) and two phenolic extracts from unroasted and roasted cocoa beans, by evaluating superoxide anion (O(2)(-)) production, cytokine release, and NF-κB activation in human monocytes stimulated by phorbol 12-myristate 13-acetate (PMA). The effects of rosmarinic acid are shown for comparison. Clovamide and rosmarinic acid inhibited PMA-induced O(2)(-) production and cytokine release (with a bell-shaped curve and maximal inhibition at 10-100 nM), as well as PMA-induced NF-κB activation; the two cocoa extracts were less effective. In all tests, clovamide was the most potent compound and also enhanced peroxisome proliferator-activated receptor-γ (PPARγ) activity, which may exert anti-inflammatory effects. These findings indicate clovamide as a possible bioactive compound with anti-inflammatory activity in human cells.

  15. Edible provenances of Jatropha curcas from Quintana Roo state of Mexico and effect of roasting on antinutrient and toxic factors in seeds.

    Science.gov (United States)

    Makkar, H P; Becker, K; Schmook, B

    1998-01-01

    Seven seed samples of J. curcas, both in raw and roasted state, sold in some villages in Quintana Roo state, Mexico for human consumption were analyzed for physical characteristics, nutrients and antinutrients. The average seed weight varied from 0.53 to 0.74 g and kernel weight as proportion of raw seed weight was from 61 to 66%. The contents of crude protein, lipid and ash of kernels from raw seeds were 27-30%, 55-62% and 3.7-5.2% respectively. The levels of antinutrients in meal from the raw seeds were: trypsin inhibitor activity (14.6-28.7 mg trypsin inhibited/g), lectin (25.6-52.2 unit; one unit is the reverse of minimum amount of mg meal/ml assay which produced haemagglutination), saponins (1.9-2.3% as diosgenin equivalent) and phytate (8.4-10%). Phorbol esters in kernels from raw seeds were not detected in four samples and in other three samples it ranged from 0.01 to 0.02 mg/g as phorbol-12-myristate 13-acetate equivalent. Roasting of seeds inactivated almost 100% of trypsin inhibitor activity. Although lectin activity reduced on roasting, it was still present in high amounts. Saponins, phytate and phorbol esters were not affected by roasting.

  16. Veronicastrum axillare Alleviates Ethanol-Induced Injury on Gastric Epithelial Cells via Downregulation of the NF-kB Signaling Pathway

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    Wei-chun Zhao

    2017-01-01

    Full Text Available We used human gastric epithelial cells (GES-1 line in an ethanol-induced cell damage model to study the protective effect of Veronicastrum axillare and its modulation to NF-κB signal pathway. The goal was to probe the molecular mechanism of V. axillare decoction in the prevention of gastric ulcer and therefore provide guidance in the clinical application of V. axillare on treating injuries from chronic nephritis, pleural effusion, gastric ulcer, and other ailments. The effects of V. axillare-loaded serums on cell viability were detected by MTT assays. Enzyme-linked immunosorbent assay (ELISA and Real-Time PCR methods were used to analyze the protein and mRNA expression of TNF-α, NF-κB, IκBα, and IKKβ. The results showed that V. axillare-loaded serum partially reversed the damaging effects of ethanol and NF-κB activator (phorbol-12-myristate-13-acetate: PMA and increased cell viability. The protein and mRNA expressions of TNF-α, NF-κB, IκBα, and IKKβ were significantly upregulated by ethanol and PMA while they were downregulated by V. axillare-loaded serum. In summary, V. axillare-loaded serum has significantly protective effect on GES-1 against ethanol-induced injury. The protective effect was likely linked to downregulation of TNF-α based NF-κB signal pathway.

  17. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    International Nuclear Information System (INIS)

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

  18. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

  19. Early age exposure to moisture damage and systemic inflammation at the age of 6 years.

    Science.gov (United States)

    Karvonen, A M; Tischer, C; Kirjavainen, P V; Roponen, M; Hyvärinen, A; Illi, S; Mustonen, K; Pfefferle, P I; Renz, H; Remes, S; Schaub, B; von Mutius, E; Pekkanen, J

    2018-05-01

    Cross-sectional studies have shown that exposure to indoor moisture damage and mold may be associated with subclinical inflammation. Our aim was to determine whether early age exposure to moisture damage or mold is prospectively associated with subclinical systemic inflammation or with immune responsiveness in later childhood. Home inspections were performed in children's homes in the first year of life. At age 6 years, subclinical systemic inflammation was measured by serum C-reactive protein (CRP) and blood leukocytes and immune responsiveness by ex vivo production of interleukin 1-beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) in whole blood cultures without stimulation or after 24 hours stimulation with phorbol 12-myristate 13-acetate and ionomycin (PI), lipopolysaccharide (LPS), or peptidoglycan (PPG) in 251-270 children. Moisture damage in child's main living areas in infancy was not significantly associated with elevated levels of CRP or leukocytes at 6 years. In contrast, there was some suggestion for an effect on immune responsiveness, as moisture damage with visible mold was positively associated with LPS-stimulated production of TNF-α and minor moisture damage was inversely associated with PI-stimulated IL-1β. While early life exposure to mold damage may have some influence on later immune responsiveness, it does not seem to increase subclinical systemic inflammation in later life. © 2018 National Institute for Health and Welfare, Finland Indoor Air published by John Wiley & Sons Ltd.

  20. Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

    Science.gov (United States)

    Żmigrodzka, M; Guzera, M; Winnicka, A

    2016-01-01

    Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

  1. Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

    Science.gov (United States)

    Seghatoleslam, Atefeh; Hemmati, Mina; Ebadat, Saeedeh; Movahedi, Bahram; Mostafavi-Pour, Zohreh

    2016-01-01

    Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at Pproteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

  2. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein......+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 n......M) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production...

  3. Inhibition of neutrophil elastase and metalloprotease-9 of human adenocarcinoma gastric cells by chamomile (Matricaria recutita L.) infusion.

    Science.gov (United States)

    Bulgari, Michela; Sangiovanni, Enrico; Colombo, Elisa; Maschi, Omar; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

    2012-12-01

    This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. Copyright © 2012 John Wiley & Sons, Ltd.

  4. PMA Induces Vaccine Adjuvant Activity by the Modulation of TLR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Dool-Ri Oh

    2014-01-01

    Full Text Available Toll-like receptor (TLR ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses. Flagellin, a TLR5 ligand, was reported to show potent mucosal vaccine adjuvant activity. To identify ligands that potentiate the adjuvant activity of flagellin, we screened a plant library using HEK293T cells transiently cotransfected with phTLR5 and pNF-κB-SEAP plasmids. The 90% EtOH extract from Croton tiglium showed significant NF-κB transactivation in a TLR5-independent manner along with the increase of a flagellin activity. We have studied to characterize an active component from Croton tiglium and to elucidate the action mechanisms. Phorbol 12-myristate 13-acetate (PMA was isolated as an active component of Croton tiglium by activity-guided fractionation, column chromatography, HPLC, NMR, and MS. PMA at a range of nM induced PKC-dependent NF-κB activation and IL-8 production in both TLR5− and TLR5+ assay systems. In in vivo mouse vaccination model, PMA induced antigen-specific IgG and IgA antibody responses and increased IL-12 production corresponding to T cell responses in spleen lymphocytes. These results suggest that PMA would serve as an efficacious mucosal vaccine adjuvant.

  5. Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells.

    Science.gov (United States)

    Pan, Yao; Wei, Xuetao; Hao, Weidong

    2015-08-28

    Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

  6. Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells

    Directory of Open Access Journals (Sweden)

    Yao Pan

    2015-08-01

    Full Text Available Trichloroethylene (TCE is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA and dichloroacetic acid (DCA, on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

  7. Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene

    International Nuclear Information System (INIS)

    Shannon, M.F.; Gamble, J.R.; Vadas, M.A.

    1988-01-01

    The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. The authors show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5', GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. They hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription

  8. Inhibition of protein kinase C induces differentiation in Neuro-2a cells

    International Nuclear Information System (INIS)

    Minana, M.D.; Felipo, V.; Grisolia, S.

    1990-01-01

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 μM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 μM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased ∼7-fold after 48 hr with 500 μM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed

  9. Gamma irradiation enhances biological activities of mulberry leaf extract

    International Nuclear Information System (INIS)

    Cho, Byoung-Ok; Che, Denis Nchang; Yin, Hong-Hua; Jang, Seon-Il

    2017-01-01

    The purpose of this study was to investigate the influence of irradiation on the anti-oxidative, anti-inflammatory and whitening effects of mulberry leaf extract. This was done by comparing the phenolic contents; 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effects; 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) radical scavenging effects; in vitro tyrosinase inhibitory effects and the production of IL-6, TNF-α, PGE 2 , and NO in lipopolysaccharide-stimulated RAW264.7 macrophages and the production of IL-6 and TNF-α in phorbol 12-myristate 13-acetate plus calcium ionophore A23187-stimulated HMC-1 cells, respectively. The results showed that irradiated mulberry leaf extract possesses more anti-oxidant, anti-inflammatory, and tyrosinase inhibitory activities than their non-irradiated counterpart, probably due to increase in phenolic contents induced by gamma irradiation at dose of 10kGy. This research stresses on the importance of irradiation in functional foods. - Highlights: • Gamma-irradiated mulberry leaf extract enhanced in vitro antioxidant activities. • Gamma-irradiated mulberry leaf extract enhanced in vitro tyrosinase inhibitory effects. • Gamma-irradiated mulberry leaf extract treatment reduced the production of IL-6, TNF-α, PGE 2 , and NO.

  10. The selective estrogen receptor modulator raloxifene inhibits neutrophil extracellular trap formation.

    Directory of Open Access Journals (Sweden)

    Roxana Flores

    2016-12-01

    Full Text Available Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effect on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA, a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs. Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERβ, known targets of raloxifene. Like raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation but not reactive oxygen species (ROS production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production.

  11. ERK-dependent and -independent pathways trigger human neural progenitor cell migration

    International Nuclear Information System (INIS)

    Moors, Michaela; Cline, Jason E.; Abel, Josef; Fritsche, Ellen

    2007-01-01

    Besides differentiation and apoptosis, cell migration is a basic process in brain development in which neural cells migrate several centimeters within the developing brain before reaching their proper positions and forming the right connections. For identifying signaling events that control neural migration and are therefore potential targets of chemicals to disturb normal brain development, we developed a human neurosphere-based migration assay based on normal human neural progenitor (NHNP) cells, in which the distance is measured that cells wander over time. Applying this assay, we investigated the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of NHNP cell migration. Exposure to model substances like ethanol or phorbol 12-myristate 13-acetate (PMA) revealed a correlation between ERK1/2 activation and cell migration. The participation of phospho-(P-) ERK1/2 was confirmed by exposure of the cells to the MEK inhibitor PD98059, which directly prohibits ERK1/2 phosphorylation and inhibited cell migration. We identified protein kinase C (PKC) and epidermal growth factor receptor (EGFR) as upstream signaling kinases governing ERK1/2 activation, thereby controlling NHNP cell migration. Additionally, treatments with src kinase inhibitors led to a diminished cell migration without affecting ERK1/2 phosphorylation. Based on these results, we postulate that migration of NHNP cells is controlled via ERK1/2-dependent and -independent pathways

  12. Thioredoxin ameliorates cutaneous inflammation by regulating the epithelial production and release of pro-Inflammatory cytokines

    Directory of Open Access Journals (Sweden)

    Hai eTian

    2013-09-01

    Full Text Available Human thioredoxin-1 (TRX is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys-. It has been demonstrated that systemic administration and transgenic overexpression of TRX ameliorate inflammation in various animal models, but its anti-inflammatory mechanism is not well characterized. We investigated the anti-inflammatory effects of topically applied recombinant human TRX (rhTRX in a murine irritant contact dermatitis (ICD induced by croton oil. Topically applied rhTRX was distributed only in the skin tissues under both non-inflammatory and inflammatory conditions, and significantly suppressed the inflammatory response by inhibiting the production of cytokines and chemokines, such as TNF-α, Il-1β, IL-6, CXCL-1, and MCP-1. In an in vitro study, rhTRX also significantly inhibited the formation of cytokines and chemokines produced by keratinocytes after exposure to croton oil and phorbol 12-myristate 13-acetate. These results indicate that TRX prevents skin inflammation via the inhibition of local formation of inflammatory cytokines and chemokines. As a promising new approach, local application of TRX may be useful for the treatment of various skin and mucosal inflammatory disorders.

  13. Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

    International Nuclear Information System (INIS)

    Ransjoe, M.; Lerner, U.H.

    1990-01-01

    Calcitonin is a well known inhibitor of osteoclastic bone resortion, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called ''escape from inhibition'' phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45 Ca. Two proteon kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45 Ca release in 120-h cultures at a concentration of 10 nmul/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any ''escape from inhibition''. This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E 2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption. (author)

  14. Detoxification of toxic phorbol esters from Malaysian Jatropha curcas Linn. kernel by Trichoderma spp. and endophytic fungi.

    Science.gov (United States)

    Najjar, Azhar; Abdullah, Norhani; Saad, Wan Zuhainis; Ahmad, Syahida; Oskoueian, Ehsan; Abas, Faridah; Gherbawy, Youssuf

    2014-02-05

    The presence of phorbol esters (PEs) with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum JQ350879.1, T. harzianum JQ517493.1, Paecilomyces sinensis JQ350881.1, Cladosporium cladosporioides JQ517491.1, Fusarium chlamydosporum JQ350882.1, F. chlamydosporum JQ517492.1 and F. chlamydosporum JQ350880.1) was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS) and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%-96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs.

  15. EBV DNA polymerase inhibition of tannins from Eugenia uniflora.

    Science.gov (United States)

    Lee, M H; Chiou, J F; Yen, K Y; Yang, L L

    2000-06-30

    Nasopharyngeal carcinoma (NPC) is one of the high population malignant tumors among Chinese in southern China and southeast Asia. Epstein-Barr virus (EBV) is a human B lymphotropic herpes virus which is known to be closely associated with NPC. EBV DNA polymerase is a key enzyme during EBV replication and is measured by its radioactivity. The addition of phorbol 12-myristate 13-acetate to Raji cell cultures led to a large increase in EBV DNA polymerase, which was purified by sequential DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. Four tannins were isolated from the active fractions of Eugenia uniflora L., which were tested for the inhibition of EBV DNA polymerase. The results showed the 50% inhibitory concentration (IC(50)) values of gallocatechin, oenothein B, eugeniflorins D(1) and D(2) were 26.5 62.3, 3.0 and 3.5 microM, respectively. Furthermore, when compared with the positive control (phosphonoacetic acid), an inhibitor of EBV replication, the IC(50) value was 16.4 microM. In view of the results, eugeniflorins D(1) and D(2) are the potency principles in the inhibition of EBV DNA polymerase from E. uniflora.

  16. Dimethyl sulfoxide-inducible cytoplasmic factor involved in erythroid differentiation in mouse erythroleukemia (Friend) cells

    International Nuclear Information System (INIS)

    Watanabe, T.; Oishi, M.

    1987-01-01

    A previous report described an intracellular factor (differentiation-inducing factor I, or DIF-I) that seem to play a role in erythroid differentiation in mouse erythroleukemia (MEL) cells. The authors have detected another erythroid-inducing factor in cell-free extracts from dimethyl sulfoxide- or hexamethylenebis(acetamide)-treated MEL cells, which acts synergistically with DIF-I. The partially purified factor (termed DIF-II) triggered erythroid differentiation when introduced into undifferentiated MEL cells that had been potentiated by the induction of DIF-I. The activity in the extracts appeared in an inducible manner after addition of dimethyl sulfoxide or hexamethylenebis(acetamide), reached a maximum at 6 hr, and then rapidly decreased. The induction was inhibited by phorbol 12-myristate 13-acetate and also by cycloheximide. No induction was observed in a mutant MEL cell line defective in erythroid differentiation. These characteristics are consistent with the supposition that DIF-II is one of the putative dimethyl sulfoxide-inducible factors detected in previously reported cell-fusion and cytoplast-fusion experiments. The role of DIF-II in MEL-cell differentiation and in vitro differentiation in general is discussed

  17. Proteomic Profiling of a Primary CD4+ T Cell Model of HIV-1 Latency Identifies Proteins Whose Differential Expression Correlates with Reactivation of Latent HIV-1.

    Science.gov (United States)

    Saha, Jamaluddin Md; Liu, Hongbing; Hu, Pei-Wen; Nikolai, Bryan C; Wu, Hulin; Miao, Hongyu; Rice, Andrew P

    2018-01-01

    The latent HIV-1 reservoir of memory CD4 + T cells that persists during combination antiviral therapy prevents a cure of infection. Insight into mechanisms of latency and viral reactivation are essential for the rational design of strategies to reduce the latent reservoir. In this study, we quantified the levels of >2,600 proteins in the CCL19 primary CD4 + T cell model of HIV-1 latency. We profiled proteins under conditions that promote latent infection and after cells were treated with phorbol 12-myristate 13-acetate (PMA) + ionomycin, which is known to efficiently induce reactivation of latent HIV-1. In an analysis of cells from two healthy blood donors, we identified 61 proteins that were upregulated ≥2-fold, and 36 proteins that were downregulated ≥2-fold under conditions in which latent viruses were reactivated. These differentially expressed proteins are, therefore, candidates for cellular factors that regulate latency or viral reactivation. Two unexpected findings were obtained from the proteomic data: (1) the interactions among the majority of upregulated proteins are largely undetermined in published protein-protein interaction networks and (2) downregulated proteins are strongly associated with Gene Ontology terms related to mitochondrial protein synthesis. This proteomic data set provides a useful resource for future mechanistic studies of HIV-1 latency.

  18. The potential protective role of taurine against experimental allergic inflammation.

    Science.gov (United States)

    Nam, Sun-Young; Kim, Hyung-Min; Jeong, Hyun-Ja

    2017-09-01

    Taurine has been widely evaluated as a potential therapeutic agent in chronic inflammatory disorders and various infections. However, the potential role of taurine in regulating allergic inflammatory responses is currently unknown. The present study was designed to evaluate the in vitro effects of taurine on the levels of thymic stromal lymphopoietin (TSLP) and other pro-inflammatory cytokines and activation of caspase-1 and nuclear factor (NF)-κB as well as the phosphorylations of c-Jun N-terminal kinase (JNK) and p38 in phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-triggered human mast cell line, HMC-1 cells. Furthermore, we assessed the therapeutic effects of taurine on ovalbumin (OVA)-induced allergic rhinitis (AR) animal models. Here, the obtained results showed that taurine dose-dependently inhibited the production and mRNA expression of TSLP and pro-inflammatory cytokines in HMC-1 cells exposed to PMACI. Taurine attenuated the phosphorylation of JNK and p38 in activated HMC-1 cells. Moreover, taurine brought a significant inhibition of the activities of NF-κB and caspase-1. In an OVA-induced AR animal model, the increased levels of nose rubbing, histamine, immunoglobulin E, TSLP, and interleukin IL-1β were dramatically reduced by the administration of taurine. In summary, taurine could serve as potential novel remedy of allergic inflammatory disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Relation between radio-adaptive response and cell to cell communication

    International Nuclear Information System (INIS)

    Keiichiro Ishii

    1996-01-01

    Ionizing radiation has been considered to cause severe damages to DNA and do harm to cells in proportion to the dose, however low it might be. In 1984, Wolff et al. showed that human peripheral lymphocytes adapted to the low-dose radiation from 3 H-TdR added in culture medium and became resistant to the subsequent irradiation with high-doses of X-rays. This response, which is called radio-adaptive response, is also induced by X-rays and gamma-rays in human lymphocytes and Chinese hamster V79 cells. However, the mechanisms of and conditions for adaptive responses to radiation have not been clarified. With an objective of clarifying the conditions for adaptive responses of cells to radiation, we examined how the cell to cell communication is involved in the adaptive responses. We irradiated normal human embryo-derived (HE) cells and cancer cells (HeLa) in culture at high density with low-dose X-ray and examined their radio-adaptive responses by measuring the changes in sensitivity to subsequent high-dose X-ray irradiation using the Trypan Blue dye-exclusion test method. We also conducted experiments to examine the effects of Ca 2+ ions and Phorbol 12-Myristate 13-Acetate (TPA) which are supposed to be involved in cell to cell communication. (author)

  20. Fisetin inhibits IL-31 production in stimulated human mast cells: Possibilities of fisetin being exploited to treat histamine-independent pruritus.

    Science.gov (United States)

    Che, Denis Nchang; Cho, Byoung Ok; Shin, Jae Young; Kang, Hyun Ju; Kim, Young-Soo; Jang, Seon Il

    2018-05-15

    Interleukin-31 (IL-31) is a recently discovered cytokine that is tightly linked to the pathogenesis of pruritus seen in atopic dermatitis. Flavonoids, like fisetin, are naturally occurring molecules with antioxidant, cytoprotective, and anti-inflammatory actions. the present study sought to investigate whether fisetin modulates IL-31 and histamine release in human mast cells (HMC-1). HMC-1 cells were pretreated with fisetin at various doses and stimulated with phorbol-12-myristate 13-acetate and calcium ionophore A23187 (PI) for different time intervals. We evaluated IL-31 production and histamine release and signaling mechanism of the action of fisetin on IL-31 production. We also investigated the effects of fisetin on scratching behaviors in mice. Fisetin decreased PI-stimulated mRNA expression and production of IL-31 in HMC-1 cells. Fisetin inhibited PI-induced phosphorylation of mitogen-activated protein kinases that further suppressed nuclear factor (NF-κB) activation and translocation to the nucleus through the inhibition of IκB-α phosphorylation. Fisetin also prevented mast cell release of histamine in HMC-1 cells. Mice in-vivo studies show that fisetin reduced scratching behaviors in mice. These pharmacological actions of fisetin provide new suggestions that fisetin can be of potential use for the treatment of pruritus that cannot be treated with histamine receptor blockers alone. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Flavonoids inhibit histamine release and expression of proinflammatory cytokines in mast cells.

    Science.gov (United States)

    Park, Hyo-Hyun; Lee, Soyoung; Son, Hee-Young; Park, Seung-Bin; Kim, Mi-Sun; Choi, Eun-Ju; Singh, Thoudam S K; Ha, Jeoung-Hee; Lee, Maan-Gee; Kim, Jung-Eun; Hyun, Myung Chul; Kwon, Taeg Kyu; Kim, Yeo Hyang; Kim, Sang-Hyun

    2008-10-01

    Mast cells participate in allergy and inflammation by secreting inflammatory mediators such as histamine and proinflammatory cytokines. Flavonoids are naturally occurring molecules with antioxidant, cytoprotective, and antiinflammatory actions. However, effect of flavonoids on the release of histamine and proinflammatory mediator, and their comparative mechanism of action in mast cells were not well defined. Here, we compared the effect of six flavonoids (astragalin, fisetin, kaempferol, myricetin, quercetin, and rutin) on the mast cell-mediated allergic inflammation. Fisetin, kaempferol, myricetin, quercetin, and rutin inhibited IgE or phorbol-12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-mediated histamine release in RBL-2H3 cells. These five flavonoids also inhibited elevation of intracellular calcium. Gene expressions and secretion of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, and IL-8 were assessed in PMACI-stimulated human mast cells (HMC-1). Fisetin, quercetin, and rutin decreased gene expression and production of all the proinflammatory cytokines after PMACI stimulation. Myricetin attenuated TNF-alpha and IL-6 but not IL-1beta and IL-8. Fisetin, myricetin, and rutin suppressed activation of NF-kappaB indicated by inhibition of nuclear translocation of NF-kappaB, NF-kappaB/DNA binding, and NF-kappaB-dependent gene reporter assay. The pharmacological actions of these flavonoids suggest their potential activity for treatment of allergic inflammatory diseases through the down-regulation of mast cell activation.

  2. Effects of Morus alba L. and Natural Products Including Morusin on In Vivo Secretion and In Vitro Production of Airway MUC5AC Mucin

    Science.gov (United States)

    Lee, Hyun Jae; Ryu, Jiho; Park, Su Hyun; Woo, Eun-Rhan; Kim, A Ryun; Lee, Sang Kook; Kim, Yeong Shik; Kim, Ju-Ock; Hong, Jang-Hee

    2014-01-01

    Background It is valuable to find the potential activity of regulating the excessive mucin secretion by the compounds derived from various medicinal plants. We investigated whether aqueous extract of the root bark of Morus alba L. (AMA), kuwanon E, kuwanon G, mulberrofuran G, and morusin significantly affect the secretion and production of airway mucin using in vivo and in vitro experimental models. Methods Effect of AMA was examined on hypersecretion of airway mucin in sulfur dioxide-induced acute bronchitis in rats. Confluent NCI-H292 cells were pretreated with ethanolic extract, kuwanon E, kuwanon G, mulberrofuran G, or morusin for 30 minutes and then stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 hours. The MUC5AC mucin secretion and production were measured by enzyme-linked immunosorbent assay. Results AMA stimulated the secretion of airway mucin in sulfur dioxide-induced bronchitis rat model; aqueous extract, ethanolic extract, kuwanon E, kuwanon G, mulberrofuran G and morusin inhibited the production of MUC5AC mucin induced by PMA from NCI-H292 cells, respectively. Conclusion These results suggest that extract of the root bark and the natural products derived from Morus alba L. can regulate the secretion and production of airway mucin and, at least in part, explains the folk use of extract of Morus alba L. as mucoregulators in diverse inflammatory pulmonary diseases. PMID:25237377

  3. Homeopathic potencies of Arnica montana L. change gene expression in a Tamm-Horsfall protein-1 cell line in vitro model: the role of ethanol as a possible confounder and statistical bias.

    Science.gov (United States)

    Chirumbolo, Salvatore; Bjørklund, Geir

    2017-07-01

    Marzotto et al. showed that homeopathic preparations of Arnica montana L. acted directly on gene expression of Tamm-Horsfall protein-1 (THP-1) monocyte/macrophage cell lines activated with phorbol12-myristate13-acetate and interleukin-4 (IL-4). A. montana homeopathic dilutions are used in complementary and alternative medicine to treat inflammation disorders and post-traumatic events as well as for wound repair. The French Pharmacopoeia of these remedies uses 0.3% ethanol in each centesimal dilution. In this paper, we discuss how ethanol-containing A. montana homeopathic centesimal dilutions can change gene expression in IL-4-treated monocyte/macrophage THP-1. We assessed the role of ethanol in the Arnica homeopathic dilutions containing this alcohol by investigating its action on gene expression of THP-1 cell. Evidence would strongly suggest that the presence of ethanol in these remedies might play a fundamental role in the dilutions ability to affect gene expression, particularly for doses from 5c to 15c. Where, rather than playing a major role in the mesoscopic structure of water, the ethanol might have a chemical-physical role in the induction of THP-1 gene expression, apoptosis, and deoxyribonucleic acid function. This evidence generates a debate about the suggestion that the use of a binary-mixed solvent in homeopathic chemistry, used by Hahnemann since 1810, may be fundamental to explain the activity of homeopathy on cell models.

  4. Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells

    International Nuclear Information System (INIS)

    Kasai, K.; Hiraiwa, M.; Emoto, T.; Akimoto, K.; Takaoka, T.; Shimoda, S.I.

    1987-01-01

    Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E 2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E 2 production by the cells in dose related fashion. PMA stimulated prostaglandin E 2 production over fifty-fold with the dose of 10 -7 M compared with control. EGF (10 -7 M) also stimulated it about ten-fold. The ED 50 values of PMA and EGF were respectively around 1 x 10 -9 M and 5 x 10 -10 M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E 2 production from 1 to 24-h incubation. The release of radioactivity from [ 3 H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E 2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells. 36 references, 2 figures, 1 table

  5. Inhibitory effects of kaempferol on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9.

    Science.gov (United States)

    Li, Chenglin; Zhao, Yuanwei; Yang, Dan; Yu, Yanyan; Guo, Hao; Zhao, Ziming; Zhang, Bei; Yin, Xiaoxing

    2015-02-01

    Matrix metalloproteinases (MMPs) have been regarded as major critical molecules assisting tumor cells during metastasis, for excessive ECM (ECM) degradation, and cancer cell invasion. In the present study, in vitro and in vivo assays were employed to examine the inhibitory effects of kaempferol, a natural polyphenol of flavonoid family, on tumor metastasis. Data showed that kaempferol could inhibit adhesion, migration, and invasion of MDA-MB-231 human breast carcinoma cells. Moreover, kaempferol led to the reduced activity and expression of MMP-2 and MMP-9, which were detected by gelatin zymography, real-time PCR, and western blot analysis, respectively. Further elucidation of the mechanism revealed that kaempferol treatment inhibited the activation of transcription factor activator protein-1 (AP-1) and MAPK signaling pathway. Moreover, kaempferol repressed phorbol-12-myristate-13-acetate (PMA)-induced MMP-9 expression and activity through suppressing the translocation of protein kinase Cδ (PKCδ) and MAPK signaling pathway. Our results also indicated that kaempferol could block the lung metastasis of B16F10 murine melanoma cells as well as the expression of MMP-9 in vivo. Taken together, these results demonstrated that kaempferol could inhibit cancer cell invasion through blocking the PKCδ/MAPK/AP-1 cascade and subsequent MMP-9 expression and its activity. Therefore, kaempferol might act as a therapeutic potential candidate for cancer metastasis.

  6. The anti-invasive effect of lucidenic acids isolated from a new Ganoderma lucidum strain.

    Science.gov (United States)

    Weng, Chia-Jui; Chau, Chi-Fai; Chen, Kuang-Dee; Chen, Deng-Hai; Yen, Gow-Chin

    2007-12-01

    Ganoderma lucidum is a well-known mushroom with various pharmacological effects that has been used for health and longevity purposes. The objective of this study was to investigate the anti-invasive effect of lucidenic acids isolated from a new G. lucidum strain (YK-02) against human hepatoma carcinoma (HepG(2)) cells. Triterpenoid components in the ethanol extract of G. lucidum (YK-02) were separated by means of a semi-preparative RP HPLC. Four major peaks were separated and crystallized from triterpenoids fraction, and were identified as lucidenic acids A, B, C, and N according to their spectroscopic values of (1)H NMR and MS. Treatment of the lucidenic acids (50 microM) in the presence of 200 nM phorbol 12-myristate 13-acetate (PMA) after 24 h of incubation all resulted in significant inhibitory effects on PMA-induced MMP-9 activity and invasion of HepG(2 )cells. The results indicate that the lucidenic acids isolated from G. lucidum (YK-02) are anti-invasive bioactive components on hepatoma cells.

  7. Mechanism of Mitochondrial Connexin43′s Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Shuai Hou

    2016-05-01

    Full Text Available We observed mitochondrial connexin43 (mtCx43 expression under cerebral ischemia-reperfusion (I/R injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO. Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD activity and malondialdehyde (MDA content. MtCx43, p-mtCx43, protein kinase C (PKC, and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX and diazoxide (DZX groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists.

  8. Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

    Science.gov (United States)

    Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

    2012-07-01

    Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

  9. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    International Nuclear Information System (INIS)

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-01-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of ≅ 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 0 C or in medium containing 5 μM colchicine or nocodazole at 37 0 C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 0 C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures

  10. Genetic variants of NOXA and MCL1 modify the risk of HPV16-associated squamous cell carcinoma of the head and neck

    International Nuclear Information System (INIS)

    Zhou, Ziyuan; Sturgis, Erich M; Liu, Zhensheng; Wang, Li-E; Wei, Qingyi; Li, Guojun

    2012-01-01

    The cooperation between phorbol 12-myristate 13-acetate induced protein 1 (NOXA) and myeloid cell leukemia 1 (MCL1) is critical in the intrinsic apoptotic pathway. Human papillomavirus 16 (HPV16), by inducing p53 and pRb-E2F degradation, may play an essential role in development of squamous cell carcinoma of the head and neck (SCCHN) through NOXA-MCL1 axis-mediated apoptosis. Therefore, genetic variants of NOXA and MCL1 may modify the SCCHN risk associated with HPV16 seropositivity. HPV16 serology was obtained by immunoadsorption assay. Four functional SNPs in the promoter of NOXA (rs9957673, rs4558496) and MCL1 (rs9803935, rs3738485) were genotyped for 380 cases and 335 frequency-matched cancer-free controls of non-Hispanic whites. Associations between the four polymorphisms and SCCHN risk were not significant, while we observed a significantly joint effect on SCCHN risk between the polymorphisms and HPV16 seropositivity. Notably, this effect modification was particularly pronounced for oropharyngeal cancer in subgroups including never smokers, never drinkers and younger subjects. Our results suggested that polymorphisms of NOXA and MCL1 may modify the risk of HPV16-associated oropharyngeal cancer. The further identification of population subgroups at higher risk provides evidence that HPV-targeting treatment may help benefit SCCHN. However, larger studies are needed to validate our findings

  11. A natural xanthone increases catalase activity but decreases NF-kappa B and lipid peroxidation in U-937 and HepG2 cell lines.

    Science.gov (United States)

    Sahoo, Binay K; Zaidi, Adeel H; Gupta, Pankaj; Mokhamatam, Raveendra B; Raviprakash, Nune; Mahali, Sidhartha K; Manna, Sunil K

    2015-10-05

    Mangiferin, a C-glycosyl xanthone, has shown anti-inflammatory, antioxidant, and anti-tumorigenic activities. In the present study, we investigated the molecular mechanism for the antioxidant property of mangiferin. Considering the role of nuclear transcription factor kappa B (NF-κB) in inflammation and tumorigenesis, we hypothesized that modulating its activity will be a viable therapeutic target in regulating the redox-sensitive ailments. Our results show that mangiferin blocks several inducers, such as tumor necrosis factor (TNF), lypopolysaccharide (LPS), phorbol-12-myristate-13-acetate (PMA) or hydrogen peroxide (H2O2) mediated NF-κB activation via inhibition of reactive oxygen species generation. In silico docking studies predicted strong binding energy of mangiferin to the active site of catalase (-9.13 kcal/mol), but not with other oxidases such as myeloperoxidase, glutathione peroxidase, or inducible nitric oxide synthase. Mangiferin increased activity of catalase by 44%, but had no effect on myeloperoxidase activity in vitro. Fluorescence spectroscopy further revealed the binding of mangiferin to catalase at the single site with binding constant and binding affinity of 3.1×10(-7) M(-1) and 1.046 respectively. Mangiferin also inhibits TNF-induced lipid peroxidation and thereby protects apoptosis. Hence, mangiferin with its ability to inhibit NF-κB and increase the catalase activity may prove to be a potent therapeutic. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Dual Stimulus-Dependent Effect of Oenothera paradoxa Extract on the Respiratory Burst in Human Leukocytes: Suppressing for Escherichia coli and Phorbol Myristate Acetate and Stimulating for Formyl-Methionyl-Leucyl-Phenylalanine

    Directory of Open Access Journals (Sweden)

    Izabela Burzynska-Pedziwiatr

    2014-01-01

    Full Text Available Although a growing body of evidence suggests that plant polyphenols can modulate human immune responses, their simultaneous action on monocyte and neutrophil oxidative burst is currently poorly understood. Based on the hypothesis that various polyphenols contained in plant extracts might affect the oxidative burst of phagocytes, we evaluated the effects of ethanolic O. paradoxa extract polyphenols on monocyte and neutrophil oxidative burst in vitro activated by different stimuli, including opsonized bacteria E. coli, phorbol 12-myristate 13-acetate (PMA, and formyl-methionyl-leucyl-phenylalanine (fMLP. Samples were analyzed by the dihydrorhodamine flow cytometry assay. Our results showed that the extract repressed significantly and dose-dependently reactive oxygen species production in both cell types stimulated with E. coli and PMA (P < 0.05 and its inhibitory efficiency was stimulus- and cell-type-dependent. Interestingly, there was significant stimulatory effect of the extract on bursting phagocytes induced by fMLP (P < 0.05. Additionally, several flavonoids and phenolic compounds as well as penta-galloyl-β-(D-glucose (PGG, the representative of hydrolyzable tannins, were identified in the 60% extract by high-performance liquid chromatography (HPLC coupled to electrospray ionization in negative ion mode. In summary, the ethanolic O. paradoxa extract, rich in flavonoids and phenolic compounds, exhibits dual stimulus-dependent effect on the respiratory burst in human leukocytes; hence, it might affect immune responses in humans.

  13. The Ameliorative Effect of Sophoricoside on Mast Cell-Mediated Allergic Inflammation in Vivo and in Vitro

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    Jae-Young Um

    2013-05-01

    Full Text Available Sophoricoside exhibits numerous pharmacological effects, including anti- inflammatory and anti-cancer actions, yet the exact mechanism that accounts for the anti-allergic effects of sophoricoside is not completely understood. The aim of the present study was to elucidate whether and how sophoricoside modulates the mast cell-mediated allergic inflammation in vitro and in vivo. We investigated the pharmacological effects of sophoricoside on both compound 48/80 or histamine-induced scratching behaviors and 2,4-dinitrochlorobenzene (DNCB-induced atopic dermatitis in mice. Additionally, to find a possible explanation for the anti-inflammatory effects of sophoricoside, we evaluated the effects of sophoricoside on the production of histamine and inflammatory cytokines and activation of nuclear factor-κB (NF-κB and caspase-1 in phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI-stimulated human mast cells (HMC-1. The finding of this study demonstrated that sophoricoside reduced compound 48/80 or histamine-induced scratching behaviors and DNCB-induced atopic dermatitis in mice. Additionally, sophoricoside inhibited the production of inflammatory cytokines as well as the activation of NF-κB and caspase-1 in stimulated HMC-1. Collectively, the findings of this study provide us with novel insights into the pharmacological actions of sophoricoside as a potential molecule for use in the treatment of allergic inflammation diseases.

  14. Parathyroid hormone blocks the stimulatory effect of insulin-like growth factor-I on collagen synthesis in cultured 21-day fetal rat calvariae

    International Nuclear Information System (INIS)

    Kream, B.E.; Petersen, D.N.; Raisz, L.G.

    1990-01-01

    We examined the interaction of parathyroid hormone (PTH) and recombinant human insulin-like growth factor I (IGF-I) on collagen synthesis in 21-day fetal rat calvariae as assessed by measuring the incorporation of [ 3 H]proline into collagenase-digestible protein. After 96 hours of culture, 10 nM PTH antagonized the stimulation of collagen synthesis and partially blocked the increase in dry weight produced by 10 nM IGF-I. The effect of PTH to block IGF-I stimulated collagen synthesis was observed in the central bone of calvariae and was mimicked by forskolin and phorbol 12-myristate 13-acetate, but not by 1,25-dihydroxyvitamin D3, transforming growth factor-alpha or dexamethasone. Our data are consistent with the concept that the direct effect of PTH is to inhibit basal CDP labeling and fully oppose IGF-I stimulated CDP labeling. The finding that this effect of PTH is mimicked by forskolin and PMA suggests that this block in IGF-I stimulation of CDP labeling involves both cAMP and protein kinase C mediated pathways

  15. Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

    Science.gov (United States)

    Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

    2018-05-01

    Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Topical Apigenin Alleviates Cutaneous Inflammation in Murine Models

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    Mao-Qiang Man

    2012-01-01

    Full Text Available Herbal medicines have been used in preventing and treating skin disorders for centuries. It has been demonstrated that systemic administration of chrysanthemum extract exhibits anti-inflammatory properties. However, whether topical applications of apigenin, a constituent of chrysanthemum extract, influence cutaneous inflammation is still unclear. In the present study, we first tested whether topical applications of apigenin alleviate cutaneous inflammation in murine models of acute dermatitis. The murine models of acute allergic contact dermatitis and acute irritant contact dermatitis were established by topical application of oxazolone and phorbol 12-myristate 13-acetate (TPA, respectively. Inflammation was assessed in both dermatitis models by measuring ear thickness. Additionally, the effect of apigenin on stratum corneum function in a murine subacute allergic contact dermatitis model was assessed with an MPA5 physiology monitor. Our results demonstrate that topical applications of apigenin exhibit therapeutic effects in both acute irritant contact dermatitis and allergic contact dermatitis models. Moreover, in comparison with the vehicle treatment, topical apigenin treatment significantly reduced transepidermal water loss, lowered skin surface pH, and increased stratum corneum hydration in a subacute murine allergic contact dermatitis model. Together, these results suggest that topical application of apigenin could provide an alternative regimen for the treatment of dermatitis.

  17. Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

    International Nuclear Information System (INIS)

    Uhlin-Hansen, L.; Eskeland, T.; Kolset, S.O.

    1989-01-01

    Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains

  18. Dual stimulus-dependent effect of Oenothera paradoxa extract on the respiratory burst in human leukocytes: suppressing for Escherichia coli and phorbol myristate acetate and stimulating for formyl-methionyl-leucyl-phenylalanine.

    Science.gov (United States)

    Burzynska-Pedziwiatr, Izabela; Bukowiecka-Matusiak, Malgorzata; Wojcik, Marzena; Machala, Waldemar; Bienkiewicz, Malgorzata; Spolnik, Grzegorz; Danikiewicz, Witold; Wozniak, Lucyna Alicja

    2014-01-01

    Although a growing body of evidence suggests that plant polyphenols can modulate human immune responses, their simultaneous action on monocyte and neutrophil oxidative burst is currently poorly understood. Based on the hypothesis that various polyphenols contained in plant extracts might affect the oxidative burst of phagocytes, we evaluated the effects of ethanolic O. paradoxa extract polyphenols on monocyte and neutrophil oxidative burst in vitro activated by different stimuli, including opsonized bacteria E. coli, phorbol 12-myristate 13-acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (fMLP). Samples were analyzed by the dihydrorhodamine flow cytometry assay. Our results showed that the extract repressed significantly and dose-dependently reactive oxygen species production in both cell types stimulated with E. coli and PMA (P < 0.05) and its inhibitory efficiency was stimulus- and cell-type-dependent. Interestingly, there was significant stimulatory effect of the extract on bursting phagocytes induced by fMLP (P < 0.05). Additionally, several flavonoids and phenolic compounds as well as penta-galloyl-β-(D)-glucose (PGG), the representative of hydrolyzable tannins, were identified in the 60% extract by high-performance liquid chromatography (HPLC) coupled to electrospray ionization in negative ion mode. In summary, the ethanolic O. paradoxa extract, rich in flavonoids and phenolic compounds, exhibits dual stimulus-dependent effect on the respiratory burst in human leukocytes; hence, it might affect immune responses in humans.

  19. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

    Science.gov (United States)

    Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

    2014-09-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines. Published by Elsevier Ltd.

  20. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2

    International Nuclear Information System (INIS)

    Burch, R.M.; Axelrod, J.

    1987-01-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E 2 (PGE 2 ) synthesis. The EC 50 values for stimulation of PGE 2 synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-[γ-thio]triphosphate stimulated PGE 2 synthesis and InsP formation, and guanosine-5'-[β-thio]diphosphate inhibited both PGE 2 synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE 2 synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE 2 synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE 2 synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE 2 synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with [ 3 H] choline, the phospholipase A 2 products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A 2 and that phospholipase A 2 is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis

  1. Inhibition of epithelial Na+ transport by atriopeptin, protein kinase c, and pertussis toxin

    International Nuclear Information System (INIS)

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A.

    1987-01-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na + by atrial natriuretic peptide and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK i . Using 22 Na + fluxes, they further investigated the modulation of Na + transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na + uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na + uptake by 93 ± 13 and 51 ± 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK i cells, inhibits 22 Na + influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na + uptake. These events may be sequentially involved in the action of atrial natriuretic peptide

  2. Activation and regulation of arachidonic acid release in rabbit peritoneal neutrophils

    International Nuclear Information System (INIS)

    Tao, W.

    1988-01-01

    Arachidonic acid release in rabbit neutrophils can be enhanced by the addition of chemotactic fMet-Leu-Phe, platelet-activating factor, PAF, or the calcium ionophore A23187. Over 80% of the release [ 3 H]arachidonic acid comes from phosphatidylcholine and phosphatidylinositol. The release is dose-dependent and increases with increasing concentration of the stimulus. The A23187-induced release increases with increasing time of the stimulation. [ 3 H]arachidonic acid release, but not the rise in the concentration of intracellular calcium, is inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The [ 3 H]arachidonic acid released by A23187 is potentiated while that release by fMET-Leu-Phe or PAF is inhibited in phorbol 12-myristate 13-acetate, PMA, treated rabbit neutrophils. The protein kinase C inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine, H-7, has no effect on the potentiation by PMA of the A23187-induced release, it prevents the inhibition by PMA of the release produced by PAF or fMet-Leu-Phe. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. The diacylglycerol kinase inhibitor R59022 increases the level of diacylglycerol in neutrophils stimulated with fMet-Leu-Phe. Furthermore, R59022 potentiates [ 3 H] arachidonic acid release produced by fMet-Leu-Phe. This potentiation is not inhibited by H-7, in fact, it is increased in H-7-treated neutrophils

  3. Anti-Allergic Activity of a Platycodon Root Ethanol Extract

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    Dong-Yeul Kwon

    2010-07-01

    Full Text Available Platycodon grandiflorum (Campanulaceae is used as traditional medicine in Asian countries. In Korean traditional medicine, Platycodon root has been widely used since ancient times as a traditional drug to treat cold, cough and asthma. However, its effects on bone marrow-derived mast cell (BMMC-mediated allergy and inflammation mechanisms remain unknown. In this study, the biological effect of Platycodon root ethanol extract (PE was evaluated in BMMC after induction of allergic mediators by phorbol 12-myristate 13-acetate (PMA plus calcium ionophore A23187 (A23187 stimulation. The effect of PE on the production of several allergic mediators, such as interleukin-6 (IL-6, prostaglandin D2 (PGD2, leukotriene C4 (LTC4, β-Hexosaminidase (β-Hex and cyclooxygenase-2 (COX-2 protein, was investigated. The results demonstrate that PE inhibits PMA + A23187 induced production of IL-6, PGD2, LTC4, β-Hexosaminidase and COX-2 protein. Taken together, these results indicate that PE has the potential for use in the treatment of allergy.

  4. Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

    Science.gov (United States)

    Park, So Young; Shrestha, Sanjeeb; Youn, Young-Jin; Kim, Jun-Kyu; Kim, Shin-Yeong; Kim, Hyun Jung; Park, So-Hee; Ahn, Won-Gyun; Kim, Shin; Lee, Myung Goo; Jung, Ki-Suck; Park, Yong Bum; Mo, Eun-Kyung; Ko, Yousang; Lee, Suh-Young; Koh, Younsuck; Park, Myung Jae; Song, Dong-Keun; Hong, Chang-Won

    2017-09-01

    Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

  5. An in vitro investigation of the anti inflammatory properties of potassium humate

    Energy Technology Data Exchange (ETDEWEB)

    Joone, G.K.; van Rensburg, C.E.J. [University of Pretoria, Pretoria (South Africa). Faculty of Health Science, Dept. of Pharmacology

    2004-06-01

    In this study the anti-inflammatory potential of potassium humate, derived from bituminous coal, has been investigated in vitro. Exposure of resting and phorbol-12-myristate-13-acetate (PMA) stimulated human neutrophils to potassium humate resulted in a decreased expression of CR3 by activated, but not resting cells, in a dose-related way. Humate also inhibited the adhesion of PMA-stimulated neutrophils to a baby hamster kidney cell line expressing ICAM1 (the CR3 ligand) (BHK331-7). Similar results were obtained using normal BHK cells indicating that this inhibition does not only target specific adhesion molecules on the neutrophil and eosinophil membrane by activated phagocytes, but also affects other mechanisms involved in cell adhesion. Opsonised Sephadex or FMLP/Cyto B-induced degranulation of neutrophils and eosinophils were also decreased by humate treatment. Inhibition of the adhesion of activated phagocytes, as well as inhibition of the release of granule polypeptides, both of which are responsible for tissue damage during inflammatory processes, are attractive targets for anti-inflammatory drugs. Because humate is well tolerated with an excellent safety profile it merits further evaluation in patients suffering from inflammatory conditions.

  6. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  7. Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus

    Science.gov (United States)

    Rubins, Kathleen H.; Hensley, Lisa E.; Relman, David A.; Brown, Patrick O.

    2011-01-01

    Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection. PMID:21267444

  8. Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

    Directory of Open Access Journals (Sweden)

    Kathleen H Rubins

    2011-01-01

    Full Text Available Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV, an emerging human pathogen, and Vaccinia virus (VAC, a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA, or poly (I-C was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

  9. Role of nuclear factor of activated T-cells and activator protein-1 in the inhibition of interleukin-2 gene transcription by cannabinol in EL4 T-cells.

    Science.gov (United States)

    Yea, S S; Yang, K H; Kaminski, N E

    2000-02-01

    We previously reported that immunosuppressive cannabinoids inhibited interleukin (IL)-2 steady-state mRNA expression and secretion by phorbol-12-myristate-13-acetate plus ionomycin-activated mouse splenocytes and EL4 murine T-cells. Here we show that inhibition of IL-2 production by cannabinol, a modest central nervous system-active cannabinoid, is mediated through the inhibition of IL-2 gene transcription. Moreover, electrophoretic mobility shift assays demonstrated that cannabinol markedly inhibited the DNA binding activity of nuclear factor of activated T-cells (NF-AT) and activator protein-1 (AP-1) in a time- and concentration-dependent manner in activated EL4 cells. The inhibitory effects produced by cannabinol on AP-1 DNA binding were quite transient, showing partial recovery by 240 min after cell activation and no effect on the activity of a reporter gene under the control of AP-1. Conversely, cannabinol-mediated inhibition of NF-AT was robust and sustained as demonstrated by an NF-AT-regulated reporter gene. Collectively, these results suggest that decreased IL-2 production by cannabinol in EL4 cells is due to the inhibition of transcriptional activation of the IL-2 gene and is mediated, at least in part, through a transient inhibition of AP-1 and a sustained inhibition of NF-AT.

  10. Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

    Science.gov (United States)

    Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

    1997-09-01

    Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

  11. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

    Science.gov (United States)

    Ku, H; Meier, K E

    2000-04-14

    Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

  12. Parathyroid hormone depresses cytosolic pH and DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Reid, I.R.; Civitelli, R.; Avioli, L.V.; Hruska, K.A.

    1988-01-01

    It has recently become apparent that a number of hormones and growth factors modulate cytosolic pH (pH i ) and there is some evidence that this in turn may influence cell growth. The authors have examined the effects of parathyroid hormone (PTH) on both these parameters in an osteoblast-like cell line, UMR 106. Preliminary studies, using the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,(6)-carboxyfluorescein indicated that these cells regulate pH i by means of an amiloride-inhibitable Na + -H + exchanger. Rat PTH-(1-34) (rPTH) caused a progressive dose-related decrease in pH i with a half-maximal effect at 10 -11 M. The diacylglycerol analogue, phorbol 12-myristate 13-acetate, increased both pH i and [ 3 H]thymidine incorporation, and amiloride reduced both indexes. However, rPTH remained a potent inhibitor of [ 3 H]thymidine incorporation in the presence of amiloride, even though it did not affect pH i in these circumstances. It is concluded that PTH decreases pH i and growth in UMR 106 cells but that these changes can be dissociated. Depression of pH i may have other important effects on bone metabolism, such as reducing cell-cell communication, and may be associated with alkalinization of the bone fluid compartment

  13. Benzoxazole derivatives suppress lipopolysaccharide-induced mast cell activation.

    Science.gov (United States)

    Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Choo, Hea-Young Park; Lee, Kyung Ho

    2018-05-01

    Mast cells are central regulators of allergic inflammation that function by releasing various proallergic inflammatory mediators, including histamine, eicosanoids and proinflammatory cytokines. Occasionally, bacterial infections may initiate or worsen allergic inflammation. A number of studies have indicated that activation of lipoxygenase in mast cells positive regulates allergic inflammatory responses by generating leukotrienes and proinflammatory cytokines. In the present study, the effects of benzoxazole derivatives on the lipopolysaccharide (LPS)‑induced expression of proinflammatory cytokines, production of histamine and surface expression of co‑stimulatory molecules on bone marrow-derived mast cells (BMMCs) were studied. The benzoxazole derivatives significantly reduced the expression of interleukin (IL)‑1β, IL‑6, IL‑13, tumor necrosis factor‑α, perilipin (PLIN) 2, and PLIN3 in BMMCs treated with LPS. Furthermore, histamine production was suppressed in BMMCs treated with LPS, or treated with phorbol-12-myristate-13-acetate/ionomycin. Benzoxazole derivatives marginally affected the surface expression of cluster of differentiation (CD)80 and CD86 on BMMCs in the presence of LPS, although LPS alone did not increase the expression of those proteins. Therefore, benzoxazole derivatives inhibited the secretion of proinflammatory cytokines in mast cells and may be potential candidate anti‑allergic agents to suppress mast cell activation.

  14. Avaliação da função de macrófagos alveolares em cavalos clinicamente sadios Evaluation of alveolar macrophage function in healthy horses

    Directory of Open Access Journals (Sweden)

    E. Mori

    2001-04-01

    Full Text Available Devido à importância dos macrófagos alveolares (MA nos mecanismos de defesa pulmonar, foram realizados estudos para avaliar a atividade desses fagócitos em cavalos hígidos. Foram realizados lavados broncoalveolares (LBA em cinco cavalos clinicamente sadios. A citologia foi realizada pela citocentrifugação das amostras e posterior confecção de lâminas coradas pelo método de Rosenfeld. Todas as amostras do LBA foram centrifugadas e a concentração celular foi ajustada para 2×10(6 células/ml, para a mensuração da atividade macrofágica (testes de espraiamento, fagocitose e liberação de peróxido de hidrogênio. A contagem diferencial das células presentes no LBA demonstrou a predominância de macrófagos (59,0± 6,9%. Os resultados obtidos nos testes de mensuração da atividade macrofágica foram: índice de espraiamento 25,1± 19,7%, índice de fagocitose 89,4± 6,2% e liberação de peróxido de hidrogênio 1,6± 0,3nmoles/2×10(5 células (sem PMA - phorbol 12-myristate 13-acetate e 1,8± 0,4nmoles/2×10(5 células (com PMA. Os resultados demonstraram um padrão de atividade para MA de cavalos hígidos, os quais apresentaram índices de ativação mesmo sem elicitação prévia, indicando que as técnicas utilizadas foram adequadas para tal propósito.Due to the importance of alveolar macrophages (AM in pulmonary defense mechanisms, studies were performed in order to evaluate the activity of these cells. Bronchoalveolar lavages (BAL were obtained from five healthy horses, and cytology was performed on glass slides after cytocentrifugation of the samples. Slides were stained by Rosenfeld. All BAL samples were centrifuged and cell concentration was adjusted to 2×10(6 cells/ml, for the measurement of AM activity (spreading, phagocytosis and hydrogen peroxide release tests. Differential counting of the BAL cells demonstrated that macrophages were the predominant type of cell (59.0± 6.9%. Measurement of AM activity presented the

  15. Production of proinflammatory cytokines without invocation of cytotoxic effects by an Epstein-Barr virus-infected natural killer cell line established from a patient with hypersensitivity to mosquito bites.

    Science.gov (United States)

    Suzuki, Daisuke; Tsuji, Kazuhide; Yamamoto, Takenobu; Fujii, Kazuyasu; Iwatsuki, Keiji

    2010-10-01

    Cumulative evidence supports that Epstein-Barr virus (EBV)-infected natural killer (NK) cells induce severe systemic and cutaneous inflammation in patients with hypersensitivity to mosquito bites (HMB). In order to understand the pathogenesis of HMB, we established an EBV-infected cell line and characterized the cytological profiles. A novel EBV-infected NK-cell line, designated NKED, was established from a patient with HMB and used for the present study along with two other NK-cell lines, KAI3 and KHYG-1. NKED expressed the latency II-related transcripts. NKED cells were positive for CD2 and CD161 antigens, and negative for CD3, CD16, CD34, CD56, and T-cell receptor α/β and γ/δ antigens. Although NKED cells contained several cytotoxic molecules, the cells had an extremely poor cytotoxic activity. The majority of NKED cells were negative for perforin, major histocompatibility complex class I-restricted NK-cell receptors, CD94 and KIR2D, and an activating receptor, NKG2D. NKED cells, however, secreted higher levels of tumor necrosis factor-α. Stimulation with phorbol 12-myristate 13-acetate or tumor necrosis factor-α induced expression of BZLF1 messenger RNA in the NKED and KAI3 cells, indicating the transition from the latent- to the lytic-cycle infection. These data suggested that NKED cells revealed a very low cytotoxic effect probably because of the low expression levels of perforin, but had the ability to release proinflammatory cytokines. NKED cells did not reflect the characteristics of HMB, as they were different from pathogenic NK cells proliferating in the HMB patient, but the difference indicated that pathogenic NK cells could change their character in the presence of interleukin-2. Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  16. Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ

    Institute of Scientific and Technical Information of China (English)

    Amy L Samuels; Alison Louw; Reza Zareie; Evan Ingley

    2017-01-01

    AIM To identify and characterize the effect of phosphorylation on the subcellular localization of Ankrd54.METHODS HEK293 T cells were treated with calyculin A, staurosporin or phorbol 12-myristate 13-acetate(PMA). Cells were transfected with eG FP-tagged Ankrd54 with or without Lyn tyrosine kinase(wild-type, Y397 F mutant, or Y508 F mutant). The subcellular localization was assessed by immunofluorescence imaging of cells, immunoblotting of subcellular fractionations. The phosphorylation of Ankrd54 was monitored using Phos-tagT M gel retardation. Phosphorylated peptides were analysed by multiplereaction-monitoring(MRM) proteomic analysis.RESULTS Activation of PKC kinases using PMA promoted nuclear export of Ankrd54 and correlated with increased Ankrd54 phosphorylation, assayed using Phos-tag TM gel retardation. Co-expression of an active form of the PKCδisoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54, while PKCδ, Akt and PKA did not. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54(Ser14, Ser17, Ser18, Ser19) reduced both PMA induced cytoplasmic localization and phosphorylation of Ankrd54. Using MRM proteomic analysis, phosphorylation of the Ser18 residue of Ankrd54 was readily detectable in response to PMA stimulation. PMA stimulation of cells co-expressing Ankrd54 and Lyn tyrosine kinase displayed increased coimmunoprecipitation and enhanced co-localization in the cytoplasm.CONCLUSION We identify phosphorylation by PKCδ as a major regulator of nuclear-cytoplasmic shuttling of Ankrd54, and its interaction with the tyrosine kinase Lyn.

  17. Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes.

    Science.gov (United States)

    Duong, Michelle; Yu, Xuejiao; Teng, Beina; Schroder, Patricia; Haller, Hermann; Eschenburg, Susanne; Schiffer, Mario

    2017-07-21

    Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of β-catenin, which promotes its degradation. β-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with β-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower β-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, β-catenin showed a reversed subcellular localization pattern: Although β-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates β-catenin expression and stabilization in a glycogen synthase kinase 3β-independent manner. Further, β-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the β-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in β-catenin that are required for full β-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and β-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of β-catenin levels and define PKCϵ as an important β-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

    Science.gov (United States)

    Price, B D; Morris, J D; Hall, A

    1989-01-01

    The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase. PMID:2690829

  19. Sirt3 deficiency does not affect venous thrombosis or NETosis despite mild elevation of intracellular ROS in platelets and neutrophils in mice.

    Directory of Open Access Journals (Sweden)

    Hideki Hayashi

    Full Text Available Inflammation is a common denominator in chronic diseases of aging. Yet, how inflammation fuels these diseases remains unknown. Neutrophils are the primary leukocytes involved in the early phase of innate immunity and inflammation. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs by releasing decondensed chromatin lined with cytotoxic proteins. NETs have been shown to induce tissue injury and thrombosis. Here, we demonstrated that Sirt3, a nicotinamide adenine dinucleotide (NAD+-dependent protein deacetylase, an enzyme linked to human longevity, was expressed in mouse neutrophils and platelets. Using Sirt3-/- mice as a model of accelerated aging, we investigated the effects of Sirt3 deficiency on NETosis and platelet function, aiming to detect enhancement of thrombosis. More mitochondrial reactive oxygen species (ROS were generated in neutrophils and platelets of Sirt3-/- mice compared to WT, when stimulated with a low concentration of phorbol 12-myristate 13-acetate (PMA and a high concentration of thrombin, respectively. There were no differences in in vitro NETosis, with or without stimulation. Platelet aggregation was mildly augmented in Sirt3-/- mice compared to WT mice, when stimulated with a low concentration of collagen. The effect of Sirt3 deficiency on platelet and neutrophil activation in vivo was examined by the venous thrombosis model of inferior vena cava stenosis. Elevation of plasma DNA concentration was observed after stenosis in both genotypes, but no difference was shown between the two genotypes. The systemic response to thrombosis was enhanced in Sirt3-/- mice with significantly elevated neutrophil count and reduced platelet count. However, no differences were observed in incidence of thrombus formation, thrombus weight and thrombin-antithrombin complex generation between WT and Sirt3-/- mice. We conclude that Sirt3 does not considerably impact NET formation, platelet function, or venous

  20. Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Scherer, N.M.; Nathanson, N.M.

    1990-01-01

    Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA - ). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA + or PKA - cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA - cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA + and PKA - cells was accompanied by desensitization of functional responses. Exposure of PKA + cells to 10 -7 M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 ± 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA + or PKA - cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA - cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA + or PKA - cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%

  1. Impact of FDA-Approved Drugs on the Prostaglandin Transporter OATP2A1/SLCO2A1.

    Science.gov (United States)

    Kamo, Shunsuke; Nakanishi, Takeo; Aotani, Rika; Nakamura, Yoshinobu; Gose, Tomoka; Tamai, Ikumi

    2017-09-01

    To understand interaction of drugs with the prostaglandin transporter OATP2A1/SLCO2A1 that regulates disposition of prostaglandins, we explored the impact of 636 drugs in an FDA-approved drug library on 6-carboxyfluorescein (6-CF) uptake by OATP2A1-expressing HEK293 cells (HEK/2A1). Fifty-one and 10 drugs were found to inhibit and enhance 6-CF uptake by more than 50%, respectively. Effect of the 51 drugs on 6-CF uptake was positively correlated with that on PGE 2 uptake (r = 0.64, p < 0.001). Among those, 5 drugs not structurally related to prostaglandins, suramin, pranlukast, zafirlukast, olmesartan medoxomil, and losartan potassium, exhibited more than 90% PGE 2 uptake inhibition. Inhibitory affinity of suramin to OATP2A1 was the highest (IC 50,2A1 of 0.17 μM), and its IC 50 values to MRP4-mediated PGE 2 transport (IC 50,MRP4 ) and PGE 2 synthesis in human U-937 cells treated with phorbol 12-myristate 13-acetate (IC 50,Syn ) were 73.6 and 336.7 times higher than IC 50,2A1 , respectively. Moreover, structure-activity relationship study in 29 nonsteroidal anti-inflammatory drugs contained in the library displayed inhibitory activities of anthranilic acid derivatives, but enhancing effects of propionic acid derivatives. These results demonstrate that suramin is a potent selective inhibitor of OATP2A1, providing a comprehensive information about drugs in clinical use that interact with OATP2A1. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  2. Antioxidant potential of selected Spirulina platensis preparations.

    Science.gov (United States)

    Dartsch, Peter C

    2008-05-01

    Recent studies suggest that Spirulina, a unicellular blue-green alga, may have a variety of health benefits and therapeutic properties and is also capable of acting as an antioxidant and antiinflammatory agent. In this study, a cell-free and a cell-based test assay were used to examine the antioxidant and antiinflammatory properties of four selected Spirulina platensis preparations: (1) Biospirulina, (2) SpiruComplex, a preparation with naturally bound selenium, chromium and zinc, (3) SpiruZink, a preparation with naturally bound zinc, (4) Zinkspirulina + Acerola, a preparation with naturally bound zinc and acerola powder. The cell-free test assay used potassium superoxide as a donor for superoxide radicals, whereas the cell-based test assay used the formation of intracellular superoxide radicals of functional neutrophils upon stimulation by phorbol-12-myristate-13-acetate as a model to investigate the potential of Spirulina preparations to inactivate superoxide radicals. In accordance with the recommended daily dosage, test concentrations ranging from 50 to 1000 microg/mL were chosen. The results showed a dose-dependent inactivation of free superoxide radicals (antioxidant effect) as well as an antiinflammatory effect characterized by a dose-dependent reduction of the metabolic activity of functional neutrophils and a dose-dependent inactivation of superoxide radicals generated during an oxidative burst. The results demonstrate that the tested Spirulina preparations have a high antioxidant and antiinflammatory potential. Especially SpiruZink and Zinkspirulina + Acerola might be useful as a supportive therapeutic approach for reducing oxidative stress and/or the generation of oxygen radicals in the course of inflammatory processes.

  3. Integration of intracellular telomerase monitoring by electrochemiluminescence technology and targeted cancer therapy by reactive oxygen species.

    Science.gov (United States)

    Zhang, Huairong; Li, Binxiao; Sun, Zhaomei; Zhou, Hong; Zhang, Shusheng

    2017-12-01

    Cancer therapies based on reactive oxygen species (ROS) have emerged as promising clinical treatments. Electrochemiluminescence (ECL) technology has also attracted considerable attention in the field of clinical diagnosis. However, studies about the integration of ECL diagnosis and ROS cancer therapy are very rare. Here we introduce a novel strategy that employs ECL technology and ROS to fill the above vacancy. Briefly, an ITO electrode was electrodeposited with polyluminol-Pt NPs composite films and modified with aptamer DNA to capture HL-60 cancer cells with high specificity. After that, mesoporous silica nanoparticles (MSNs) filled with phorbol 12-myristate 13-acetate (PMA) were closed by the telomerase primer DNA (T-primer DNA) and aptamer. After aptamer on MSN@PMA recognized and combined with the HL-60 cancer cells with high specificity, T-primer DNA on MSN@PMA could be moved away from the MSN@PMA surface after extension by telomerase in the HL-60 cancer cells and PMA was released to induce the production of ROS by the HL-60 cancer cells. After that, the polyluminol-Pt NPs composite films could react with hydrogen peroxide (a major ROS) and generate an ECL signal. Thus the intracellular telomerase activity of the HL-60 cancer cells could be detected in situ . Besides, ROS could induce apoptosis in the HL-60 cancer cells with high efficacy by causing oxidative damage to the lipids, protein, and DNA. Above all, the designed platform could not only detect intracellular telomerase activity instead of that of extracted telomerase, but could also kill targeted tumors by ECL technology and ROS.

  4. Helicobacter pylori induces IL-1β and IL-18 production in human monocytic cell line through activation of NLRP3 inflammasome via ROS signaling pathway.

    Science.gov (United States)

    Li, Xiang; Liu, Sheng; Luo, Jingjing; Liu, Anyuan; Tang, Shuangyang; Liu, Shuo; Yu, Minjun; Zhang, Yan

    2015-06-01

    This study investigated whether Helicobacter pylori could activate the nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome in human macrophages and the involvement of reactive oxygen species (ROS) in inflammasome activation. Phorbol-12-myristate-13-acetate (PMA)-differentiated human acute monocytic leukemia cell line THP-1 was infected with H. pylori. The levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in supernatant were measured by ELISA. Intracellular ROS level was analyzed by flow cytometry. Quantitative real-time PCR and western blot analysis were employed to determine the mRNA and protein expression levels of NLRP3 and caspase-1 in THP-1 cells, respectively. Our results showed that H. pylori infection could induce IL-1β and IL-18 production in PMA-differentiated THP-1 cells in a dose- and time-dependent manner. Moreover, secretion of IL-1β and IL-18 in THP-1 cells following H. pylori infection was remarkably reduced by NLRP3-specific small interfering RNA treatment. In addition, the intracellular ROS level was elevated by H. pylori infection, which could be eliminated by the ROS scavenger N-acetylcysteine (NAC). Furthermore, NAC treatment could inhibit NLRP3 inflammasome formation and caspase-1 activation and suppress the release of IL-1β and IL-18 from H. pylori-infected THP-1 cells. These findings provide novel insights into the innate immune response against H. pylori infection, which could potentially be used for the prevention and treatment of H. pylori-related diseases. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study

    Science.gov (United States)

    Westera, Liset; van Viegen, Tanja; Jeyarajah, Jenny; Azad, Azar; Bilsborough, Janine; van den Brink, Gijs R; Cremer, Jonathan; Danese, Silvio; D'Haens, Geert; Eckmann, Lars; Faubion, William; Filice, Melissa; Korf, Hannelie; McGovern, Dermot; Panes, Julian; Salas, Azucena; Sandborn, William J; Silverberg, Mark S; Smith, Michelle I; Vermeire, Severine; Vetrano, Stefania; Shackelton, Lisa M; Stitt, Larry; Jairath, Vipul; Levesque, Barrett G; Spencer, David M; Feagan, Brian G; Vande Casteele, Niels

    2017-01-01

    Objectives: Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor. Results: Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4–102.1% LGCS, 10.9–65.6% CG, 1.8–20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG. Conclusions: Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays. PMID:29095427

  6. Alterations in polyamine levels induced by phorbol diesters and other agents that promote differentiation in human promyelocytic leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Weeks, C.; Herrmann, A.; Callaham, M.; Slaga, T.

    1981-02-01

    Polyamine levels were evaluated in human HL-60 promyelocytic leukemia cells after treatment with inducers of terminal differentiation. Differentiation in these cells was determined by increases in the percentage of morphologically mature cells and in lysozyme activity. Treatment of the HL-60 cells with phorbol 12-myristate-13-acetate (PMA), phorbol 12,13-didecanoate or other inducers of terminal differentiation such as dimethylsulfoxide and retinoic acid resulted in increased levels of putrescine. However, no increase in putrescine could be detected after PMA treatment of a HL-60 cell variant that exhibited a decreased susceptibility to PMA-induced terminal differentiation. Similarly, no increase in putrescine was observed with two nontumor-promoters (phorbol 12,13-diacetate and 4-O-methyl-PMA) or with anthralin, a non-phorbol tumor promoter. In addition to enhancing putrescine levels, PMA also increased the amount of spermidine and decreased the amount of spermine. The increase in putrescine and spermidine preceded the expression of the various differentiation markers. Unlike the changes observed in the polyamine levels after PMA treatment, the activities of ornithine and S-adenosylmethionine decarboxylases, which are polyamine biosynthetic enzymes, did not significantly change. ..cap alpha..-Methylornithine and ..cap alpha..-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), which are inhibitors of the polyamine biosynthetic enzymes, did not affect differentiation in control or PMA-treated cells. Because of these observations, we suggest that the change in polyamine levels involve biochemical pathways other than the known biosynthetic ones. By-products of these pathways may perhaps be the controlling factors involved in the induction of terminal differentiation in the HL-60 and other cell types as well.

  7. Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection: A New Mechanism of Host Defense.

    Science.gov (United States)

    Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Miller, Derek; Unkel, Ronald; Shaman, Majid; Jacques, Suzanne M; Panaitescu, Bogdan; Garcia-Flores, Valeria; Hassan, Sonia S

    2017-08-01

    Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

  8. Regulation of insulin-like growth factor I receptors on vascular smooth muscle cells by growth factors and phorbol esters.

    Science.gov (United States)

    Ververis, J J; Ku, L; Delafontaine, P

    1993-06-01

    Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells. To characterize regulation of vascular IGF I receptors, we performed radioligand displacement experiments using rat aortic smooth muscle cells (RASMs). Serum deprivation for 48 hours caused a 40% decrease in IGF I receptor number. Exposure of quiescent RASMs to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or angiotensin II (Ang II) caused a 1.5-2.0-fold increase in IGF I receptors per cell. After FGF exposure, there was a marked increase in the mitogenic response to IGF I. IGF I downregulated its receptors in the presence of platelet-poor plasma. Stimulation of protein kinase C (PKC) by exposure of quiescent RASMs to phorbol 12-myristate 13-acetate caused a biphasic response in IGF I binding; there was a 42% decrease in receptor number at 45 minutes and a 238% increase at 24 hours. To determine the role of PKC in growth factor-induced regulation of IGF I receptors, we downregulated PKC by exposing RASMs to phorbol 12,13-dibutyrate (PDBu) for 48 hours. PDGF- and FGF- but not Ang II-mediated upregulation of IGF I receptors was completely inhibited in PDBu-treated cells. Thus, acute PKC activation by phorbol esters inhibits IGF I binding, whereas chronic PKC activation increases IGF I binding. PDGF and FGF but not Ang II regulate vascular IGF I receptors through a PKC-dependent pathway. These data provide new insights into the regulation of vascular smooth muscle cell IGF I receptors in vitro and are of potential importance in characterizing vascular proliferative responses in vivo.

  9. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    International Nuclear Information System (INIS)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-01-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

  10. Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

    Science.gov (United States)

    Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

    1994-01-01

    Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

  11. The Role of FAK in the Secretion of MMP9 after CD147 Stimulation in Macrophages.

    Science.gov (United States)

    Yu, Chen; Lixia, Yang; Ruiwei, Guo; Yankun, Shi; Jinshan, Ye

    2018-03-30

    To investigate whether focal adhesion kinase (FAK) can participate in the secretion of matrix metalloproteinase 9 (MMP9) after CD147 stimulation in THP-1 induced macrophages; thus, to explore the potential treatment perspectives for acute coronary syndrome (ACS).Phorbol-12-myristate-13-acetate (PMA) was used to induce THP-1 cells to differentiate into macrophages. To confirm the peak mRNA and protein expression of FAK and MMP9 after the stimulation of CD147, the macrophages were divided into 5 groups (0, 3, 6, 9, and 12 hours), with 0 hours group as control group. To investigate the role of FAK in the secretion of MMP9, with stimulation of CD147 for 9 hours, FAK inhibitor 14 was used to inhibit FAK Y397 phosphorylation. The mRNA and protein expressions were quantified by qRT-PCR and western blotting, respectively. (1) Relative mRNA expression of FAK and MMP9 were both significantly up-regulated (all P CD147, FAK peaked at 9 hours (3.908 ± 0.106 versus 1, P CD147 stimulation (all P CD147 up-regulates FAK, pFAK, and MMP9 mRNA and protein expressions in a dose-dependent manner. (4) FAK inhibitor 14 significantly reduced the relative protein expression level of pFAK (0.077 ± 0.012 versus 1, P CD147 stimulation.The results demonstrated that FAK Y397 phosphorylation was involved in the secretion of MMP9 after CD147 stimulation in macrophages and may play a role in the regulation of ACS.

  12. Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

    Science.gov (United States)

    Kamoshida, Go; Kikuchi-Ueda, Takane; Nishida, Satoshi; Tansho-Nagakawa, Shigeru; Ubagai, Tsuneyuki; Ono, Yasuo

    2018-01-01

    Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections. PMID:29467765

  13. Cajaninstilbene acid relaxes rat renal arteries: roles of Ca2+ antagonism and protein kinase C-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Dong-Mei Zhang

    Full Text Available Cajaninstilbene acid (CSA is a major active component present in the leaves of Cajanus cajan (L. Millsp. The present study explores the underlying cellular mechanisms for CSA-induced relaxation in rat renal arteries. Vascular reactivity was examined in arterial rings that were suspended in a Multi Myograph System and the expression of signaling proteins was assessed by Western blotting method. CSA (0.1-10 µM produced relaxations in rings pre-contracted by phenylephrine, serotonin, 9, 11-dideoxy-9α, 11α-epoxymethanoprostaglandin F(2α (U46619, and 60 mM KCl. CSA-induced relaxations did not show difference between genders and were unaffected by endothelium denudation, nor by treatment with N(G-nitro-L-arginine methyl ester, indomethacin, ICI-182780, tetraethylammonium ion, BaCl(2, glibenclamide, 4-aminopyridine or propranolol. CSA reduced contraction induced by CaCl(2 (0.01-5 mM in Ca(2+-free 60 mM KCl solution and by 30 nM (--Bay K8644 in 15 mM KCl solution. CSA inhibited 60 mM KCl-induced Ca(2+ influx in smooth muscle of renal arteries. In addition, CSA inhibited contraction evoked by phorbol 12-myristate 13-acetate (PMA, protein kinase C agonist in Ca(2+-free Krebs solution. Moreover, CSA reduced the U46619- and PMA-induced phosphorylation of myosin light chain (MLC at Ser19 and myosin phosphatase target subunit 1 (MYPT1 at Thr853 which was associated with vasoconstriction. CSA also lowered the phosphorylation of protein kinase C (PKCδ at Thr505. In summary, the present results suggest that CSA relaxes renal arteries in vitro via multiple cellular mechanisms involving partial inhibition of calcium entry via nifedipine-sensitive calcium channels, protein kinase C and Rho kinase.

  14. Resveratrol Targeting of Carcinogen-Induced Brain Endothelial Cell Inflammation Biomarkers MMP-9 and COX-2 is Sirt1-Independent

    Directory of Open Access Journals (Sweden)

    Borhane Annabi

    2012-01-01

    Full Text Available The occurrence of a functional relationship between the release of metalloproteinases (MMPs and the expression of cyclooxygenase (COX-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB, increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Tissue scan microarray analysis of grade I–IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I–III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA, a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IκB phosphorylation in siSirt1-transfected HBMEC was however observed. Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.

  15. Chrysin inhibits tumor promoter-induced MMP-9 expression by blocking AP-1 via suppression of ERK and JNK pathways in gastric cancer cells.

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    Yong Xia

    Full Text Available Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9 is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.

  16. Micronutrient Synergy in the Fight against Hepatocellular Carcinoma

    International Nuclear Information System (INIS)

    Roomi, M. Waheed; Roomi, Nusrath W.; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2012-01-01

    The incidence of hepatocellular carcinoma (HCC), once thought to be a rare tumor in North America, has rapidly increased in recent years in the United States. Current treatment modalities to halt the progression of this disease are only marginally effective. The mainstay treatment is liver transplantation, which is often confronted with donor shortage. Invasion, metastasis and recurrence contribute to the high mortality rate of this disease. Matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) have been associated with the progression, invasion and metastasis of the disease. We have developed strategies to strengthen the ECM collagen and inhibit MMPs through micronutrients such as lysine, proline and ascorbic acid. Addition of epigallocatechin gallate or green tea extract to these micronutrients synergistically enhanced anti-carcinogenic activity in HepG2 cells. Addition of certain other micronutrients, such as N-acetylcysteine, selenium, copper and zinc (NM) synergistically enhanced the anticancer activity of the mixture in a model of hepatocellular carcinoma using HepG2 cells. In vitro studies using HepG2 demonstrated that NM was very effective in inhibiting cell proliferation (by MTT assay), MMPs secretion (by gelatinase zymography), cell invasion (through Matrigel) and induction of apoptosis (by live green caspase). In addition, NM was shown to down-regulate urokinase plasminogen activator (by fibrin zymography) and up-regulate tissue inhibitors of metalloproteinases (by reverse zymography) in another HCC cell line, SK-Hep-1. MMP-2 and MMP-9 activities were further modulated by phorbol 12-myristate 13-acetate (PMA) induction and inhibited by NM. In previous studies, NM inhibited Sk-Hep-1 xenografts in nude mice and also inhibited hepatic metastasis of B16FO melanoma cells. Our results suggest that NM is an excellent candidate for therapeutic use in the treatment HCC by inhibiting critical parameters in cancer development and progression

  17. A newly synthesized macakurzin C-derivative attenuates acute and chronic skin inflammation: The Nrf2/heme oxygenase signaling as a potential target

    Energy Technology Data Exchange (ETDEWEB)

    Akram, Muhammad [College of Pharmacy Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan (Korea, Republic of); Shin, Iljin [College of Pharmacy and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon (Korea, Republic of); Kim, Kyeong-A; Noh, Dabi [College of Pharmacy Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan (Korea, Republic of); Baek, Seung-Hoon; Chang, Sun-Young [College of Pharmacy and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon (Korea, Republic of); Kim, Hyoungsu, E-mail: hkimajou@ajou.ac.kr [College of Pharmacy and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon (Korea, Republic of); Bae, Ok-Nam, E-mail: onbae@hanyang.ac.kr [College of Pharmacy Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan (Korea, Republic of)

    2016-09-15

    Impaired immune responses in skin play a pivotal role in the development and progression of chemical-associated inflammatory skin disorders. In this study, we synthesized new flavonoid derivatives from macakurzin C, and identified in vitro and in vivo efficacy of a potent anti-inflammatory flavonoid, Compound 14 (CPD 14), with its underlying mechanisms. In lipopolysaccharide (LPS)-stimulated murine macrophages and IFN-γ/TNF-α-stimulated human keratinocytes, CPD 14 significantly inhibited the release of inflammatory mediators including nitric oxide (NO), prostaglandins, and cytokines (IC{sub 50} for NO inhibition in macrophages: 4.61 μM). Attenuated NF-κB signaling and activated Nrf2/HO-1 pathway were responsible for the anti-inflammatory effects of CPD 14. The in vivo relevance was examined in phorbol 12-myristate 13-acetate (TPA)-induced acute skin inflammation and oxazolone-induced atopic dermatitis models. Topically applied CPD 14 significantly protected both irritation- and sensitization-associated skin inflammation by suppressing the expression of inflammatory mediators. In summary, we demonstrated that a newly synthesized flavonoid, CPD 14, has potent inhibitory effects on skin inflammation, suggesting it is a potential therapeutic candidate to treat skin disorders associated with excessive inflammation. - Highlights: • An anti-inflammatory flavonoid CPD 14 was newly synthesized from macakurzin C. • CPD 14 potently inhibited inflammatory reaction in keratinocytes and macrophages. • Dermal toxicity by irritation or sensitization in rats was protected by CPD 14. • Attenuated NF-κB and activated Nrf2/HO-1 were main mechanisms of CPD 14 action.

  18. Docosahexaenoic acid ester of phloridzin inhibit lipopolysaccharide-induced inflammation in THP-1 differentiated macrophages.

    Science.gov (United States)

    Sekhon-Loodu, Satvir; Ziaullah; Rupasinghe, H P Vasantha

    2015-03-01

    Phloridzin or phlorizin (PZ) is a predominant phenolic compound found in apple and also used in various natural health products. Phloridzin shows poor absorption and cellular uptake due to its hydrophilic nature. The aim was to investigate and compare the effect of docosahexaenoic acid (DHA) ester of PZ (PZ-DHA) and its parent compounds (phloridzin and DHA), phloretin (the aglycone of PZ) and cyclooxygenase inhibitory drugs (diclofenac and nimesulide) on production of pro-inflammatory biomarkers in inflammation-induced macrophages by lipopolysaccharide (LPS)-stimulation. Human THP-1 monocytes were seeded in 24-well plates (5×10(5)/well) and treated with phorbol 12-myristate 13-acetate (PMA, 0.1μg/mL) for 48h to induce macrophage differentiation. After 48h, the differentiated macrophages were washed with Hank's buffer and treated with various concentrations of test compounds for 4h, followed by the LPS-stimulation (18h). Pre-exposure of PZ-DHA ester was more effective in reducing tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) protein levels compared to DHA and nimesulide. However, diclofenac was the most effective in reducing prostaglandin (PGE2) level by depicting a dose-dependent response. However, PZ-DHA ester and DHA were the most effective in inhibiting the activation of nuclear factor-kappa B (NF-κB) among other test compounds. Our results suggest that PZ-DHA ester might possess potential therapeutic activity to treat inflammation related disorders such as type 2 diabetes, asthma, atherosclerosis and inflammatory bowel disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Screening for oxidative damage by engineered nanomaterials: a comparative evaluation of FRAS and DCFH

    Science.gov (United States)

    Pal, Anoop K.; Hsieh, Shu-Feng; Khatri, Madhu; Isaacs, Jacqueline A.; Demokritou, Philip; Gaines, Peter; Schmidt, Daniel F.; Rogers, Eugene J.; Bello, Dhimiter

    2014-02-01

    Several acellular assays are routinely used to measure oxidative stress elicited by engineered nanomaterials (ENMs), yet little comparative evaluations of such methods exist. This study compares for the first time the performance of the dichlorofluorescein (DCFH) assay which measures reactive oxygen species (ROS) generation, to that of the ferric-reducing ability of serum (FRAS) assay, which measures biological oxidant damage in serum. A diverse set of 28 commercially important and extensively characterized ENMs were tested on both the assays. Intracellular oxidative stress was also assessed on a representative subset of seven ENMs in THP-1 (phorbol 12-myristate 13-acetate matured human monocytes) cells. Associations between assay responses and ENM physicochemical properties were assessed via correlation and regression analysis. DCFH correlated strongly with FRAS after dose normalization for mass ( R 2 = 0.78) and surface area ( R 2 = 0.68). Only 10/28 ENMs were positive in DCFH versus 21/28 in FRAS. Both assays were strongly associated with specific surface area and transition metal content. Qualitatively, a similar response ranking was observed for acellular FRAS and intracellular reduced:oxidized glutathione ratio (GSH:GSSG) in cells. Quantitatively, weak correlation was found between intracellular GSSG and FRAS or DCFH ( R 2 < 0.25) even after calculating effective dose to cells. The FRAS assay was more sensitive than DCFH, especially for ENMs with low to moderate oxidative damage potential, and may serve as a more biologically relevant substitute for acellular ROS measurements of ENMs. Further in vitro and in vivo validations of FRAS against other toxicological endpoints with larger datasets are recommended.

  20. Death of Monocytes through Oxidative Burst of Macrophages and Neutrophils: Killing in Trans.

    Directory of Open Access Journals (Sweden)

    Viviane Ponath

    Full Text Available Monocytes and their descendants, macrophages, play a key role in the defence against pathogens. They also contribute to the pathogenesis of inflammatory diseases. Therefore, a mechanism maintaining a balance in the monocyte/macrophage population must be postulated. Our previous studies have shown that monocytes are impaired in DNA repair, rendering them vulnerable to genotoxic stress while monocyte-derived macrophages are DNA repair competent and genotoxic stress-resistant. Based on these findings, we hypothesized that monocytes can be selectively killed by reactive oxygen species (ROS produced by activated macrophages. We also wished to know whether monocytes and macrophages are protected against their own ROS produced following activation. To this end, we studied the effect of the ROS burst on DNA integrity, cell death and differentiation potential of monocytes. We show that monocytes, but not macrophages, stimulated for ROS production by phorbol-12-myristate-13-acetate (PMA undergo apoptosis, despite similar levels of initial DNA damage. Following co-cultivation with ROS producing macrophages, monocytes displayed oxidative DNA damage, accumulating DNA single-strand breaks and a high incidence of apoptosis, reducing their ability to give rise to new macrophages. Killing of monocytes by activated macrophages, termed killing in trans, was abolished by ROS scavenging and was also observed in monocytes co-cultivated with ROS producing activated granulocytes. The data revealed that monocytes, which are impaired in the repair of oxidised DNA lesions, are vulnerable to their own ROS and ROS produced by macrophages and granulocytes and support the hypothesis that this is a mechanism regulating the amount of monocytes and macrophages in a ROS-enriched inflammatory environment.

  1. Zinc oxide nanoparticles, a novel candidate for the treatment of allergic inflammatory diseases.

    Science.gov (United States)

    Kim, Min-Ho; Seo, Jun-Ho; Kim, Hyung-Min; Jeong, Hyun-Ja

    2014-09-05

    Zinc (Zn) is an essential trace metal for eukaryotes. The roles of Zn in the numerous physiological functions have been elucidated. Bamboo salt contains Zn that was shown to have anti-inflammatory effect and other health benefits. Nanoparticles of various types have found application in the biology, medicine, and physics. Here we synthesized tetrapod-like, zinc oxide nanoparticles (ZO-NP; diameter 200 nm, source of Zn) using a radio frequency thermal plasma system and investigated its effects on mast cell-mediated allergic inflammatory reactions. ZO-NP was found to inhibit the productions and mRNA expressions of inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α on the phorbol 12-myristate 13-acetate plus A23187 (PMACI)-stimulated human mast cell line, HMC-1 cells. In these stimulated cells, caspase-1 and nuclear factor-κB activations were abolished by ZO-NP, and the expressions of receptor interacting protein2 (RIP2) and IκB kinaseβ (IKKβ) induced by PAMCI were reduced. On the other hand, ZO-NP alone increased the expressions of RIP2 and IKKβ in normal condition. ZO-NP inhibited the phosphorylation of extracellular signal-regulated protein kinase in the PMACI-stimulated HMC-1 cells. Furthermore, ZO-NP significantly inhibited passive cutaneous anaphylaxis activated by anti-dinitrophenyl IgE. These findings indicate that ZO-NP effectively ameliorates mast cell-mediated allergic inflammatory reaction, and suggest that ZO-NP be considered a potential therapeutic for the treatment of mast cell-mediated allergic diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Role of ATM in bystander signaling between human monocytes and lung adenocarcinoma cells.

    Science.gov (United States)

    Ghosh, Somnath; Ghosh, Anu; Krishna, Malini

    2015-12-01

    The response of a cell or tissue to ionizing radiation is mediated by direct damage to cellular components and indirect damage mediated by radiolysis of water. Radiation affects both irradiated cells and the surrounding cells and tissues. The radiation-induced bystander effect is defined by the presence of biological effects in cells that were not themselves in the field of irradiation. To establish the contribution of the bystander effect in the survival of the neighboring cells, lung carcinoma A549 cells were exposed to gamma-irradiation, 2Gy. The medium from the irradiated cells was transferred to non-irradiated A549 cells. Irradiated A549 cells as well as non-irradiated A549 cells cultured in the presence of medium from irradiated cells showed decrease in survival and increase in γ-H2AX and p-ATM foci, indicating a bystander effect. Bystander signaling was also observed between different cell types. Phorbol-12-myristate-13-acetate (PMA)-stimulated and gamma-irradiated U937 (human monocyte) cells induced a bystander response in non-irradiated A549 (lung carcinoma) cells as shown by decreased survival and increased γ-H2AX and p-ATM foci. Non-stimulated and/or irradiated U937 cells did not induce such effects in non-irradiated A549 cells. Since ATM protein was activated in irradiated cells as well as bystander cells, it was of interest to understand its role in bystander effect. Suppression of ATM with siRNA in A549 cells completely inhibited bystander effect in bystander A549 cells. On the other hand suppression of ATM with siRNA in PMA stimulated U937 cells caused only a partial inhibition of bystander effect in bystander A549 cells. These results indicate that apart from ATM, some additional factor may be involved in bystander effect between different cell types. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Peripheral blood CD161+ T cells from asthmatic patients are activated during asthma attack and predominantly produce IFN-gamma.

    Science.gov (United States)

    González-Hernández, Y; Pedraza-Sánchez, S; Blandón-Vijil, V; del Río-Navarro, B E; Vaughan, G; Moreno-Lafont, M; Escobar-Gutiérrez, A

    2007-04-01

    In humans, T cells expressing the CD161 molecule NKR-P1A constitute around 20% of the circulating CD3(+) cells and are potentially immunoregulatory in several diseases. Their role in asthma is not well known, but they could participate in asthma attacks. To determinate whether activation of CD161(+) T cells and their cytokine production correlate with clinical status of asthma, we analysed blood samples from asthma attack patients (AAP) and stable asthma patients (SAP) in comparison with healthy non-atopic controls (HC). There was a significant higher baseline expression of CD69 on T cells from AAP and the difference was more notorious on CD161(+) T cells; upregulation of CD69 was observed on both CD161(-) and CD161(+) T cells driven by Dermatophagoides pteronyssinus crude extract, whereas polyclonal stimulation with phorbol 12-myristate 13-acetate plus ionomycin predominantly induced IFN-gamma but no IL-4, IL-5 and IL-13 by CD161(+) T cells in all groups; upon polyclonal stimulation, there were more CD161(+) T cells producing IFN-gamma and less CD161(-) T cells producing this cytokine, contrasting with the opposite results observed in SAP and HC groups. Our results indicate that, during asthma attack, CD161(+) T cells are activated and are able to produce predominantly IFN-gamma but no Th2 cytokines. We hypothesize that during an asthma attack, IFN-gamma produced by CD161(+) T cells could help to reestablish the Th1/Th2 equilibrium. These observations may contribute to the understanding of the immune mechanisms involved in asthma attacks.

  4. Influence of the protein kinase C activator phorbol myristate acetate on the intracellular activity of antibiotics against hemin- and menadione-auxotrophic small-colony variant mutants of Staphylococcus aureus and their wild-type parental strain in human THP-1 cells.

    Science.gov (United States)

    Garcia, Laetitia G; Lemaire, Sandrine; Kahl, Barbara C; Becker, Karsten; Proctor, Richard A; Tulkens, Paul M; Van Bambeke, Françoise

    2012-12-01

    In a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700-3711, 2012), we evaluated the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistant Staphylococcus aureus strain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, its hemB mutant, and the genetically complemented strain in PMA-activated cells and against the menD strain in both activated and nonactivated cells. This effect was inhibited when cells were incubated with N-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H(2)O(2). In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition of N-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H(2)O(2). Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

  5. Polyphenol Content and Modulatory Activities of Some Tropical Dietary Plant Extracts on the Oxidant Activities of Neutrophils and Myeloperoxidase

    Directory of Open Access Journals (Sweden)

    Thierry Franck

    2012-01-01

    Full Text Available Young leaves of Manihot esculenta Crantz (Euphorbiaceae, Abelmoschus esculentus (Malvaceae, Hibiscus acetosella (Malvaceae and Pteridium aquilinum (Dennstaedtiaceae are currently consumed as green vegetables by peoples in sub-Saharan Africa, Latin America, Asia and their migrants living in Western Europe. Sub-Saharan peoples use Manihot, Abelmoschus and Hibiscus also in the folk medicine to alleviate fever and pain, in the treatment of conjunctivitis, rheumatism, hemorrhoid, abscesses, ... The present study investigates the effects of aqueous extracts of those plants on the production of reactive oxygen species (ROS and the release of myeloperoxidase (MPO by equine neutrophils activated with phorbol 12-myristate 13-acetate (PMA. The ROS production was measured by lucigenin-enhanced chemiluminescence (CL, and the release of total MPO by an ELISA method. The study also investigates the effect of the extracts on the activity of MPO by studying its nitration activity on tyrosine and by using a new technique called SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection that allows studying the direct interaction of compounds with the enzyme. In all experiments, the aqueous extracts of the plants developed concentration-dependent inhibitory effects. A moderate heat treatment did not significantly modify the inhibitory capacity of the extracts in comparison to not heated ones. Total polyphenol and flavonoid contents were determined with an HPLC-UV/DAD analysis and a spectroscopic method using Folin-Ciocalteu reagent. Some polyphenols with well-known antioxidant activities (caffeic acid, chlorogenic acid, hyperoside, rosmarinic acid and rutin were found in the extracts and may partly explain the inhibitory activities observed. The role of those dietary and medicinal plants in the treatment of ROS-dependent inflammatory diseases could have new considerations for health.

  6. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    Science.gov (United States)

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  7. Activation of p44/42 MAPK plays a role in the TBT-induced loss of human natural killer (NK) cell function.

    Science.gov (United States)

    Dudimah, Fred D; Griffey, Denisha; Wang, Xiaofei; Whalen, Margaret M

    2010-10-01

    Natural killer (NK) cells destroy (lyse) tumor cells, virally infected cells, and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen-activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen Toxicology 209:263-277, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function ((51)Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1-h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1-h exposure to 5 nM PMA caused a sixfold increase in phospho-p44/42 levels. Previous studies showed a fivefold increase in phospho-p44/42 in response to a 1-h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function.

  8. Activation of p44/42 MAPK Plays a Role in the TBT-induced Loss of Human Natural Killer (NK) Cell Function

    Science.gov (United States)

    Dudimah, Fred D.; Griffey, Denisha; Wang, Xiaofei; Whalen, Margaret M.

    2009-01-01

    Natural Killer (NK) cells destroy (lyse) tumor cells, virally infected cells and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as Phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function (51Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1 h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1 h exposure to 5 nM PMA caused a 6 fold increase in phospho-p44/42 levels. Previous studies showed a 5 fold increase in phospho-p44/42 in response to a 1 h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function. PMID:20213532

  9. The dual action of poly(ADP-ribose polymerase -1 (PARP-1 inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

    Directory of Open Access Journals (Sweden)

    Slava eRom

    2015-08-01

    Full Text Available The transcription of HIV-1 (HIV is regulated by complex mechanisms involving various cellular factors and virus-encoded transactivators. Poly(ADP-ribose polymerase 1 (PARP-1 inhibition has emerged recently as a potent anti-inflammatory tool, since PARP-1 is involved in the regulation of some genes through its interaction with various transcription factors. We propose a novel approach to diminish HIV replication via PARP-1 inhibition using human primary monocyte-derived macrophages (MDM as an in vitro model system. PARP-1 inhibitors were able to reduce HIV replication in MDM by 60-80% after 7 days infection. Long Terminal Repeat (LTR acts as a switch in virus replication and can be triggered by several agents such as: Tat, tumor necrosis factor α (TNFα, and phorbol 12-myristate 13-acetate (PMA. Overexpression of Tat in MDM transfected with an LTR reporter plasmid led to a 4.2-fold increase in LTR activation; PARP inhibition resulted in 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%. MDM treated with PARP inhibitors showed 90% reduction in NFκB activity (known to mediate PMA- and TNFα-induced HIV LTR activation. Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These findings suggest that HIV replication in MDM could be suppressed by PARP inhibition via NFκB suppression, diminution of LTR activation and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide a potent approach to treatment of HIV infection.

  10. Cornuside inhibits mast cell-mediated allergic response by down-regulating MAPK and NF-κB signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Li, Liangchang [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Yanbian University, Yanji, 133002 (China); Jin, Guangyu [Yanbian University Hospital, Medicine College, Yanbian University, Yanji, 133000 (China); Jiang, Jingzhi [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Yanbian University, Yanji, 133002 (China); Zheng, Mingyu; Jin, Yan [College of Pharmacy, Yanbian University, Yanji, 133002 (China); Lin, Zhenhua [Department of Pathology & Cancer Research Center, Yanbian University Medical College, Yanji, 133002 (China); Li, Guangzhao [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Yanbian University, Yanji, 133002 (China); Choi, Yunho, E-mail: why76@jbnu.ac.kr [Department of Anatomy, Medical School, Institute for Medical Sciences, Chonbuk National University, Jeonju, 561-756 (Korea, Republic of); Yan, Guanghai, E-mail: ghyan2015@sina.com [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Yanbian University, Yanji, 133002 (China)

    2016-04-29

    Aims: The present study is to investigate the effect of cornuside on mast cell-mediated allergic response, as well as its possible mechanisms of action. Methods: To test the anti-allergic effects of cornuside in vivo, local extravasation was induced by local injection of anti-dinitrophenyl immunoglobulin E (IgE) followed by intravenous antigenic challenge in passive cutaneous anaphylaxis model rats. Mast cell viability was determined using MTT assay. Histamine content from rat peritoneal mast cells was measured by the radioenzymatic method. To investigate the mechanisms by which cornuside affects the reduction of histamine release, the levels of calcium uptake were measured. To examine whether cornuside affects the expression of pro-inflammatory cytokines, Western blotting and ELISA were carried out. Results: Oral administration of cornuside inhibited passive cutaneous anaphylaxis in rats. Presence of cornuside attenuated IgE-induced histamine release from rat peritoneal mast cells. The inhibitory effect of cornuside on histamine release was mediated by the modulation of intracellular calcium. In addition, cornuside decreased phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated production and secretion of pro-inflammatory cytokines such as TNF-α and IL-6 in human mast cells. The inhibitory effect of cornuside on pro-inflammatory cytokines was dependent on nuclear factor-κB and p38 mitogen-activated protein kinase. Conclusions: The present study provides evidence that cornuside inhibits mast cell-derived inflammatory allergic reactions by blocking histamine release and pro-inflammatory cytokine expression. Furthermore, in vivo and in vitro anti-allergic effects of cornuside suggest a possible therapeutic application of this agent in inflammatory allergic diseases.

  11. Cornuside inhibits mast cell-mediated allergic response by down-regulating MAPK and NF-κB signaling pathways

    International Nuclear Information System (INIS)

    Li, Liangchang; Jin, Guangyu; Jiang, Jingzhi; Zheng, Mingyu; Jin, Yan; Lin, Zhenhua; Li, Guangzhao; Choi, Yunho; Yan, Guanghai

    2016-01-01

    Aims: The present study is to investigate the effect of cornuside on mast cell-mediated allergic response, as well as its possible mechanisms of action. Methods: To test the anti-allergic effects of cornuside in vivo, local extravasation was induced by local injection of anti-dinitrophenyl immunoglobulin E (IgE) followed by intravenous antigenic challenge in passive cutaneous anaphylaxis model rats. Mast cell viability was determined using MTT assay. Histamine content from rat peritoneal mast cells was measured by the radioenzymatic method. To investigate the mechanisms by which cornuside affects the reduction of histamine release, the levels of calcium uptake were measured. To examine whether cornuside affects the expression of pro-inflammatory cytokines, Western blotting and ELISA were carried out. Results: Oral administration of cornuside inhibited passive cutaneous anaphylaxis in rats. Presence of cornuside attenuated IgE-induced histamine release from rat peritoneal mast cells. The inhibitory effect of cornuside on histamine release was mediated by the modulation of intracellular calcium. In addition, cornuside decreased phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated production and secretion of pro-inflammatory cytokines such as TNF-α and IL-6 in human mast cells. The inhibitory effect of cornuside on pro-inflammatory cytokines was dependent on nuclear factor-κB and p38 mitogen-activated protein kinase. Conclusions: The present study provides evidence that cornuside inhibits mast cell-derived inflammatory allergic reactions by blocking histamine release and pro-inflammatory cytokine expression. Furthermore, in vivo and in vitro anti-allergic effects of cornuside suggest a possible therapeutic application of this agent in inflammatory allergic diseases.

  12. Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.

    Science.gov (United States)

    Ojima, Yoshihiro; Duncan, Mark Thompson; Nurhayati, Retno Wahyu; Taya, Masahito; Miller, William Martin

    2013-08-15

    The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Differential stimulation of luminol-enhanced chemiluminescence (CL) and arachidonic acid metabolism in rat peritoneal neutrophils

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    Sturm, R.J.; Adams, L.M.; Cullinan, C.A.; Berkenkopf, J.W.; Weichman, B.M.

    1986-03-05

    Phorbol 12-myristate, 13-acetate (PMA) induced the production of radical oxygen species (ROS) from rat peritoneal neutrophils as assessed by CL. ROS generation occurred in a time- (maximum at 13.5 min) and dose- (concentration range of 1.7-498 nM) related fashion. However, 166 nM PMA did not induce either cyclooxygenase (CO) or lipoxygenase (LPO) product formation by 20 min post-stimulation. Conversely, A23187, at concentrations between 0.1 and 10 ..mu..M, stimulated both pathways of arachidonic acid metabolism, but had little or no effect upon ROS production. When suboptimal concentrations of PMA (5.5 nM) and A23187 (0.1-1 ..mu..M) were coincubated with the neutrophils, a synergistic ROS response was elicited. However, arachidonic acid metabolism in the presence of PMA was unchanged relative to A12187 alone. Nordihydroguaiaretic acid (NDGA) inhibited both PMA-induced CL (IC/sub 50/ = 0.9 ..mu..M) and A23187-induced arachidonic acid metabolism (IC/sub 50/ = 1.7 ..mu..M and 6.0 ..mu..M for LPO and CO, respectively). The mixed LPO-CO inhibitor, BW755C, behaved in a qualitatively similar manner to NDGA, whereas the CO inhibitors, indomethacin, piroxicam and naproxen had no inhibitory effect on ROS generation at concentrations as high as 100 ..mu..M. These results suggest that NDGA and BW755C may inhibit CL and arachidonic acid metabolism by distinct mechanisms in rat neutrophils.

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

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

    1990-01-01

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

  15. Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

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    Kikuchi, Hidetomo; Yuan, Bo; Nishimura, Yoshio; Imai, Masahiko; Furutani, Ryota; Kamoi, Saki; Seno, Misako; Fukushima, Shin; Hazama, Shingo; Hirobe, Chieko; Ohyama, Kunio; Hu, Xiao-Mei; Takagi, Norio; Hirano, Toshihiko; Toyoda, Hiroo

    2013-12-01

    We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D₃ (VD₃)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

  16. Curcumin inhibits EMMPRIN and MMP-9 expression through AMPK-MAPK and PKC signaling in PMA induced macrophages.

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    Cao, Jiatian; Han, Zhihua; Tian, Lei; Chen, Kan; Fan, Yuqi; Ye, Bozhi; Huang, Weijian; Wang, Changqian; Huang, Zhouqing

    2014-09-21

    In coronary arteries, plaque disruption, the major acute clinical manifestations of atherosclerosis, leads to a subsequent cardiac event, such as acute myocardial infarction (AMI) and unstable angina pectoris (UA). Numerous reports have shown that high expression of MMP-9 (matrix metalloproteinase-9), MMP-13 (matrix metalloproteinase-13) and EMMPRIN (extracellular matrix metalloproteinase induce) in monocyte/macrophage results in the plaque progression and destabilization. Curcumin exerts well-known anti-inflammatory and antioxidant effects and probably has a protective role in the atherosclerosis. The purpose of our study was to investigate the molecular mechanisms by which curcumin affects MMP-9, MMP13 and EMMPRIN in PMA (phorbol 12-myristate 13-acetate) induced macrophages. Human monocytic cells (THP-1 cells) were pretreated with curcumin or compound C for 1 h, and then induced by PMA for 48 h. Total RNA and proteins were collected for real-time PCR and Western blot analysis, respectively. In the present study, the exposure to curcumin resulted in attenuated JNK, p38, and ERK activation and decreased expression of MMP-9, MMP-13 and EMMPRIN in PMA induced macrophages. Moreover, we demonstrated that AMPK (AMP-activated protein kinase) and PKC (Protein Kinase C) was activated by PMA during monocyte/macrophage differentiation. Furthermore, curcumin reversed PMA stimulated PKC activation and suppressed the chronic activation of AMPK, which in turn reduced the expression of MMP-9, MMP-13 and EMMPRIN. Therefore, it is suggested that curcumin by inhibiting AMPK-MAPK (mitogen activated protein kinase) and PKC pathway may led to down-regulated EMMPRIN, MMP-9 and MMP-13 expression in PMA-induced THP-1 cells.

  17. Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages.

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    Wang, Qi-Ming; Wang, Hao; Li, Ya-Fei; Xie, Zhi-Yong; Ma, Yao; Yan, Jian-Jun; Gao, Yi Fan Wei; Wang, Ze-Mu; Wang, Lian-Sheng

    2016-01-01

    It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer) and MMPs (matrix metalloproteinases) by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG) has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR) has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. We showed that EGCG (10-50µmol/L) significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque. © 2016 The Author(s) Published by S. Karger AG, Basel.

  18. Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages

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    Qi-Ming Wang

    2016-11-01

    Full Text Available Background/Aims: It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer and MMPs (matrix metalloproteinases by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA. Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. Results: We showed that EGCG (10-50µmol/L significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2, p38 and c-Jun N-terminal kinase (JNK in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Conclusion: Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque.

  19. Leukocyte-associated immunoglobulin-like receptor-1 is expressed on human megakaryocytes and negatively regulates the maturation of primary megakaryocytic progenitors and cell line

    International Nuclear Information System (INIS)

    Xue, Jiangnan; Zhang, Xiaoshu; Zhao, Haiya; Fu, Qiang; Cao, Yanning; Wang, Yuesi; Feng, Xiaoying; Fu, Aili

    2011-01-01

    Research highlights: → LAIR-1 is expressed on human megakaryocytes from an early stage. → Up-regulation of LAIR-1 negatively regulates megakaryocytic differentiation of cell line. → LAIR-1 negatively regulates the differentiation of primary megakaryocytic progenitors. -- Abstract: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory collagen receptor which belongs to the immunoglobulin (Ig) superfamily. Although the inhibitory function of LAIR-1 has been extensively described in multiple leukocytes, its role in megakaryocyte (MK) has not been explored so far. Here, we show that LAIR-1 is expressed on human bone marrow CD34 + CD41a + and CD41a + CD42b + cells. LAIR-1 is also detectable in a fraction of human cord blood CD34 + cell-derived MK that has morphological characteristics of immature MK. In megakaryoblastic cell line Dami, the membrane protein expression of LAIR-1 is up-regulated significantly when cells are treated with phorbol ester phorbol 12-myristate 13-acetate (PMA). Furthermore, cross-linking of LAIR-1 in Dami cells with its natural ligand or anti-LAIR-1 antibody leads to the inhibition of cell proliferation and PMA-promoted differentiation when examined by the MK lineage-specific markers (CD41a and CD42b) and polyploidization. In addition, we also observed that cross-linking of LAIR-1 results in decreased MK generation from primary human CD34 + cells cultured in a cytokines cocktail that contains TPO. These results suggest that LAIR-1 is a likely candidate for an early marker of MK differentiation, and provide initial evidence indicating that LAIR-1 serves as a negative regulator of megakaryocytopoiesis.

  20. Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils

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    Paino I.M.M.

    2005-01-01

    Full Text Available The release of reactive oxygen specie (ROS by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM, indomethacin (12 µM, naproxen (160 µM, piroxicam (13 µM, and tenoxicam (30 µM were incubated at 37ºC in PBS (10 mM, pH 7.4, for 30 min with rat neutrophils (1 x 10(6 cells/ml stimulated by phorbol-12-myristate-13-acetate (100 nM. The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2% diclofenac, 90 ± 2% indomethacin, 33 ± 3% piroxicam, and 45 ± 6% tenoxicam (N = 6. For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5% and diclofenac showed amplification in the light emission of 181 ± 60% (N = 6. Using the myeloperoxidase (MPO/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3%, indomethacin (97 ± 2, 100 ± 1%, naproxen (56 ± 8, 76 ± 3%, piroxicam (77 ± 5, 99 ± 1%, and tenoxicam (90 ± 2, 100 ± 1%, respectively (N = 3. These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.

  1. Anti-hyperalgesic activity of the aqueous and methanol extracts of the leaves of Pittosporum mannii Hook on CFA-induced persistent inflammatory pain.

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    Wandji, Bibiane Aimée; Bomba, Francis Desire Tatsinkou; Nkeng-Efouet, Pepin Alango; Piegang, Basile Nganmegne; Kamanyi, Albert; Nguelefack, Télesphore Benoît

    2018-02-01

    Previous study showed that aqueous (AEPM) and methanol (MEPM) extracts from the leaves of Pittosporum mannii have analgesic effects in acute pain models. The present study evaluates the acute and chronic anti-hypernociceptive and anti-inflammatory effects of AEPM and MEPM in a model of persistent inflammatory pain. The third day after induction of inflammatory pain by subplantar injection of 100 µL of CFA in Wistar rats, AEPM and MEPM were administered orally (75, 150 and 300 mg/kg/day) and their anti-hyperalgesic and anti-inflammatory effects were follow in acute (1-24 h) and chronic (for 14 days) treatments. At the end of the chronic treatment, oxidative stress and liver parameters were assessed. Effects of plant extracts were also evaluated on nociception induced by Phorbol 12-Myristate 13-Acetate (PMA) and 8-bromo 3',5'-cAMP (8-Br-cAMP) in mice. AEPM and MEPM significantly reversed the mechanical hyperalgesia caused by CFA in acute and chronic treatment. Moreover, AEPM and MEPM also significantly reduced the nociception caused by PMA (60%) and 8-Br-cAMP (87%). Nevertheless, AEPM and MEPM failed to inhibit the paw edema caused by CFA. Plant extracts significantly reduced the nitric oxide content in the spinal cord and the plasmatic concentration of alanine aminotransferase. MEPM also significantly increased the glutathione content in the spinal cord. AEPM and MEPM given orally are effective in inhibiting mechanical hyperalgesia in persistent inflammatory pain caused by CFA. Their mechanisms of action seem to involve an interaction with PKC, PKA and nitric oxide pathways. These extracts might be devoid of hepatotoxic effects.

  2. Alveolar macrophage phagocytosis is enhanced after blunt chest trauma and alters the posttraumatic mediator release.

    Science.gov (United States)

    Seitz, Daniel H; Palmer, Annette; Niesler, Ulrike; Fröba, Janine S; Heidemann, Vera; Rittlinger, Anne; Braumüller, Sonja T; Zhou, Shaoxia; Gebhard, Florian; Knöferl, Markus W

    2011-12-01

    Blunt chest trauma is known to induce a pulmonary invasion of short-lived polymorphonuclear neutrophils and apoptosis of alveolar epithelial type 2 (AT2) cells. Apoptotic cells are removed by alveolar macrophages (AMΦ). We hypothesized that chest trauma alters the phagocytic response of AMΦ as well as the mediator release of AMΦ during phagocytosis. To study this, male Sprague-Dawley rats were subjected to blunt chest trauma. Phagocytosis assays were performed in AMΦ isolated 2 or 24 h after trauma with apoptotic cells or opsonized beads. Phagocytosis of apoptotic AT2 cells by unstimulated AMΦ was significantly increased 2 h after trauma. At 24 h, AMΦ from traumatized animals, stimulated with phorbol-12-myristate-13-acetate, ingested significantly more apoptotic polymorphonuclear neutrophils than AMΦ from sham animals. Alveolar macrophages after trauma released significantly higher levels of tumor necrosis factor α, macrophage inflammatory protein 1α, and cytokine-induced neutrophil chemoattractant 1 when they incorporated latex beads, but significantly lower levels of interleukin 1β and macrophage inflammatory protein 1α when they ingested apoptotic cells. In vivo, phagocytosis of intratracheally instilled latex beads was decreased in traumatized rats. The bronchoalveolar lavage concentrations of the phagocytosis-supporting surfactant proteins A and D after blunt chest trauma were slightly decreased, whereas surfactant protein D mRNA expression in AT2 cells was significantly increased after 2 h. These findings indicate that chest trauma augments the phagocytosis of apoptotic cells by AMΦ. Phagocytosis of opsonized beads enhances and ingestion of apoptotic cells downregulates the immunologic response following lung contusion. Our data emphasize the important role of phagocytosis during posttraumatic inflammation after lung contusion.

  3. Micronutrient Synergy in the Fight against Hepatocellular Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Roomi, M. Waheed; Roomi, Nusrath W.; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra, E-mail: a.niedz@drrath.com; Rath, Matthias [Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050 (United States)

    2012-03-23

    The incidence of hepatocellular carcinoma (HCC), once thought to be a rare tumor in North America, has rapidly increased in recent years in the United States. Current treatment modalities to halt the progression of this disease are only marginally effective. The mainstay treatment is liver transplantation, which is often confronted with donor shortage. Invasion, metastasis and recurrence contribute to the high mortality rate of this disease. Matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) have been associated with the progression, invasion and metastasis of the disease. We have developed strategies to strengthen the ECM collagen and inhibit MMPs through micronutrients such as lysine, proline and ascorbic acid. Addition of epigallocatechin gallate or green tea extract to these micronutrients synergistically enhanced anti-carcinogenic activity in HepG2 cells. Addition of certain other micronutrients, such as N-acetylcysteine, selenium, copper and zinc (NM) synergistically enhanced the anticancer activity of the mixture in a model of hepatocellular carcinoma using HepG2 cells. In vitro studies using HepG2 demonstrated that NM was very effective in inhibiting cell proliferation (by MTT assay), MMPs secretion (by gelatinase zymography), cell invasion (through Matrigel) and induction of apoptosis (by live green caspase). In addition, NM was shown to down-regulate urokinase plasminogen activator (by fibrin zymography) and up-regulate tissue inhibitors of metalloproteinases (by reverse zymography) in another HCC cell line, SK-Hep-1. MMP-2 and MMP-9 activities were further modulated by phorbol 12-myristate 13-acetate (PMA) induction and inhibited by NM. In previous studies, NM inhibited Sk-Hep-1 xenografts in nude mice and also inhibited hepatic metastasis of B16FO melanoma cells. Our results suggest that NM is an excellent candidate for therapeutic use in the treatment HCC by inhibiting critical parameters in cancer development and progression

  4. Macropinocytosis is responsible for the uptake of pathogenic and non-pathogenic mycobacteria by B lymphocytes (Raji cells

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    García-Pérez Blanca Estela

    2012-10-01

    Full Text Available Abstract Background The classical roles of B cells include the production of antibodies and cytokines and the generation of immunological memory, these being key factors in the adaptive immune response. However, their role in innate immunity is currently being recognised. Traditionally, B cells have been considered non-phagocytic cells; therefore, the uptake of bacteria by B cells is not extensively documented. In this study, we analysed some of the features of non-specific bacterial uptake by B lymphocytes from the Raji cell line. In our model, B cells were infected with Mycobacterium tuberculosis (MTB, Mycobacterium smegmatis (MSM, and Salmonella typhimurium (ST. Results Our observations revealed that the Raji B cells were readily infected by the three bacteria that were studied. All of the infections induced changes in the cellular membrane during bacterial internalisation. M. smegmatis and S. typhimurium were able to induce important membrane changes that were characterised by abundant filopodia and lamellipodia formation. These membrane changes were driven by actin cytoskeletal rearrangements. The intracellular growth of these bacteria was also controlled by B cells. M. tuberculosis infection also induced actin rearrangement-driven membrane changes; however, the B cells were not able to control this infection. The phorbol 12-myristate 13-acetate (PMA treatment of B cells induced filopodia and lamellipodia formation, the production of spacious vacuoles (macropinosomes, and the fluid-phase uptake that is characteristic of macropinocytosis. S. typhimurium infection induced the highest fluid-phase uptake, although both mycobacteria also induced fluid uptake. A macropinocytosis inhibitor such as amiloride was used and abolished the bacterial uptake and the fluid-phase uptake that is triggered during the bacterial infection. Conclusions Raji B cells can internalise S. typhimurium and mycobacteria through an active process, such as

  5. Detoxification of Toxic Phorbol Esters from Malaysian Jatropha curcas Linn. Kernel by Trichoderma spp. and Endophytic Fungi

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    Azhar Najjar

    2014-02-01

    Full Text Available The presence of phorbol esters (PEs with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum JQ350879.1, T. harzianum JQ517493.1, Paecilomyces sinensis JQ350881.1, Cladosporium cladosporioides JQ517491.1, Fusarium chlamydosporum JQ350882.1, F. chlamydosporum JQ517492.1 and F. chlamydosporum JQ350880.1 was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p < 0.05 removed PEs with percentage losses of 96.9%–99.7%, while F. chlamydosporum strains showed percentage losses of 88.9%–92.2%. All fungal strains could utilize the PEs-rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%–96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs.

  6. Curcumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications.

    Science.gov (United States)

    Balasubramanyam, M; Koteswari, A Adaikala; Kumar, R Sampath; Monickaraj, S Finny; Maheswari, J Uma; Mohan, V

    2003-12-01

    There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric, Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca2+ influx suggest that a rise in cytosolic Ca2+ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca2+ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.

  7. The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

    Science.gov (United States)

    Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

    2009-01-01

    Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

  8. Flavonoids targeting of IκB phosphorylation abrogates carcinogen-induced MMP-9 and COX-2 expression in human brain endothelial cells

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    Tahanian E

    2011-05-01

    Full Text Available Elizabeth Tahanian¹, Luis Arguello Sanchez¹, Tze Chieh Shiao², René Roy², Borhane Annabi¹¹Centre de Recherche BioMED, ²Centre de Recherche PharmaQAM, Département de chimie, Université du Québec à Montréal, QC, CanadaAbstract: Brain endothelial cells play an essential role as structural and functional components of the blood–brain barrier (BBB. Increased BBB breakdown and brain injury are associated with neuroinflammation and are thought to trigger mechanisms involving matrix metalloproteinase upregulation. Emerging evidence also indicates that cyclooxygenase (COX inhibition limits BBB disruption, but the mechanisms linking metalloproteinase to COX remain unknown. In this study, we sought to investigate the nuclear factor-kappa B (NF-κB signaling pathway, a common pathway in both the regulation of matrix metalloproteinase-9 (MMP-9 and COX-2 expression, and the inhibitory properties of several chemopreventive flavonoids. Human brain microvascular endothelial cells were treated with a combination of phorbol 12-myristate 13-acetate (PMA, a carcinogen documented to increase MMP-9 and COX-2 through NF-κB, and several naturally occurring flavonoids. Among the molecules tested, we found that fisetin, apigenin, and luteolin specifically and dose-dependently antagonized PMA-induced COX-2 and MMP-9 gene and protein expressions as assessed by qRT-PCR, immunoblotting, and zymography respectively. We further demonstrate that flavonoids impact on IκK-mediated phosphorylation activity as demonstrated by the inhibition of PMA-induced IκB phosphorylation levels. Our results suggest that BBB disruption during neuroinflammation could be pharmacologically reduced by a specific class of flavonoids acting as NF-κB signal transduction inhibitors.Keywords: blood–brain barrier, flavonoids, neuroinflammation, NF-κB signal transduction inhibitors

  9. Effects of extremely low frequency electromagnetic field (ELF-EMF) on catalase, cytochrome P450 and nitric oxide synthase in erythro-leukemic cells.

    Science.gov (United States)

    Patruno, Antonia; Tabrez, Shams; Pesce, Mirko; Shakil, Shazi; Kamal, Mohammad A; Reale, Marcella

    2015-01-15

    Extremely low frequency electromagnetic fields (ELF-EMFs) are widely employed in electrical appliances and different equipment such as television sets, mobile phones, computers and microwaves. The molecular mechanism through which ELF-EMFs can influence cellular behavior is still unclear. A hypothesis is that ELF-EMFs could interfere with chemical reactions involving free radical production. Under physiologic conditions, cells maintain redox balance through production of ROS/RNS and antioxidant molecules. The altered balance between ROS generation and elimination plays a critical role in a variety of pathologic conditions including neurodegenerative diseases, aging and cancer. Actually, there is a disagreement as to whether there is a causal or coincidental relationship between ELF-EMF exposure and leukemia development. Increased ROS levels have been observed in several hematopoietic malignancies including acute and chronic myeloid leukemias. In our study, the effect of ELF-EMF exposure on catalase, cytochrome P450 and inducible nitric oxide synthase activity and their expression by Western blot analysis in myelogenous leukemia cell line K562 was evaluated. A significant modulation of iNOS, CAT and Cyt P450 protein expression was recorded as a result of ELF-EMF exposure in both phorbol 12-myristate 13-acetate (PMA)-stimulated and non-stimulated cell lines. Modulation in kinetic parameters of CAT, CYP-450 and iNOS enzymes in response to ELF-EMF indicates an interaction between the ELF-EMF and the enzymological system. These new insights might be important in establishing a mechanistic framework at the molecular level within which the possible effects of ELF-EMF on health can be understood. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

    Science.gov (United States)

    Almami, Ibtesam; Dickenson, John M; Hargreaves, Alan J; Bonner, Philip L R

    2014-01-01

    BACKGROUND AND PURPOSE Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (β-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2O2-induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases. PMID:24821315

  11. The investigation of minoxidil-induced [Ca2+]i rises and non-Ca2+-triggered cell death in PC3 human prostate cancer cells.

    Science.gov (United States)

    Chen, I-Shu; Chou, Chiang-Ting; Liu, Yuan-Yuarn; Yu, Chia-Cheng; Liang, Wei-Zhe; Kuo, Chun-Chi; Shieh, Pochuen; Kuo, Daih-Huang; Chen, Fu-An; Jan, Chung-Ren

    2017-02-01

    Minoxidil is clinically used to prevent hair loss. However, its effect on Ca 2+ homeostasis in prostate cancer cells is unclear. This study explored the effect of minoxidil on cytosolic-free Ca 2+ levels ([Ca 2+ ] i ) and cell viability in PC3 human prostate cancer cells. Minoxidil at concentrations between 200 and 800 μM evoked [Ca 2+ ] i rises in a concentration-dependent manner. This Ca 2+ signal was inhibited by 60% by removal of extracellular Ca 2+ . Minoxidil-induced Ca 2+ influx was confirmed by Mn 2+ -induced quench of fura-2 fluorescence. Pre-treatment with the protein kinase C (PKC) inhibitor GF109203X, PKC activator phorbol 12-myristate 13 acetate (PMA), nifedipine and SKF96365 inhibited minoxidil-induced Ca 2+ signal in Ca 2+ containing medium by 60%. Treatment with the endoplasmic reticulum Ca 2+ pump inhibitor 2,5-ditert-butylhydroquinone (BHQ) in Ca 2+ -free medium abolished minoxidil-induced [Ca 2+ ] i rises. Conversely, treatment with minoxidil abolished BHQ-induced [Ca 2+ ] i rises. Inhibition of phospholipase C (PLC) with U73122 abolished minoxidil-evoked [Ca 2+ ] i rises. Overnight treatment with minoxidil killed cells at concentrations of 200-600 μM in a concentration-dependent fashion. Chelation of cytosolic Ca 2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent minoxidil's cytotoxicity. Together, in PC3 cells, minoxidil induced [Ca 2+ ] i rises that involved Ca 2+ entry through PKC-regulated store-operated Ca 2+ channels and PLC-dependent Ca 2+ release from the endoplasmic reticulum. Minoxidil-induced cytotoxicity in a Ca 2+ -independent manner.

  12. N,N'-Dimethylthiourea dioxide formation from N,N'-dimethylthiourea reflects hydrogen peroxide concentrations in simple biological systems

    International Nuclear Information System (INIS)

    Curtis, W.E.; Muldrow, M.E.; Parker, N.B.; Barkley, R.; Linas, S.L.; Repine, J.E.

    1988-01-01

    The authors hypothesized that measurement of a specific product from reaction of N,N'-dimethylthiourea (Me 2 TU) and H 2 O 2 would provide a good indication of the H 2 O 2 scavenging and protection seen after addition of Me 2 TU to biological systems. They found that addition of H 2 O 2 to Me 2 TU yielded a single stable product, Me 2 TU dioxide. Me 2 TU dioxide formation correlated with Me 2 TU consumption as a function of added H 2 O 2 concentration and was prevented by simultaneous addition of catalase (but not boiled catalase), superoxide dismutase, dimethyl sulfoxide, mannitol, or sodium benzoate. Me 2 TU dioxide formation, Me 2 TU consumption, and H 2 O 2 concentration increases occurred in mixtures containing phorbol 12-myristate 13-acetate (PMA) and normal human neutrophils but not in mixtures containing PMA and neutrophils from patients with chronic granulomatous disease or in mixtures containing PMA and normal neutrophils and catalase. Me 2 TU dioxide formation also occurred in isolated rat lungs perfused with Me 2 TU and H 2 O 2 but not in lungs perfused with Me 2 TU and elastase, histamine, or oleic acid. In contrast, Me 2 TU dioxide formation did not occur after exposure of Me 2 TU to 60 Co-generated hydroxyl radical or hypochlorous acid in the presence of catalase. The results indicate that reaction of Me 2 TU with H 2 O 2 selectively forms Me 2 TU may be useful for assessing the presence and significance of H 2 O 2 in biological systems

  13. Albizia lebbeck suppresses histamine signaling by the inhibition of histamine H1 receptor and histidine decarboxylase gene transcriptions.

    Science.gov (United States)

    Nurul, Islam Mohammed; Mizuguchi, Hiroyuki; Shahriar, Masum; Venkatesh, Pichairajan; Maeyama, Kazutaka; Mukherjee, Pulok K; Hattori, Masashi; Choudhuri, Mohamed Sahabuddin Kabir; Takeda, Noriaki; Fukui, Hiroyuki

    2011-11-01

    Histamine plays major roles in allergic diseases and its action is mediated mainly by histamine H(1) receptor (H1R). We have demonstrated that histamine signaling-related H1R and histidine decarboxylase (HDC) genes are allergic diseases sensitive genes and their expression level affects severity of the allergic symptoms. Therefore, compounds that suppress histamine signaling should be promising candidates as anti-allergic drugs. Here, we investigated the effect of the extract from the bark of Albizia lebbeck (AL), one of the ingredients of Ayruvedic medicines, on H1R and HDC gene expression using toluene-2,4-diisocyanate (TDI) sensitized allergy model rats and HeLa cells expressing endogenous H1R. Administration of the AL extract significantly decreased the numbers of sneezing and nasal rubbing. Pretreatment with the AL extract suppressed TDI-induced H1R and HDC mRNA elevations as well as [(3)H]mepyramine binding, HDC activity, and histamine content in the nasal mucosa. AL extract also suppressed TDI-induced up-regulation of IL-4, IL-5, and IL-13 mRNA. In HeLa cells, AL extract suppressed phorbol-12-myristate-13-acetate- or histamine-induced up-regulation of H1R mRNA. Our data suggest that AL alleviated nasal symptoms by inhibiting histamine signaling in TDI-sensitized rats through suppression of H1R and HDC gene transcriptions. Suppression of Th2-cytokine signaling by AL also suggests that it could affect the histamine-cytokine network. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. In vivo application of a small molecular weight antifungal protein of Penicillium chrysogenum (PAF)

    Energy Technology Data Exchange (ETDEWEB)

    Palicz, Zoltán; Jenes, Ágnes; Gáll, Tamás [Department of Physiology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Miszti-Blasius, Kornél [Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Kollár, Sándor; Kovács, Ilona [Department of Pathology, Kenézy Hospital LTD, Debrecen (Hungary); Emri, Miklós; Márián, Teréz [Department of Nuclear Medicine, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Leiter, Éva; Pócsi, István [Department of Microbial Biotechnology and Cell Biology, Faculty of Science and Technology, Centre of Arts, Humanities and Sciences, University of Debrecen, Debrecen (Hungary); Csősz, Éva; Kalló, Gergő [Proteomics Core Facility, Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Hegedűs, Csaba; Virág, László [Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Csernoch, László [Department of Physiology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Szentesi, Péter, E-mail: szentesi.peter@med.unideb.hu [Department of Physiology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary)

    2013-05-15

    The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 μg·kg{sup −1} daily, for 2 weeks. Even at the highest concentration – a concentration highly toxic in vitro for all affected molds – used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy. - Highlights: • PAF, the antifungal protein of Penicillium chrysogenum, was not toxic in mice. • Its intranasal application didn't induce pathological reactions in the lung. • PAF retained its antifungal activity in lung extracts. • Its application on the skin did not cause inflammation.

  15. In vivo application of a small molecular weight antifungal protein of Penicillium chrysogenum (PAF)

    International Nuclear Information System (INIS)

    Palicz, Zoltán; Jenes, Ágnes; Gáll, Tamás; Miszti-Blasius, Kornél; Kollár, Sándor; Kovács, Ilona; Emri, Miklós; Márián, Teréz; Leiter, Éva; Pócsi, István; Csősz, Éva; Kalló, Gergő; Hegedűs, Csaba; Virág, László; Csernoch, László; Szentesi, Péter

    2013-01-01

    The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 μg·kg −1 daily, for 2 weeks. Even at the highest concentration – a concentration highly toxic in vitro for all affected molds – used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy. - Highlights: • PAF, the antifungal protein of Penicillium chrysogenum, was not toxic in mice. • Its intranasal application didn't induce pathological reactions in the lung. • PAF retained its antifungal activity in lung extracts. • Its application on the skin did not cause inflammation

  16. The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation*

    Science.gov (United States)

    Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob; Gether, Ulrik; Jørgensen, Trine Nygaard

    2014-01-01

    The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation. PMID:24973209

  17. Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini*

    Science.gov (United States)

    Vuorenpää, Anne; Jørgensen, Trine N.; Newman, Amy H.; Madsen, Kenneth L.; Scheinin, Mika

    2016-01-01

    The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the “long loop” recycling marker Rab11, whereas less overlap was seen with the “short loop” recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements in the intracellular N terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function. PMID:26786096

  18. Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker

    Science.gov (United States)

    Yoshizaki, G.; Patino, R.; Thomas, P.; Bolamba, D.; Chang, Xiaotian

    2001-01-01

    A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown /maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker. ?? 2001 Elsevier Science.

  19. Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

    Science.gov (United States)

    Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

    2010-03-23

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

  20. A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364

    Energy Technology Data Exchange (ETDEWEB)

    Littrell, BobbiJo R [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2018-04-16

    The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364

  1. Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

    Science.gov (United States)

    Pavarotti, Martín; Capmany, Anahí; Vitale, Nicolas; Colombo, María Isabel; Damiani, María Teresa

    2012-02-01

    Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  2. Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

    Directory of Open Access Journals (Sweden)

    Go Kamoshida

    2018-02-01

    Full Text Available Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs, has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA, and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

  3. Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation

    International Nuclear Information System (INIS)

    Moretta, A.; Poggi, A.; Olive, D.; Bottino, C.; Fortis, C.; Pantaleo, G.; Moretta, L.

    1987-01-01

    A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3 + , Ti + , T44 + , T11 + , T40 + ) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of γ-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, negative cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 - Ti - T44 + Leu1 + T11 + T40 + 4F2 + HLA class I + surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3 - Ti - T44 - Leu1 - T11 - T40 - 4F2 - HLA class I + phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate

  4. Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

    International Nuclear Information System (INIS)

    Ishikawa, S.; Okada, K.; Saito, T.

    1988-01-01

    The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ([Ca2+]i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased [Ca2+] significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of [Ca2+]i depended on the intracellular Ca pool, since an AVP-induced rise in [Ca2+]i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased 45 Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells

  5. Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin.

    Science.gov (United States)

    Johnson, Adam S; García, Dana M

    2007-12-19

    Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.

  6. Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin

    Directory of Open Access Journals (Sweden)

    García Dana M

    2007-12-01

    Full Text Available Abstract Background Inside bluegill (Lepomis macrochirus retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. Results Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. Conclusion A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms, our evidence does not support a significant role for PKC.

  7. Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway.

    Science.gov (United States)

    Lim, Jung Hwa; Jung, Cho-Rok; Lee, Chan-Hee; Im, Dong-Soo

    2008-11-01

    E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.

  8. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide

    International Nuclear Information System (INIS)

    Genin, Marie; Clement, Francois; Fattaccioli, Antoine; Raes, Martine; Michiels, Carine

    2015-01-01

    Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis

  9. Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

    Science.gov (United States)

    Yoshimura, T; Mayumi, M; Yorifuji, T; Kim, K M; Heike, T; Miyanomae, T; Shinomiya, K; Mikawa, H

    1987-09-01

    A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

  10. DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line.

    Science.gov (United States)

    Takahashi, S; Maecker, H T; Levy, R

    1989-10-01

    An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

  11. Development and Application of High-Content Biological Screening for Modulators of NET Production

    Directory of Open Access Journals (Sweden)

    Ilaria J. Chicca

    2018-03-01

    Full Text Available Neutrophil extracellular traps (NETs are DNA-based antimicrobial web-like structures whose release is predominantly mediated by reactive oxygen species (ROS; their purpose is to combat infections. However, unbalanced NET production and clearance is involved in tissue injury, circulation of auto-antibodies and development of several chronic diseases. Currently, there is lack of agreement regarding the high-throughput methods available for NET investigation. This study, therefore, aimed to develop and optimize a high-content analysis (HCA approach, which can be applied for the assay of NET production and for the screening of compounds involved in the modulation of NET release. A suitable paraformaldehyde fixation protocol was established to enable HCA of neutrophils and NETs. Bespoke and in-built bioinformatics algorithms were validated by comparison with standard low-throughput approaches for application in HCA of NETs. Subsequently, the optimized protocol was applied to high-content screening (HCS of a pharmaceutically derived compound library to identify modulators of NETosis. Of 56 compounds assessed, 8 were identified from HCS for further characterization of their effects on NET formation as being either inducers, inhibitors or biphasic modulators. The effects of these compounds on naïve neutrophils were evaluated by using specific assays for the induction of ROS and NET production, while their modulatory activity was validated in phorbol 12-myristate 13-acetate-stimulated neutrophils. Results indicated the involvement of glutathione reductase, Src family kinases, molecular-target-of-Rapamycin, and mitogen-activated-protein-kinase pathways in NET release. The compounds and pathways identified may provide targets for novel therapeutic approaches for treating NET-associated pathologies.

  12. Micronutrient Synergy in the Fight against Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Aleksandra Niedzwiecki

    2012-03-01

    Full Text Available The incidence of hepatocellular carcinoma (HCC, once thought to be a rare tumor in North America, has rapidly increased in recent years in the United States. Current treatment modalities to halt the progression of this disease are only marginally effective. The mainstay treatment is liver transplantation, which is often confronted with donor shortage. Invasion, metastasis and recurrence contribute to the high mortality rate of this disease. Matrix metalloproteinases (MMPs that degrade the extracellular matrix (ECM have been associated with the progression, invasion and metastasis of the disease. We have developed strategies to strengthen the ECM collagen and inhibit MMPs through micronutrients such as lysine, proline and ascorbic acid. Addition of epigallocatechin gallate or green tea extract to these micronutrients synergistically enhanced anti-carcinogenic activity in HepG2 cells. Addition of certain other micronutrients, such as N-acetylcysteine, selenium, copper and zinc (NM synergistically enhanced the anticancer activity of the mixture in a model of hepatocellular carcinoma using HepG2 cells. In vitro studies using HepG2 demonstrated that NM was very effective in inhibiting cell proliferation (by MTT assay, MMPs secretion (by gelatinase zymography, cell invasion (through Matrigel and induction of apoptosis (by live green caspase. In addition, NM was shown to down-regulate urokinase plasminogen activator (by fibrin zymography and up-regulate tissue inhibitors of metalloproteinases (by reverse zymography in another HCC cell line, SK-Hep-1. MMP-2 and MMP-9 activities were further modulated by phorbol 12-myristate 13-acetate (PMA induction and inhibited by NM. In previous studies, NM inhibited Sk-Hep-1 xenografts in nude mice and also inhibited hepatic metastasis of B16FO melanoma cells. Our results suggest that NM is an excellent candidate for therapeutic use in the treatment HCC by inhibiting critical parameters in cancer development and

  13. Anti-inflammatory and apoptotic effects of the polyphenol curcumin on human fibroblast-like synoviocytes.

    Science.gov (United States)

    Kloesch, Burkhard; Becker, Tatjana; Dietersdorfer, Elisabeth; Kiener, Hans; Steiner, Guenter

    2013-02-01

    It has recently been reported that the polyphenol curcumin has pronounced anti-carcinogenic, anti-inflammatory and pro-apoptotic properties. This study investigated possible anti-inflammatory and apoptotic effects of curcumin on the human synovial fibroblast cell line MH7A, and on fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA). MH7A cells and RA-FLS were stimulated either with interleukin (IL)-1β or phorbol 12-myristate 13 acetate (PMA), and treated simultaneously or sequentially with increasing concentrations of curcumin. Release of interleukin (IL)-6 and vascular endothelial growth factor (VEGF)-A was quantified by enzyme-linked immunosorbent assays (ELISAs). In MH7A cells, modulation of the transcription factor nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinases (MAPKs) such as p38 and extracellular-signal regulated kinase (ERK1/2) were analysed by a reporter gene assay and Western blot, respectively. Pro-apoptotic events were monitored by Annexin-V/7-AAD based assay. Cleavage of pro-caspase-3 and -7 was checked with specific antibodies. Curcumin effectively blocked IL-1β and PMA-induced IL-6 expression both in MH7A cells and RA-FLS. VEGF-A expression could only be detected in RA-FLS and was induced by PMA, but not by IL-1β. Furthermore, curcumin inhibited activation of NF-κB and induced dephosphorylation of ERK1/2. Treatment of FLS with high concentrations of curcumin was associated with a decrease in cell viability and induction of apoptosis. The natural compound curcumin represents strong anti-inflammatory properties and induces apoptosis in FLS. This study provides an insight into possible molecular mechanisms of this substance and suggests it as a natural remedy for the treatment of chronic inflammatory diseases like RA. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

    Science.gov (United States)

    Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

    1996-09-01

    We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

  15. Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

    Science.gov (United States)

    Salton, S R; Fischberg, D J; Dong, K W

    1991-05-01

    Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

  16. Cortical delta-opioid receptors potentiate K+ homeostasis during anoxia and oxygen-glucose deprivation.

    Science.gov (United States)

    Chao, Dongman; Donnelly, David F; Feng, Yin; Bazzy-Asaad, Alia; Xia, Ying

    2007-02-01

    Central neurons are extremely vulnerable to hypoxic/ischemic insult, which is a major cause of neurologic morbidity and mortality as a consequence of neuronal dysfunction and death. Our recent work has shown that delta-opioid receptor (DOR) is neuroprotective against hypoxic and excitotoxic stress, although the underlying mechanisms remain unclear. Because hypoxia/ischemia disrupts ionic homeostasis with an increase in extracellular K(+), which plays a role in neuronal death, we asked whether DOR activation preserves K(+) homeostasis during hypoxic/ischemic stress. To test this hypothesis, extracellular recordings with K(+)-sensitive microelectrodes were performed in mouse cortical slices under anoxia or oxygen-glucose deprivation (OGD). The main findings in this study are that (1) DOR activation with [D-Ala(2), D-Leu(5)]-enkephalinamide attenuated the anoxia- and OGD-induced increase in extracellular K(+) and decrease in DC potential in cortical slices; (2) DOR inhibition with naltrindole, a DOR antagonist, completely abolished the DOR-mediated prevention of increase in extracellular K(+) and decrease in DC potential; (3) inhibition of protein kinase A (PKA) with N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide dihydrochloride had no effect on the DOR protection; and (4) inhibition of protein kinase C (PKC) with chelerythrine chloride reduced the DOR protection, whereas the PKC activator (phorbol 12-myristate 13-acetate) mimicked the effect of DOR activation on K(+) homeostasis. These data suggest that activation of DOR protects the cortex against anoxia- or ODG-induced derangement of potassium homeostasis, and this protection occurs via a PKC-dependent and PKA-independent pathway. We conclude that an important aspect of DOR-mediated neuroprotection is its early action against derangement of K(+) homeostasis during anoxia or ischemia.

  17. Immunosuppressive effect of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through the inhibition of T-lymphocyte proliferation and IL-2 production

    International Nuclear Information System (INIS)

    Yun, Cheol-Heui; Son, Chang Gue; Jung, Uhee; Han, Seung Hyun

    2006-01-01

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the predominant heterocyclic amine formed in cooked meat and fish and causes cancers in the colon, the mammary glands, and the lymphoid organs. In the present study, we investigated the immunological impact of PhIP using thymocytes isolated from Balb/c mice and a murine thymocyte-derived cell line, EL4. Treatment of the thymocytes with PhIP moderately inhibited T-cell mitogen-induced cell proliferation and interleukin (IL)-2 secretion. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that PhIP attenuated IL-2 mRNA expression in the thymocytes and EL4 cells stimulated with phytohemagglutinin (PHA) plus phorbol 12-myristate 13-acetate (PMA). In vitro transient transfection assay using a reporter gene construct containing IL-2 promoter showed that the decrease in the steady-state IL-2 mRNA level by PhIP is partially due to the attenuation of IL-2 mRNA synthesis at the transcriptional level. Furthermore, an electrophoretic mobility shift assay showed that PhIP inhibited DNA binding activity of nuclear factor for immunoglobulin κ chain in B cells (NF-κB), activator protein-1 (AP-1) and nuclear factor of activated T cells (NF-AT), which are known to be responsible for IL-2 transcriptional activation. Concomitantly, PhIP inhibited the PMA/PHA-induced generation of reactive oxygen species (ROS) involved in activation of the transcription factors. These results suggest that PhIP has potential immunosuppressive effects by inhibiting T-cell proliferation and IL-2 expression through down regulation of ROS generation and thereby inhibiting NF-κB, AP-1 and NF-AT activation

  18. Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

    Science.gov (United States)

    Condie, R; Herring, A; Koh, W S; Lee, M; Kaminski, N E

    1996-05-31

    Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

  19. Differential expression of FAK and Pyk2 in metastatic and non-metastatic EL4 lymphoma cell lines.

    Science.gov (United States)

    Zhang, Zhihong; Knoepp, Stewart M; Ku, Hsun; Sansbury, Heather M; Xie, Yuhuan; Chahal, Manpreet S; Tomlinson, Stephen; Meier, Kathryn E

    2011-08-01

    The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases FAK and Pyk2 in EL4 phenotype were examined, with a particular emphasis on the role of these proteins in metastasis. FAK is expressed only in PMA-resistant (or intermediate phenotype) EL4 cells, correlating with enhanced cell-substrate adherence, while Pyk2 is more highly expressed in non-adherent PMA-sensitive cells. PMA treatment causes modulation of mRNA for FAK (up-regulation) and Pyk2 (down-regulation) in PMA-sensitive but not PMA-resistant EL4 cells. The increase in Pyk2 mRNA is correlated with an increase in Pyk2 protein expression. The roles of FAK in cell phenotype were further explored using transfection and knockdown experiments. The results showed that FAK does not play a major role in modulating PMA-induced Erk activation in EL4 cells. However, the knockdown studies demonstrated that FAK expression is required for proliferation and migration of PMA-resistant cells. In an experimental metastasis model using syngeneic mice, only FAK-expressing (PMA-resistant) EL4 cells form liver tumors. Taken together, these studies suggest that FAK expression promotes metastasis of EL4 lymphoma cells.

  20. Mechanism of phorbol ester-mediated protein kinase C activation in EL4 thymoma cells

    International Nuclear Information System (INIS)

    Huang, F.L.; Arora, P.K.; Hanna, E.E.; Huang, K.P.

    1987-01-01

    Mouse thymoma EL4 cells respond to phorbol 12-myristate 13-acetate (PMA) in interleukin-2 secretion and growth inhibition. A rapid translocation of protein kinase C (PKC) from cytosol to the particulate fraction and followed by proteolytic degradation occur when EL4 cells are incubated with PMA. In the present study the translocated membrane-associated PKC (PP-PKC) was solubilized by buffer containing NP-40 and its behavior on column chromatography, molecular weight, and kinetic properties were compared to the cytosolic PKC (CS-PKC) from untreated cells. From DE-52 cellulose column, CS-PKC could be eluted by buffer containing 0.1 M KCl, whereas PP-PKC was eluted with buffer containing 0.25 M KCl and 0.2% NP-40. On gel filtration the partially purified PP-PKC from DE-52 was separated into two species: a high Mr species, which was a complex of 82KDa PKC, PMA, and lipid as evidenced by immunoblot analysis and labeling with [ 3 H]PMA and [ 3 H]myristic acid, and a 82KDa species, which was free of PMA and lipid. This 82KDa PP-PKC, though similar to the CS-PKC in molecular weight, is distinguishable from the CS-PKC in having lower Ka values for both Ca 2+ and PS and no longer requires diacylglycerol for maximum activation. These results indicate that upon PMA treatment of EL4 cells, the CS-PKC was modified through enhancing the kinase activity and affinity for membrane lipid. The modification results in the translocation and complexing of PKC with membrane lipid and PMA and subsequent degradation

  1. Integrated modulation of phorbol ester-induced Raf activation in EL4 lymphoma cells.

    Science.gov (United States)

    Han, Shujie; Meier, Kathryn E

    2009-05-01

    The EL4 murine lymphoma cell line exists in variant phenotypes that differ with respect to responses to the tumor promoter phorbol 12-myristate 13-acetate (PMA1). Previous work showed that "PMA-sensitive" cells, characterized by a high magnitude of PMA-induced Erk activation, express RasGRP, a phorbol ester receptor that directly activates Ras. In "PMA-resistant" and "intermediate" EL4 cell lines, PMA induces Erk activation to lesser extents, but with a greater response in intermediate cells. In the current study, these cell lines were used to examine mechanisms of Raf-1 modulation. Phospho-specific antibodies were utilized to define patterns and kinetics of Raf-1 phosphorylation on several sites. Further studies showed that Akt is constitutively activated to a greater extent in PMA-resistant than in PMA-sensitive cells, and also to a greater extent in resistant than intermediate cells. Akt negatively regulates Raf-1 activation (Ser259), partially explaining the difference between resistant and intermediate cells. Erk activation exerts negative feedback on Raf-1 (Ser289/296/301), thus resulting in earlier termination of the signal in cells with a higher level of Erk activation. RKIP, a Raf inhibitory protein, is expressed at higher levels in resistant cells than in sensitive or intermediate cells. Knockdown of RKIP increases Erk activation and also negative feedback. In conclusion, this study delineates Raf-1 phosphorylation events occurring in response to PMA in cell lines with different extents of Erk activation. Variations in the levels of expression and activation of multiple signaling proteins work in an integrated fashion to modulate the extent and duration of Erk activation.

  2. Exposure to 4-tert-octylphenol, an environmentally persistent alkylphenol, enhances interleukin-4 production in T cells via NF-AT activation

    International Nuclear Information System (INIS)

    Lee, Mi H.; Kim, Eugene; Kim, Tae S.

    2004-01-01

    4-tert-Octylphenol (OP) is a representative endocrine disruptor that may have adverse effects on human health. The influence of this compound on allergic immune responses remains unclear. In this study, we have examined the effects of OP on production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune responses. OP significantly enhanced IL-4 production in antigen-primed T cells in a dose-dependent manner. Treatment with OP in vivo resulted in significant increase of IL-4 production in T cells and of IgE levels in sera of antigen-primed mice. Furthermore, OP enhanced the activation of IL-4 gene promoter in EL4 T cells transiently transfected with IL-4 promoter/reporter constructs, and the enhancing effect mapped to a region in the IL-4 promoter containing binding sites for nuclear factor of activated T cell (NF-AT). Activation of T cells by phorbol-12-myristate-13-acetate (PMA) resulted in markedly enhanced binding activities to the NF-AT site, which significantly increased upon addition of OP, indicating that the transcription factor NF-AT was involved in the enhancing effect of OP on IL-4 production. The enhancement of IL-4 production by OP was blocked by FK506, a calcineurin inhibitor, but not by the estrogen receptor (ER) antagonist ICI 182 780. FK506 inhibited the NF-AT-DNA binding activity and IL-4 gene promoter activity enhanced by OP in a dose-dependent manner. These findings demonstrate that OP enhances IL-4 production in T cells via the stimulation of calcineurin-dependent NF-AT activation

  3. RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

    Science.gov (United States)

    Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

    2007-01-01

    The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

  4. PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

    International Nuclear Information System (INIS)

    Seo, Kang-Sik; Hwang, Byung-Doo; Kim, Jong-Seok; Park, Ji-Hoon; Song, Kyoung-Sub; Yun, Eun-Jin; Park, Jong-Il; Kweon, Gi Ryang; Yoon, Wan-Hee; Lim, Kyu

    2014-01-01

    Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G 1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. These results suggest that the synergy between PMA and apicularen A is involved by

  5. PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

    Science.gov (United States)

    Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

    2018-02-01

    To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

  6. Human tissue factor: cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    Scarpati, E.M.; Wen, D.; Broze, G.J. Jr.; Miletich, J.P.; Flandermeyer, R.R.; Siegel, N.R.; Sadler, J.E.

    1987-01-01

    A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

  7. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

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    Yi-zhou Ye

    2012-12-01

    Full Text Available Abstract Background Administration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C. However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM, a constituent of HDL, was affected by dihydrotestosterone (DHT. Methods HepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC, phorbol-12-myristate-13-acetate (PMA, blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis. Results Addition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice. Conclusions DHT directly and selectively down-regulated the level of ApoM mRNA and the

  8. The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells

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    Vaclav Janovec

    2018-02-01

    Full Text Available Recent studies have reported that the crosslinking of regulatory receptors (RRs, such as blood dendritic cell antigen 2 (BDCA-2 (CD303 or ILT7 (CD85g, of plasmacytoid dendritic cells (pDCs efficiently suppresses the production of type I interferons (IFN-I, α/β/ω and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9 ligands. The exact mechanism of how this B cell receptor (BCR-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that

  9. Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses.

    Science.gov (United States)

    Goodwin, B J; Moore, J O; Weinberg, J B

    1984-02-01

    Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12-myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5-10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of

  10. Antioxidant Effects of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius) on Multiple Free Radical and Reducing Power Assays, Especially on Different Superoxide Anion Radical Generation Systems.

    Science.gov (United States)

    Sugahara, Shintaro; Ueda, Yuto; Fukuhara, Kumiko; Kamamuta, Yuki; Matsuda, Yasushi; Murata, Tatsuro; Kuroda, Yasuhiro; Kabata, Kiyotaka; Ono, Masateru; Igoshi, Keiji; Yasuda, Shin

    2015-11-01

    Yacon (Smallanthus sonchifolius), a native Andean plant, has been cultivated as a crop and locally used as a traditional folk medicine for the people suffering from diabetes and digestive/renal disorders. However, the medicinal properties of this plant and its processed foods have not been completely established. This study investigates the potent antioxidative effects of herbal tea leaves from yacon in different free radical models and a ferric reducing model. A hot-water extract exhibited the highest yield of total polyphenol and scavenging effect on 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical among four extracts prepared with hot water, methanol, ethanol, and ethylacetate. In addition, a higher reducing power of the hot-water extract was similarly demonstrated among these extracts. Varying concentrations of the hot-water extract resulted in different scavenging activities in four synthetic free radical models: DPPH radical (EC50 28.1 μg/mL), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (EC50 23.7 μg/mL), galvinoxyl radical (EC50 3.06 μg/mL), and chlorpromazine cation radical (EC50 475 μg/mL). The yacon tea-leaf extract further demonstrated superoxide anion (O2(-)) radical scavenging effects in the phenazine methosulfate-NADH-nitroblue tetrazolium (EC50 64.5 μg/mL) and xanthine oxidase assay systems (EC50 20.7 μg/mL). Subsequently, incubating human neutrophilic cells in the presence of the tea-leaf extract could suppress the cellular O2(-) radical generation (IC50 65.7 μg/mL) in a phorbol 12-myristate 13-acetate-activated cell model. These results support yacon tea leaves may be a good source of natural antioxidants for preventing O2(-) radical-mediated disorders. Yacon has been considered to be a potent alternative food source for patients who require a dietary cure in regional area, while the leaf part has been provided and consumed as an herbal tea in local markets. We demonstrated here potent antioxidative effects of the tea

  11. 2',3-dihydroxy-5-methoxybiphenyl suppresses fMLP-induced superoxide anion production and cathepsin G release by targeting the β-subunit of G-protein in human neutrophils.

    Science.gov (United States)

    Liao, Hsiang-Ruei; Chen, Ih-Sheng; Liu, Fu-Chao; Lin, Shinn-Zhi; Tseng, Ching-Ping

    2018-06-15

    This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between β subunit of G-protein (Gβ) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gβ-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC 50 :2.57 ± 0.22 μM), cathepsin G release (IC 50 :18.72 ± 3.76 μM) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gβ-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47 ph ° x and P40 ph ° x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gβ-protein with PLCβ2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47 ph ° x and P40 ph ° x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gβ-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47 ph ° x and p40 phox . Copyright © 2018 Elsevier B.V. All rights reserved.

  12. High Uric Acid Activates the ROS-AMPK Pathway, Impairs CD68 Expression and Inhibits OxLDL-Induced Foam-Cell Formation in a Human Monocytic Cell Line, THP-1

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    Chaohuan Luo

    2016-11-01

    Full Text Available Background/Aims: Hyperuricemia is part of the metabolic-syndrome cluster of abdominal obesity, impaired glucose tolerance, insulin resistance, dyslipidemia, and hypertension. Monocytes/macrophages are critical in the development of metabolic syndrome, including gout, obesity and atherosclerosis. However, how high uric acid (HUA exposure affects monocyte/macrophage function remains unclear. In this study, we investigated the molecular mechanism of HUA exposure in monocytes/macrophages and its impact on oxidized low-density lipoprotein (oxLDL-induced foam-cell formation in a human monocytic cell line, THP-1. Methods: We primed THP-1 cells with phorbol-12-myristate-13-acetate (PMA for differentiation, then exposed cells to HUA and detected the production of reactive oxygen species (ROS and analyzed the level of phospho-AMPKα. THP-1 cells were pre-incubated with Compound C, an AMPK inhibitor, or N-acetyl-L-cysteine (NAC, a ROS scavenger, or HUA before PMA, to assess CD68 expression and phospho-AMPKα level. PMA-primed THP-1 cells were pre-treated with oxLDL before Compound C and HUA treatment. Western blot analysis was used to examine the levels of phospho-AMPKα, CD68, ABCG1, ABCA1, cyclooxygenase-2 (COX-2 and NF-κB (p65. Flow cytometry was used to assess ROS production and CD68 expression in live cells. Oil-red O staining was used to observe oxLDL uptake in cells. Results: HUA treatment increased ROS production in PMA-primed THP-1 cells; NAC blocked HUA-induced oxidative stress. HUA treatment time-dependently increased phospho-AMPKα level in PMA-primed THP-1 cells. The HUA-induced oxidative stress increased phospho-AMPKα levels, which was blocked by NAC. HUA treatment impaired CD68 expression during cell differentiation by activating the AMPK pathway, which was reversed by Compound C treatment. Finally, HUA treatment inhibited oxLDL uptake in the formation of foam cells in THP-1 cells, which was blocked by Compound C treatment. HUA treatment

  13. FC-99 ameliorates sepsis-induced liver dysfunction by modulating monocyte/macrophage differentiation via Let-7a related monocytes apoptosis.

    Science.gov (United States)

    Zhao, Yarong; Zhu, Haiyan; Wang, Haining; Ding, Liang; Xu, Lizhi; Chen, Dai; Shen, Sunan; Hou, Yayi; Dou, Huan

    2018-03-13

    The liver is a vital target for sepsis-related injury, leading to inflammatory pathogenesis, multiple organ dysfunction and high mortality rates. Monocyte-derived macrophage transformations are key events in hepatic inflammation. N 1 -[(4-methoxy)methyl]-4-methyl-1,2-benzenediamine (FC-99) previously displayed therapeutic potential on experimental sepsis. However, the underlying mechanism of this protective effect is still not clear. FC-99 treatment attenuated the liver dysfunction in septic mice that was accompanied with reduced numbers of pro-inflammatory Ly6C hi monocytes in the peripheral blood and CD11b + F4/80 lo monocyte-derived macrophages in the liver. These effects were attributed to the FC-99-induced apoptosis of CD11b + cells. In PMA-differentiated THP-1 cells, FC-99 repressed the expression of CD11b, CD14 and caspase3 and resulted in a high proportion of Annexin V + cells. Moreover, let-7a-5p expression was abrogated upon CLP stimulation in vivo , whereas it was restored by FC-99 treatment. TargetScan analysis and luciferase assays indicated that the anti-apoptotic protein BCL-XL was targeted by let-7a-5p. BCL-XL was inhibited by FC-99 in order to induce monocyte apoptosis, leading to the impaired monocyte-to-macrophage differentiation. Murine acute liver failure was generated by caecal ligation puncture surgery after FC-99 administration; Blood samples and liver tissues were collected to determine the monocyte/macrophage subsets and the induction of apoptosis. Human acute monocytic leukemia cell line (THP-1) cells were pretreated with FC-99 followed by phorbol-12-myristate-13-acetate (PMA) stimulation, in order to induce monocyte-to-macrophage differentiation. The target of FC-99 and the mechanistic analyses were conducted by microarrays, qRT-PCR validation, TargetScan algorithms and a luciferase report assay. FC-99 exhibits potential therapeutic effects on CLP-induced liver dysfunction by restoring let-7a-5p levels.

  14. Convenience versus Biological Significance: Are PMA-Differentiated THP-1 Cells a Reliable Substitute for Blood-Derived Macrophages When Studying in Vitro Polarization?

    Science.gov (United States)

    Tedesco, Serena; De Majo, Federica; Kim, Jieun; Trenti, Annalisa; Trevisi, Lucia; Fadini, Gian Paolo; Bolego, Chiara; Zandstra, Peter W; Cignarella, Andrea; Vitiello, Libero

    2018-01-01

    Human peripheral-blood monocytes are used as an established in vitro system for generating macrophages. For several reasons, monocytic cell lines such as THP-1 have been considered as a possible alternative. In view of their distinct developmental origins and phenotypic attributes, we set out to assess the extent to which human monocyte-derived macrophages (MDMs) and phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells were overlapping across a variety of responses to activating stimuli. Resting (M0) macrophages were polarized toward M1 or M2 phenotypes by 48-h incubation with LPS (1 μg/ml) and IFN-γ (10 ng/ml) or with IL-4 (20 ng/ml) and IL-13 (5 ng/ml), respectively. At the end of stimulation, MDMs displayed more pronounced changes in marker gene expression than THP-1. Upon assaying an array of 41 cytokines, chemokines and growth factors in conditioned media (CM) using the Luminex technology, secretion of 29 out of the 41 proteins was affected by polarized activation. While in 12 of them THP-1 and MDM showed comparable trends, for the remaining 17 proteins their responses to activating stimuli did markedly differ. Quantitative comparison for selected analytes confirmed this pattern. In terms of phenotypic activation markers, measured by flow cytometry, M1 response was similar but the established MDM M2 marker CD163 was undetectable in THP-1 cells. In a beads-based assay, MDM activation did not induce significant changes, whereas M2 activation of THP-1 decreased phagocytic activity compared to M0 and M1. In further biological activity tests, both MDM and THP-1 CM failed to affect proliferation of mouse myogenic progenitors, whereas they both reduced adipogenic differentiation of mouse fibro-adipogenic progenitor cells (M2 to a lesser extent than M1 and M0). Finally, migration of human umbilical vein endothelial cells was enhanced by CM irrespective of cell type and activation state except for M0 CM from MDMs. In summary, PMA-differentiated THP-1

  15. Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression

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    Hai Van Le

    2016-06-01

    Full Text Available Toll-like receptor 10 (TLR10 is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1, lipopolysaccharide (LPS, and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8, Interleukin-1 beta (IL-1β, Tumor necrosis factor-alpha (TNF-α and Chemokine (C–C Motif Ligand 20 (CCL20 expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

  16. Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression.

    Science.gov (United States)

    Le, Hai Van; Kim, Jae Young

    2016-06-01

    Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C-C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

  17. [Acetyl-11-keto-beta-boswellic acid and arsenic trioxide regulate the productions and activities of matrix metalloproteinases in human skin fibroblasts and human leukemia cell line THP-1].

    Science.gov (United States)

    Liang, Ya-hui; Li, Ping; Zhao, Jing-xia; Liu, Xin; Huang, Qi-fu

    2010-11-01

    In order to reveal the treatment mechanism of Chinese medicine with the effect of activating blood and resolving putridity, we selected acetyl-11-keto-beta-boswellic acid (AKBA) and arsenic trioxide (ATO), the main monomeric components of frankincense and arsenolite which are two most commonly used Chinese medicine with effect of activating blood and resolving putridity. We combined AKBA and ATO as a compound, and explored its regulatory role in productions and activities of matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 in human skin fibroblasts (HSFbs) and human acute monocytic leukemia cell line THP-1 in inflammatory state. In order to simulate the inflammatory micro-environment of chronic wounds, we established 3 cell models: HSFb model activated by tumor necrosis factor-alpha (TNF-α), THP-1 cell model activated by phorbol-12-myristate-13-acetate (PMA) and HSFb-THP-1 cell coculture system. AKBA and ATO were cocultured with these cell models. Enzyme-linked immunosorbent assay (ELISA), gelatin zymography assay and reverse transcription-polymerase chain reaction (RT-PCR) were used to test the secretions, activities and mRNA expressions of MMP-1, MMP-2 and MMP-9. In the study of the regulatory mechanism of AKBA and ATO on MMPs, AKBA and ATO were cocultured with the cell models. ELISA was used to test the secretions of TNF-α and interleukin-1beta (IL-β) and Western blot was used to test the phosphorylation levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated proteinkinase (p38MAPK). Compound of AKBA and ATO inhibited MMP-1, MMP-2 and MMP-9 mRNA expressions, secretions and activities respectively in HSFbs and THP-1 cells in inflammatory state (PTHP-1 cells and cell coculture system (PTHP-1 cells (P<0.05, P<0.01). The combined use of AKBA and ATO which in line with the rule of activating blood and resolving putridity inhibits fibroblasts and inflammatory cells in producing MMPs in inflammatory state through inhibiting the

  18. Sp1/3 and NF-1 mediate basal transcription of the human P2X1 gene in megakaryoblastic MEG-01 cells

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    Ennion Steven J

    2006-03-01

    Full Text Available Abstract Background P2X1 receptors play an important role in platelet function as they can induce shape change, granule centralization and are also involved in thrombus formation. As platelets have no nuclei, the level of P2X1 expression depends on transcriptional regulation in megakaryocytes, the platelet precursor cell. Since nothing is known about the molecular mechanisms regulating megakaryocytic P2X1 expression, this study aimed to identify and functionally characterize the P2X1 core promoter utilized in the human megakaryoblastic cell line MEG-01. Results In order to identify cis-acting elements involved in the transcriptional regulation of P2X1 expression, the ability of 4.7 kb P2X1 upstream sequence to drive luciferase reporter gene expression was tested. Low promoter activity was detected in proliferating MEG-01 cells. This activity increased 20-fold after phorbol-12-myristate-13-acetate (PMA induced differentiation. A transcription start site was detected 365 bp upstream of the start codon by primer extension. Deletion analysis of reporter constructs indicated a core promoter located within the region -68 to +149 bp that contained two Sp1 sites (named Sp1a and Sp1b and an NF-1 site. Individual mutations of Sp1b or NF-1 binding sites severely reduced promoter activity whereas triple mutation of Sp1a, Sp1b and NF-1 sites completely abolished promoter activity in both untreated and PMA treated cells. Sp1/3 and NF-1 proteins were shown to bind their respective sites by EMSA and interaction of Sp1/3, NF-1 and TFIIB with the endogenous P2X1 core promoter in MEG-01 cells was demonstrated by chromatin immunoprecipitation. Alignment of P2X1 genes from human, chimp, rat, mouse and dog revealed consensus Sp1a, Sp1b and NF-1 binding sites in equivalent positions thereby demonstrating evolutionary conservation of these functionally important sites. Conclusion This study has identified and characterized the P2X1 promoter utilized in MEG-01 cells and

  19. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Science.gov (United States)

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  20. Protein kinase C δ is activated in mouse ovarian surface epithelial cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    International Nuclear Information System (INIS)

    Williams, Shalmica R.; Son, Deok-Soo; Terranova, Paul F.

    2004-01-01

    Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24 h of treatment, and pre-treatment with (1 μM) of either a general PKC inhibitor (BisI) or PKCδ-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCα, β, ε, τ, λ and RACK1. PKCγ, η, θ and DGKθ were not detected. TCDD (1 nM) increased PKCδ protein approximately eight-fold after 24 h of treatment and this effect was dose-dependent (0.1-100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCδ, from the cytoplasm and the nucleus to the perinuclear area after 2 h of treatment, however, after 24 h of treatment PKCδ was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCδ-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCδ-specific activities increased after 1-2 h of treatment. Then TCDD increased the total PKC activity again after 12 h of treatment, whereas, PKCδ-specific activity resurged at 24 h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCδ protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCδ as an effector molecule for TCDD-mediated biological events in this ovarian

  1. Gateways to clinical trials.

    Science.gov (United States)

    Bayés, M; Rabasseda, X; Prous, J R

    2006-10-01

    -globulin, ivabradine hydrochloride, ixabepilone; LA-419, lacosamide, landiolol, lanthanum carbonate, lidocaine/prilocaine, liposomal cisplatin, lutropin alfa; Matuzumab, MBP(82-98), mecasermin, MGCD-0103, MMR-V, morphine hydrochloride, mycophenolic acid sodium salt; Natalizumab, NCX-4016, neridronic acid, nesiritide, nilotinib, NSC-330507; O6-benzylguanine, olanzapine/fluoxetine hydrochloride, omalizumab; Panitumumab, parathyroid hormone (human recombinant), parecoxib sodium, PEG-filgrastim, peginterferon alfa-2a, peginterferon alfa-2b, pegvisomant, pemetrexed disodium, perospirone hydrochloride, pexelizumab, phorbol 12-myristate 13-acetate, pneumococcal 7-valent conjugate vaccine, posaconazole, pramiconazole, prasugrel, pregabalin, prilocaine; rAAV-GAD65, raclopride, rasagiline mesilate, retapamulin, rosuvastatin calcium, rotigotine, rufinamide; SarCNU, SB-743921, SHL-749, sirolimus-eluting stent, sitaxsentan sodium, sorafenib; TachoSil, tadalafil, talampanel, Taxus, tegaserod maleate, telithromycin, telmisartan/hydrochlorothiazide, temsirolimus, tenatoprazole, teriflunomide, tetrathiomolybdate, ticilimumab, timcodar dimesilate, tipifarnib, tirapazamine, TPI, tramiprosate, trifluridine/TPI, trimethoprim; Ularitide, Urocortin 2; Valdecoxib, valganciclovir hydrochloride, valproate magnesium, valspodar, vardenafil hydrochloride hydrate, vitespen, vofopitant hydrochloride, volociximab, vorinostat; Yttrium 90 (90Y) ibritumomab tiuxetan; Ziprasidone hydrochloride, zotarolimus, zotarolimus-eluting stent.

  2. Rac1 GTPase regulates 11β hydroxysteroid dehydrogenase type 2 and fibrotic remodeling.

    Science.gov (United States)

    Lavall, Daniel; Schuster, Pia; Jacobs, Nadine; Kazakov, Andrey; Böhm, Michael; Laufs, Ulrich

    2017-05-05

    The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression ( r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression ( r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling. © 2017 by The American Society for

  3. Enhanced interleukin-8 production in THP-1 human monocytic cells by lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Baqui, A A; Meiller, T F; Falkler, W A

    1999-10-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-8 (IL-8) plays an important role in macrophage mediated inflammatory processes including exacerbation of periodontal diseases, one of the most common complications in GM-CSF receiving cancer patients. The effect of GM-CSF supplementation on IL-8 production was investigated in a human monocyte cell line THP-1, stimulated with lipopolysaccharide extracted from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. Resting THP-1 cells were treated with lipopolysaccharide (1 microgram/ml) of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) for varying time periods. The production of IL-8 in THP-1 cells was measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). A very low level of the cytokine IL-8 was produced constitutive in THP-1 cells. Starting from 8 h of treatment and afterwards GM-CSF alone significantly increased IL-8 production in THP-1 cells. Lipopolysaccharide (1 microgram/ml) extracts from either F. nucleatum or P. gingivalis amplified IL-8 production 500-800 times in comparison to resting THP-1 cells. When lipopolysaccharide of F. nucleatum or P. gingivalis was supplemented with 50 IU/ml of GM-CSF, there was a statistically significant enhanced production of IL-8 by THP-1 cells after 1 day to 7 days of treatment as compared with lipopolysaccharide treatment alone. GM-CSF (50 IU/ml) also significantly increased IL-8 production from 2-7 days of treatment of THP-1 cells when supplemented with a positive control, phorbol-12-myristate-13 acetate (PMA), as compared to PMA treatment alone. These investigations using the in vitro THP-1 human monocyte cell model indicate that there may be an increase in the response on a cellular level to oral endotoxin following GM-CSF therapy as evidenced by enhanced production of the tissue-reactive inflammatory cytokine, IL-8.

  4. S6K1 is involved in polyploidization through its phosphorylation at Thr421/Ser424.

    Science.gov (United States)

    Ma, Dongchu; Yu, Huiying; Lin, Di; Sun, Yinghui; Liu, Liping; Liu, Yage; Dai, Bing; Chen, Wei; Cao, Jianping

    2009-04-01

    Studies on polyploidization of megakaryocytes have been hampered by the lack of synchronized polyploid megakaryocytes. In this study, a relatively synchronized polyploid cell model was successfully established by employing Dami cells treated with nocodazole. In nocodazole-induced cells, cyclin B expression oscillated normally as in diploid cells and polyploid megakaryocytes. By using the nocodazole-induced Dami cell model, we found that 4E-BP1 and Thr421/Ser424 of ribosomal S6 kinase 1(S6K1) were phosphorylated mostly at M-phase in cytoplasm and oscillated in nocodazole-induced polyploid Dami cells, concomitant with increased expression of p27 and cyclin D3. However, phosphorylation of 4E-BP1 and S6K1 on Thr421/Ser424 was significantly decreased in differentiated Dami cells induced by phorbol 12-myristate 13-acetate (PMA), concomitant with increased expression of cyclin D1 and p21 and cyclin D3. Overexpression of the kinase dead form of S6K1 containing the mutation Lys 100 --> Gln in PMA-induced Dami cells increased ploidy whereas overexpression of rapamycin-resistant form of S6K1 containing the mutations Thr421 --> Glu and Ser424 --> Asp significantly dephosphorylated 4E-BP1 and reduced expression of cyclin D1, cyclin D3, p21 and p27, and slightly decreased the ploidy of PMA-induced Dami cells, compared with treatment with PMA alone. Moreover, overexpression of rapamycin-resistant form of S6K1 significantly reversed polyploidization of nocodazole-induced Dami cells. Furthermore, MAP (a novel compound synthesized recently) partly blocked the phosphorylation of S6K1 on Thr421/Ser424 and decreased the expression of p27 and polyploidization in nocodazole-induced Dami cells. Taken together, these data suggested that S6K1/4E-BP1 pathway may play an important role in polyploidization of megakaryocytes. (c) 2008 Wiley-Liss, Inc.

  5. Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases - Anti-inflammatory Effects of Rutin on Neutrophils -

    Directory of Open Access Journals (Sweden)

    Bahareh Abd Nikfarjam

    2017-03-01

    Full Text Available Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO and myeloperoxidase (MPO. These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor (TNF-α productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI medium, pre-incubated with or without rutin (25 μM for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA. Then, the TNF-α, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA, Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT, and neutrophils were treated with various concentrations of rutin (1 - 100 μM, after which MTT was appended and incubated at 37ºC for 4 hour. Results: Rutin at concentrations up to 100 μM did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and TNF-α productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001. Also, MPO activity was significantly reduced by rutin (P < 0.001. Conclusion: In this in vitro study, rutin had an anti-inflammatory effect

  6. Protein Kinase Cα and P-Type Ca2+ Channel CaV2.1 in Red Blood Cell Calcium Signalling

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    Lisa Wagner-Britz

    2013-06-01

    Full Text Available Background/Aims: Protein kinase Cα (PKCα is activated by an increase in cytosolic Ca2+ in red blood cells (RBCs. Previous work has suggested that PKCα directly stimulates the CaV2.1 channel, whereas other studies revealed that CaV2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca2+ concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA led to two distinct populations of cells with an increase in intracellular Ca2+: a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca2+ elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the CaV2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca2+ entry processes: the first is independent of CaV2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of CaV2.1. Further studies using lysophosphatidic acid (LPA as a stimulation agent have provided additional evidence that PKCα and CaV2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and CaV2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and CaV2.1.

  7. Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts.

    Science.gov (United States)

    Raveh, Avi; Schultz, Pamela J; Aschermann, Lauren; Carpenter, Colleen; Tamayo-Castillo, Giselle; Cao, Shugeng; Clardy, Jon; Neubig, Richard R; Sherman, David H; Sjögren, Benita

    2014-10-01

    Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCβ-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  8. Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

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    Schudt Christian

    2005-07-01

    Full Text Available Abstract Background The sources and measurement of reactive oxygen species (ROS in intact organs are largely unresolved. This may be related to methodological problems associated with the techniques currently employed for ROS detection. Electron spin resonance (ESR with spin trapping is a specific method for ROS detection, and may address some these technical problems. Methods We have established a protocol for the measurement of intravascular ROS release from isolated buffer-perfused and ventilated rabbit and mouse lungs, combining lung perfusion with the spin probe l-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH and ESR spectroscopy. We then employed this technique to characterize hypoxia-dependent ROS release, with specific attention paid to NADPH oxidase-dependent superoxide formation as a possible vasoconstrictor pathway. Results While perfusing lungs with CPH over a range of inspired oxygen concentrations (1–21 %, the rate of CP• formation exhibited an oxygen-dependence, with a minimum at 2.5 % O2. Addition of superoxide dismutase (SOD to the buffer fluid illustrated that a minor proportion of this intravascular ROS leak was attributable to superoxide. Stimulation of the lungs by injection of phorbol-12-myristate-13-acetate (PMA into the pulmonary artery caused a rapid increase in CP• formation, concomitant with pulmonary vasoconstriction. Both the PMA-induced CPH oxidation and the vasoconstrictor response were largely suppressed by SOD. When the PMA challenge was performed at different oxygen concentrations, maximum superoxide liberation and pulmonary vasoconstriction occurred at 5 % O2. Using a NADPH oxidase inhibitor and NADPH-oxidase deficient mice, we illustrated that the PMA-induced superoxide release was attributable to the stimulation of NADPH oxidases. Conclusion The perfusion of isolated lungs with CPH is suitable for detection of intravascular ROS release by ESR spectroscopy. We employed this technique to

  9. Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@med.monash.edu.au [Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Vic. 3800 (Australia); Tochon-Danguy, Natalie; Ian Smith, A. [Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Vic. 3800 (Australia)

    2010-07-23

    Research highlights: {yields} PKC activation increases the trafficking of ECE-1 to the cell surface. {yields} This in turn leads to an increase in the amount of ECE-1 shed. {yields} Only the catalytically active C-terminal region is shed from the cell surface. -- Abstract: This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 {mu}M phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 {+-} 32.3% of control, n = 5). The ECE-1 activity (expressed as {mu}M substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor (CGS35066). The stimulation of cells by PMA (1 {mu}M, 6 h) significantly increased the ECE-1 activity (0.28 {+-} 0.02; n = 3) compared to the control (0.07 {+-} 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 {mu}M for 1 h; 0.10 {+-} 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 {+-} 0.01; n = 3) compared to control (0.08 {+-} 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.

  10. Protein kinase C regulates human pluripotent stem cell self-renewal.

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    Masaki Kinehara

    Full Text Available The self-renewal of human pluripotent stem (hPS cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2 appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells.In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC, GF109203X (GFX, increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β, suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2 synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells.Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK, PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though h

  11. Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

    Science.gov (United States)

    Kinehara, Masaki; Kawamura, Suguru; Tateyama, Daiki; Suga, Mika; Matsumura, Hiroko; Mimura, Sumiyo; Hirayama, Noriko; Hirata, Mitsuhi; Uchio-Yamada, Kozue; Kohara, Arihiro; Yanagihara, Kana; Furue, Miho K.

    2013-01-01

    Background The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. Methodology/Principal Findings In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Conclusions/Significance Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long

  12. Molecular and immunological tools for the evaluation of the cellular immune response in the neotropical monkey Saimiri sciureus, a non-human primate model for malaria research.

    Science.gov (United States)

    Riccio, Evelyn K P; Pratt-Riccio, Lilian R; Bianco-Júnior, Cesare; Sanchez, Violette; Totino, Paulo R R; Carvalho, Leonardo J M; Daniel-Ribeiro, Cláudio Tadeu

    2015-04-18

    The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus. Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences. The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-γ. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-β, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed

  13. An HIV-1 encoded peptide mimics the DNA binding loop of NF-κB and binds thioredoxin with high affinity

    International Nuclear Information System (INIS)

    Su Guoping; Wang Min; Taylor, Ethan Will

    2005-01-01

    Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-κB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-κB via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-κB/Rel family of transcription factors by the putative HIV-1 pro-fs protein

  14. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Directory of Open Access Journals (Sweden)

    Teruhito Yamashita

    Full Text Available Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA, a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the

  15. Enhanced biological activities of gamma-irradiated persimmon leaf extract.

    Science.gov (United States)

    Cho, Byoung-Ok; Nchang Che, Denis; Yin, Hong-Hua; Jang, Seon-Il

    2017-09-01

    The aim of this study was to compare the anti-oxidative and anti-inflammatory activities of gamma-irradiated persimmon leaf extract (GPLE) with those of non-irradiated persimmon leaf extract (PLE). Ethanolic extract of persimmon leaf was exposed to gamma irradiation at a dose of 10 kGy. After gamma irradiation, the color of the extract changed from dark brown to light brown. The anti-oxidative and anti-inflammatory activities of GPLE and PLE were assessed from: total polyphenol and total flavonoid contents; 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay; 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay, and levels of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The total polyphenol contents of GPLE and PLE were determined to be 224.44 ± 1.54 and 197.33 ± 5.81 mg gallic acid equivalents (GAE)/g, respectively, and the total flavonoid contents of GPLE and PLE were 206.27 ± 1.15 and 167.60 ± 2.00 mg quercetin equivalents (QUE)/g, respectively. The anti-oxidant activities of GPLE and PLE as measured by DPPH assays were 338.33 ± 30.19 μg/ml (IC50) and 388.68 ± 8.45 μg/ml (IC50), respectively, and those measured by ABTS assays were 510.49 ± 15.12 μg/ml (IC50) and 731.30 ± 10.63 μg/ml (IC50), respectively. IC50 is the inhibitor concentration that reduces the response by 50%. GPLE strongly inhibited the production of NO, PGE2 and IL-6 compared with PLE in lipopolysaccharide-stimulated RAW264.7 macrophages. Furthermore, GPLE significantly inhibited the production of TNF-α and IL-6 cytokines compared with PLE in phorbol 12-myristate 13-acetate (PMA) plus A23187-stimulated HMC-1 human mast cells. These results indicate that gamma irradiation of PLE can enhance its anti-oxidative and anti-inflammatory activities through elevation of the phenolic contents. Therefore, gamma-irradiated PLE has potential for use in the food and cosmetic

  16. T-cell activation and early gene response in dogs.

    Directory of Open Access Journals (Sweden)

    Sally-Anne Mortlock

    Full Text Available T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR, and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA (5μg/ml, including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2, early growth response 1 (EGR1, growth arrest and DNA damage-inducible gene (GADD45B, phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS, early growth response 2 (EGR2, hemogen (HEMGN, polo-like kinase 2 (PLK2 and polo-like kinase 3 (PLK3. Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in

  17. Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

    Directory of Open Access Journals (Sweden)

    A. Mouithys-Mickalad

    1997-01-01

    Full Text Available Sphingolipids inhibit the activation of the neutrophil (PMN NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR and chemiluminescence (CL, we studied the effects of sphingosine (SPN and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5 × 10-7M stimulated PMN (6 × 106 cells. By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide, we showed that SPN (5 to 8 × 10-6M, C2-ceramide (N-acetyl SPN and C6-ceramide (N-hexanoyl SPN at the final concentration of 2 × 10-5 and 2 × 10-4M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5 × 10-6M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8 × 10-6M has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA. SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2 × 10-6 to 10-5M, but N-hexanoyl SPN was less active (from 2 × 10-5 to 2 × 10-4M. These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.

  18. Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation.

    Science.gov (United States)

    Gou, Xiangbo; Wang, Wenying; Zou, Sihao; Qi, Yajuan; Xu, Yanfang

    2018-03-01

    The slowly activating delayed rectifier K + current (I Ks ) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates I Ks through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of I Ks by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record I Ks in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited I Ks in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1-2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human I Ks in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCβ) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on I Ks and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism

  19. Low-dose gamma-rays and simulated solar particle event protons modify splenocyte gene and cytokine expression patterns

    International Nuclear Information System (INIS)

    Rizvi, A.; Pecaut, M.J.; Gridley, D.S.

    2011-01-01

    The goal was to investigate the T helper (Th) response in splenocytes of mice exposed to low-dose/low-dose-rate (LDR) γ-rays, simulated solar particle event protons (sSPE), or combination of both. C57BL/6 mice were exposed to LDR γ-radiation ( 57 Co) to a total dose of 0.05 Gray (Gy) at 0.024 cGy/h, either with or without subsequent exposure to 2 Gy sSPE protons. Expression of genes related to Th cells was evaluated immediately after exposure (day 0). On day 21, intra- and extracellular cytokine production was assessed after activation with anti-CD3 monoclonal antibodies (mAb) or phorbol 12-myristate 13-acetate/ionophore (PMA/I). Five genes were significantly modulated on day 0 in one or more of the irradiated groups compared to controls (p<0.05): Ccl11, Ccr5, Cd80, Inha, and Il9. On day 21, numbers of cells positive for interferon-γ were high in the LDR + sSPE group versus 0 Gy and LDR γ-rays (p<0.05), but there was no difference in interleukin (IL)-2 and tumor necrosis factor (TNF)-α. Levels of secreted cytokines after anti-CD3 mAb activation were high for 5 (maximum intensity projection (MIP)-1α, GM-CSF, interferon (IFN)-γ, TNF-α, IL-13) and low for 2 (IL-7, IL-9) in all irradiated groups. Priming with LDR photons had a significant effect on IFN-γ and IL-17 compared to sSPE protons alone; IL-2 was low only in the LDR + sSPE group. The cytokine patterns after anti-phorbol myristate acetate (PMA)/ionomycin (I) activation were different compared to anti-CD3 mAb and with fewer differences among groups. The data show that total-body exposure to space-relevant radiation has profound effects on Th cell status and that priming with LDR γ-rays can in some cases modulate the response to sSPE. (author)

  20. ChpK and MazF of the toxin-antitoxin modules are involved in the virulence of Leptospira interrogans during infection.

    Science.gov (United States)

    Komi, Komi Koukoura; Ge, Yu-Mei; Xin, Xiao-Yang; Ojcius, David M; Sun, Dexter; Hu, Wei-Lin; Zhao, Xin; Lin, Xu'ai; Yan, Jie

    2015-01-01

    Pathogenic Leptospira species are the causative agents of leptospirosis, a global zoonotic infectious disease. Toxin-antitoxin (TA) modules have been confirmed as stress-response elements that induce prokaryotic and eukaryotic cell-growth arrest or death, but their role in the virulence of Leptospira has not been reported. Here, we confirmed that all the tested leptospiral strains had the chpIK and mazEF TA modules with highly-conserved sequences. The transcription and expression of the chpI, chpK, mazE, and mazF genes of Leptospira interrogans strain Lai were significantly increased during infection of phorbol 12-myristate 13-acetate-induced human THP-1 macrophages. The toxic ChpK and MazF but not the antitoxic ChpI and MazE proteins were detectable in the cytoplasmic fraction of leptospire-infected THP-1 cells, indicating the external secretion of ChpK and MazF during infection. Transfection of the chpK or mazF gene caused decreased viability and necrosis in THP-1 cells, whereas the chpI or mazE gene transfection did not affect the viability of THP-1 cells but blocked the ChpK or MazF-induced toxicity. Deletion of the chpK or mazF gene also decreased the late-apoptotic and/or necrotic ratios of THP-1 cells at the late stages of infection. The recombinant protein MazF (rMazF) cleaved the RNAs but not the DNAs from Leptospira and THP-1 cells, and this RNA cleavage was blocked by rMazE. However, the rChpK had no RNA or DNA-degrading activity. All these findings indicate that the ChpK and MazF proteins in TA modules are involved in the virulence of L. interrogans during infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  1. Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

    Science.gov (United States)

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  2. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

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    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  3. Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

    Science.gov (United States)

    Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

    2001-01-01

    Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

  4. Regulation of Ca2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca2+ channels.

    Science.gov (United States)

    Sena, C M; Santos, R M; Boarder, M R; Rosário, L M

    1999-02-05

    Phorbol esters reduce depolarization-evoked Ca2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K+-evoked rises in cytosolic free Ca2+ concentration ([Ca2+]i) and the early component of the depolarization-evoked Mn2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1-1 microM) inhibited the early and late phases of the ionomycin (2 microM)-evoked [Ca2+]i transients by 14-23%. Omega-agatoxin IVA, a blocker of P/Q-type Ca2+ channels, inhibited high K+-evoked [Ca2+]i rises in a dose-dependent fashion (IC50 = 50 nM). In contrast, 0.1 microM omega-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (< 45%) component of early Ca2+ influx persisted in the combined presence of omega-agatoxin IVA (100 nM) and nitrendipine (1 microM). Simultaneous exposure to omega-agatoxin IVA and PMA inhibited both the early [Ca2+]i transients and Mn2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca2+]i transients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of protein kinase C inhibits preferentially L-type VSCCs over P/Q type channels in adrenal chromaffin cells. However, the possibility cannot be ruled out that dihydropyridine-resistant, non-P/Q type channels might also be negatively regulated by protein kinase C. This may represent an important pathway for the specific control of VSCCs by protein kinase C

  5. Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

    Science.gov (United States)

    Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

    2005-03-01

    In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of

  6. Formation of neutrophil extracellular traps under low oxygen level

    Directory of Open Access Journals (Sweden)

    Katja Branitzki-Heinemann

    2016-11-01

    Full Text Available Since their discovery, neutrophil extracellular traps (NETs have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia inducible factor 1α (HIF-1α by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21% oxygen level and compared to hypoxic (1% conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA or S. aureus wildtype and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was

  7. Characterization of αβ and γδ T cell subsets expressing IL-17A in ruminants and swine.

    Science.gov (United States)

    Elnaggar, Mahmoud M; Abdellrazeq, Gaber S; Dassanayake, Rohana P; Fry, Lindsay M; Hulubei, Victoria; Davis, William C

    2018-08-01

    As part of our ongoing program to expand immunological reagents available for research in cattle, we developed a monoclonal antibody (mAb) to bovine interleukin-17A (IL-17A), a multifunctional cytokine centrally involved in regulating innate and adaptive immune responses. Initial comparative studies demonstrated the mAb recognizes a conserved epitope expressed on orthologues of IL-17A in sheep, goats and pigs. Comparative flow cytometric analyses of lymphocyte subsets stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin revealed differences in expression of IL-17A by CD4, CD8, and γδ T cells across ruminants and swine species. Results in cattle showed the largest proportion of IL-17A + cells were CD4 + followed by γδ and CD8 + T cells. Further analysis revealed the IL-17A + γδ T cell subset was comprised of WC1.1 + , WC1.2 + , and WC1 - subsets. Analysis of the IL-17A + CD8 + T cell subset revealed it was comprised of αβ and γδ T cell subsets. Results in sheep and goats revealed IL-17A is expressed mainly by CD4 + and CD8 + T cells, with little expression by γδ T cells. Analysis of IL-17A + CD8 + T cells showed the majority were CD8 + αβ in sheep, whereas they were CD8 + γδ in goats. The majority of the sheep and goat IL-17A + γδ T cells were WC1 + . Results obtained in swine showed expression of IL-17A by CD4, CD8, and γδ T cell subsets were similar to results reported in other studies. Comparison of expression of IL-17A with IFN-γ revealed subsets co-expressed IL-17A and IFN-γ in cattle, sheep, and goats. The new mAb expands opportunities for immunology research in ruminants and swine. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Four CISH paralogues are present in rainbow trout Oncorhynchus mykiss: differential expression and modulation during immune responses and development.

    Science.gov (United States)

    Maehr, Tanja; Vecino, Jose L González; Wadsworth, Simon; Wang, Tiehui; Secombes, Christopher J

    2014-11-01

    Suppressor of cytokine signalling (SOCS) family members are crucial in the control and attenuation of cytokine induced responses via activation of the JAK/STAT, TLR and NF-kB signalling pathways. SOCS proteins orchestrate the termination of many types of immune responses and are often the targets of microbial pathogens exploiting SOCS mechanisms to evade the host's immune response. Through whole and lineage specific genome duplication events, the teleost cytokine/SOCS network is complex. Not only are the orthologues of all mammalian SOCS members present, namely cytokine inducible Src homology 2 (SH2)-containing protein (CISH) and SOCS-1 to -7, but multiple gene copies exist that may potentially become functionally divergent. In this paper we focus on the CISH genes in rainbow trout (Oncorhynchus mykiss), and have cloned two further paralogues, CISHa2 and CISHb2, additional to the known CISHa1 and CISHb1 genes. We present for the first time a comparative expression analysis of these four paralogues, to establish whether subfunctionalisation is apparent. In vivo examination of gene expression revealed a higher constitutive expression level of CISHa paralogues compared to CISHb expression in adult trout tissues. All CISHs were relatively highly abundant in immune tissues but CISHa2 and CISHb2 had highest expression in the heart and muscle. An inverse picture of CISH abundance during trout ontogeny was seen, and further hints at differential roles of the four genes in immune regulation and development. Stimulation of head kidney (HK) leukocytes with trout recombinant interleukin (rIL)-15 and rIL-21 had a major effect on CISHa2 and to a lesser extent CISHa1 expression. In HK macrophages rIL-1β, phytohemagglutinin, and phorbol 12-myristate 13-acetate also had a strong impact on CISHa2 expression. Yersinia ruckeri infection caused a temporally and spatially differential onset of CISH expression that may be viewed in the context of pathogen evasion strategies. These data

  9. In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes.

    Science.gov (United States)

    Vermeer, Joop E M; van Wijk, Ringo; Goedhart, Joachim; Geldner, Niko; Chory, Joanne; Gadella, Theodorus W J; Munnik, Teun

    2017-07-01

    Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP-C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP-C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    International Nuclear Information System (INIS)

    Bencherif, M.; Lukas, R.J.

    1991-01-01

    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate [ 3 H-QNB]. The rank order potency of selective antagonists that inhibit specific 3 HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2[2-[(diethylamino)methyl]-1-[piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of 3 HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA

  11. Orexin-A potentiates L-type calcium/barium currents in rat retinal ganglion cells.

    Science.gov (United States)

    Liu, F; Weng, S-J; Yang, X-L; Zhong, Y-M

    2015-10-01

    Two neuropeptides, orexin-A and orexin-B (also called hypocretin-1 and -2), have been implicated in sleep/wake regulation, feeding behaviors via the activation of two subtypes of G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). While the expression of orexins and orexin receptors is immunohistochemically revealed in retinal neurons, the function of these peptides in the retina is largely unknown. Using whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that orexin-A increased L-type-like barium currents (IBa,L) in ganglion cells (GCs), and the effect was blocked by the selective OX1R antagonist SB334867, but not by the OX2R antagonist TCS OX2 29. The orexin-A effect was abolished by intracellular dialysis of GDP-β-S/GPAnt-2A, a Gq protein inhibitor, suggesting the mediation of Gq. Additionally, during internal dialysis of the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor U73122, orexin-A did not change the IBa,L of GCs, whereas the orexin-A effect persisted in the presence of the phosphatidylcholine (PC)-PLC inhibitor D609. The orexin-A-induced potentiation was not seen with internal infusion of Ca(2+)-free solution or when inositol 1,4,5-trisphosphate (IP3)-sensitive Ca(2+) release from intracellular stores was blocked by heparin/xestospongins-C. Moreover, the orexin-A effect was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, but was eliminated when PKC was inhibited by bisindolylmaleimide IV (Bis-IV)/Gö6976. Neither adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) nor guanosine 3',5'-cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway was likely involved, as orexin-A persisted to potentiate the IBa,L of GCs no matter these two pathways were activated or inhibited. These results suggest that, by activating OX1R, orexin-A potentiates the IBa,L of rat GCs through a distinct Gq/PI-PLC/IP3/Ca(2+)/PKC signaling pathway. Copyright

  12. Regulatory cells, cytokine pattern and clinical risk factors for asthma in infants and young children with recurrent wheeze.

    Science.gov (United States)

    Borrego, L M; Arroz, M J; Videira, P; Martins, C; Guimarães, H; Nunes, G; Papoila, A L; Trindade, H

    2009-08-01

    Several risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. This study was designed to assess and compare the immunological status during the first 2 years in steroid-naive young children with >or= three episodes of physician-confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age-matched healthy controls (n=30). Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) T cells and their cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN-gamma, TGF-beta and IL-10), CTLA-4 and Foxp3 mRNA expression were evaluated (real-time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12-myristate 13-acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). Flow cytometry results showed a significant reduction in the absolute number of CD4(+)CD25(high) and the absolute and percentage numbers of CD4(+)CD25(+)CTLA-4(+) in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4(+)CD25(+) (P=0.01) and CD4(+)CD25(high) (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA-4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA-4 (P=0.03) and IFN-gamma (P=0.04) expression was diminished in wheezy children compared with healthy children. High-risk children had lower expression of IFN-gamma (P=0.03) compared with low-risk and healthy children and lower expression of CTLA-4 (P=0.01) compared with healthy

  13. Anti-TNF-alpha antibody attenuates subarachnoid hemorrhage-induced apoptosis in the hypothalamus by inhibiting the activation of Erk

    Directory of Open Access Journals (Sweden)

    Ma L

    2018-02-01

    Full Text Available Ling Ma,1 Yong Jiang,2 Yanan Dong,2 Jun Gao,2 Bin Du,2 Dianwei Liu2 1Department of Clinical Laboratory, The Second Hospital of Shandong University, Jinan, Shandong, People’s Republic of China; 2Department of Neurosurgery, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, People’s Republic of China Background: Subarachnoid hemorrhage (SAH can induce apoptosis in many regions of the brain including the cortex and hippocampus. However, few studies have focused on apoptosis in the hypothalamus after SAH. Although some antiapoptotic strategies have been developed for SAH, such as anti-tumor necrosis factor-alpha (TNF-α antibody, the molecular mechanisms underlying this condition have yet to be elucidated. Therefore, the purpose of this study was to evaluate whether SAH could induce apoptosis in the hypothalamus and identify the potential molecular mechanisms underlying the actions of anti-TNF-α antibody, as a therapeutic regimen, upon apoptosis. Materials and methods: SAH was induced in a rat model. Thirty minutes prior to SAH, anti-TNF-α antibody or U0126, an extracellular signal-regulated kinase (Erk inhibitor, was microinjected into the left lateral cerebral ventricle. In addition, phorbol-12-myristate-13-acetate was injected intraperitoneally immediately after the anti-TNF-α antibody microinjection. Then, real-time polymerase chain reaction, Western blotting and immunohistochemistry were used to detect the expression of caspase-3, bax, bcl-2, phosphorylated Erk (p-Erk and Erk. Finally, anxiety-like behavior was identified by using open field. Results: Levels of caspase-3, bax and bcl-2, all showed a temporary rise after SAH in the hypothalamus, indicating the induction of apoptosis in this brain region. Interestingly, we found that the microinjection of anti-TNF-α antibody could selectively block the elevated levels of bax, suggesting the potential role of anti-TNF-α antibody in the inhibition of SAH

  14. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    Energy Technology Data Exchange (ETDEWEB)

    Bencherif, M.; Lukas, R.J. (Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (USA))

    1991-06-01

    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.

  15. Characterization of Microvesicles Released from Human Red Blood Cells

    Directory of Open Access Journals (Sweden)

    Duc Bach Nguyen

    2016-03-01

    Full Text Available Background/Aims: Extracellular vesicles (EVs are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS, scanning electron microscopy (SEM, atomic force microscopy (AFM and dynamic light scattering (DLS. The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control, lysophosphatidic acid (LPA, or phorbol-12 myristate-13 acetate (PMA in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 m

  16. Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

    Directory of Open Access Journals (Sweden)

    Massimo Conese

    Full Text Available In seeking more specific biomarkers of the cystic fibrosis (CF lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, hydrogen voltage-gated channel 1 (HVCN1, and β-arrestin 1 (ARRB1. The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils

  17. Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

    Science.gov (United States)

    Conese, Massimo; Castellani, Stefano; Lepore, Silvia; Palumbo, Orazio; Manca, Antonio; Santostasi, Teresa; Polizzi, Angela Maria; Copetti, Massimiliano; Di Gioia, Sante; Casavola, Valeria; Guerra, Lorenzo; Diana, Anna; Montemurro, Pasqualina; Mariggiò, Maria Addolorata; Gallo, Crescenzio; Maffione, Angela Bruna; Carella, Massimo

    2014-01-01

    In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and

  18. T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity

    Science.gov (United States)

    Bradley, Jillian H.; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P.; Gregg, Randal K.

    2017-11-01

    Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When

  19. Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction.

    Science.gov (United States)

    De Lorenzo, Silvana; Veggetti, Mariela; Muchnik, Salomón; Losavio, Adriana

    2004-05-01

    1. At the mouse neuromuscular junction, adenosine (AD) and the A(1) agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A(1) AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L- and N-type VDCCs), we measured the miniature end-plate potential (mepp) frequency in mouse diaphragm muscles. 2. Blockade of VDCCs by Cd(2+) prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not omega-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3. As A(1) receptors are coupled to a G(i/o) protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4. The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester epsilon6TDelta-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca(2+)-calmodulin. 5. To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K(+) concentrations. The effect of CCPA on ACh secretion evoked by 10 mm K(+) was prevented by the P/Q-type VDCC antagonist omega-agatoxin IVA. 6. CCPA failed to

  20. T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity.

    Science.gov (United States)

    Bradley, Jillian H; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P; Gregg, Randal K

    2017-11-01

    Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter

  1. Liberação de peróxido de hidrogênio por fagócitos de glândulas mamárias bovinas hígidas e infectadas Hydrogen peroxide release by phagocytes from healthy and infected bovine mammary gland

    Directory of Open Access Journals (Sweden)

    Fernanda Alves Brasil

    2012-04-01

    O2 release by phagocytes from infected quarters, as well as, a negative correlation between the H2O2 release by phagocytes and SCC (r=-0.34; P=0.0025 and percentage of neutrophils (r=-0.24; P=0.04. Furthermore, it was observed a tendency toward a lower H2O2 release by phagocytes stimulated by phorbol 12-myristate 13-acetate in the infected quarters. However, a higher H2O2 release by phagocytes was observed in milk samples from infected quarters by the estimation of the H2O2 release by phagocytes in 1mL of milk according to SCC mL-1 of each sample. The present study showed a decrease in H2O2 release by the infected quarters which indicate lower microbicidal activity. However, the higher H2O2 release by phagocytes in 1mL of milk in the infected quarters may contribute to recruitment of leukocytes to the site of infection and the persistence of infection.

  2. A systematic review of p53 regulation of oxidative stress in skeletal muscle.

    Science.gov (United States)

    Beyfuss, Kaitlyn; Hood, David A

    2018-12-01

    transcription factor 4; ATM: ATM serine/threonine kinase; Bax: BCL2 associated X, apoptosis regulator; Bcl-2: B cell Leukemia/Lymphoma 2 apoptosis regulator; Bhlhe40: basic helix-loop-helix family member e40; BH3: Borane; Bim: bcl-2 interacting mediator of cell death; Bok: Bcl-2 related ovarian killer; COX-IV: cytochrome c oxidase IV; cGMP: Cyclic guanosine monophosphate; c-myc: proto-oncogene protein; Cpt1b: carnitine palmitoyltransferase 1B; Dr5: death receptor 5; eNOS: endothelial nitric oxide synthase; ERK: extracellular regulated MAP kinase; Fas: Fas Cell surface death receptor; FDXR: Ferredoxin Reductase; FOXO3a: forkhead box O3; Gadd45a: growth arrest and DNA damage-inducible 45 alpha; GLS2: glutaminase 2; GLUT 1 and 4: glucose transporter 1(endothelial) and 4 (skeletal muscle); GSH: Glutathione; Hes1: hes family bHLH transcription factor 1; Hey1: hes related family bHLH transcription factor with YRPW motif 1; HIFI-α: hypoxia-inducible factor 1, α-subunit; HK2: Hexokinase 2; HSP70: Heat Shock Protein 70; H 2 O 2 : Hydrogen Peroxide; Id2: inhibitor of DNA-binding 2; IGF-1-BP3: Insulin-like growth factor binding protein 3; IL-1β: Interleukin 1 beta; iNOS: inducible nitric oxide synthase; IRS-1: Insulin receptor substrate 1; JNK: c-Jun N-terminal kinases; LY-83583: 6-anilino-5,8-quinolinedione; inhibitor of soluble guanylate cyclase and of cGMP production; Mdm 2/ 4: Mouse double minute 2 homolog (mouse) Mdm4 (humans); mtDNA: mitochondrial DNA; MURF1: Muscle RING-finger protein-1; MyoD: Myogenic differentiation 1; MyoG: myogenin; Nanog: Nanog homeobox; NF-kB: Nuclear factor-κB; NO: nitric oxide; NoxA: phorbol-12-myristate-13-acetate-induced protein 1 (Pmaip1); NRF-1: nuclear respiratory factor 1; Nrf2: Nuclear factor erythroid 2-related factor 2; P21: Cdkn1a cyclin-dependent kinase inhibitor 1A (P21); P38 MAPK: mitogen-activated protein kinases; p53R2: p53 inducible ribonucleotide reductase gene; P66Shc: src homology 2 domain-containing transforming protein C1; PERP: p

  3. Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in Vivo

    Science.gov (United States)

    Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Steven W. Newell; richard W. Hemingway; Vittorio Bottari; Jean-Pierre Perchellet

    1995-01-01

    m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce ODC activity at least as much as 3 ug of 12-O-tetradecanoylphorbol-13-acetate (TPA). ODC induction peaks...

  4. Protective effects of black rice bran against chemically-induced inflammation of mouse skin

    Science.gov (United States)

    We investigated the inhibitory effects of black rice (cv. LK1-3-6-12-1-1) bran against 12-O-tetradecanolylphorbol-13-acetate (TPA)-induced skin edema and 2,4-dinitroflurobenzene (DNFB)-induced allergic contact dermatitis (ACD) in inflammatory mouse models. We also determined the effects of the bran...

  5. Characterization of the tumor-promoting activity of m-chloroperoxybenzoic acid in SENCAR mouse skin and its inhibition by gallotannin, oligomeric proanthocyanidin, and their monomeric units

    Science.gov (United States)

    Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Fatima K. Johnson; Amy W. Davis; Steven W. Newell; Richard W. Hemingway; Vittorio Bottari; Jean-Pierre Perchellett

    1996-01-01

    m-Chloroperoxybenzoic acid (CPBA). Which induces ornithine decarboxylase activity as much as 12-0- terradecanoyIp horbol-13-acetate (TPA ). was tested for its ability to induce DNA synthesis. bydroperoxide (HPx) production. and tumor promotion in mouse epidermis in vivo. After an early inhibition. CPBA stimulates DNA synthesis. A response which is maintained between 16...

  6. Effects of protease inhibitors on radiation transformation in vitro

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Little, J.B.

    1981-01-01

    We have investigated the effects of three protease inhibitors, antipain, leupeptin, and soybean trypsin inhibitor, on the induction of oncogenic transformation in mouse C3H10T 1/2 cells by X-rays. The patterns of inhibition by the three protease inhibitors were different. Antipain was the most effective, having the ability to suppress completely radiation transformation as well as radiation transformation enhanced by the phorbol ester promoting agent 12-O-tetradecanoylphorbol-13-acetate. The fact that antipain could suppress transformation when present for only 1 day following irradiation suggests that an effect on a DNA repair process might be important in its action. Leupeptin was less effective than antipain in its inhibition of radiation transformation. Soybean trypsin inhibitor suppressed only the promotional effects of 12-O-tetradecanoylphorbol-13-acetate on transformation. Our results suggest that there may be more than one protease involved in carcinogenesis

  7. Micronucleus formation in cultured human keratinocytes: Involvement of intercellular bioactivation.

    Science.gov (United States)

    van Pelt, F N; Haring, R M; Weterings, P J

    1991-01-01

    Micronucleus formation in cultured human keratinocytes was studied after exposure to benzo[a]pyrene, cyclophosphamide and 12-O-tetradecanoylphorbol-13-acetate without the addition of an exogenous metabolizing system. The first two agents need bioactivation by specific isoenzymes of cytochrome P-450 to form genotoxic intermediates. Benzo[a]pyrene induced the micronucleus formation in both uninduced and Aroclor 1254-pretreated cultures. Clastogenic effects of cyclophosphamide were observed only in Aroclor 1254-pretreated cells. The tumour promotor 12-O-tetradecanoylphorbol-13-acetate did not affect the frequency of micronuclei in human keratinocytes. The data indicate that cultured human keratinocytes can be used to study the tissue-specific response to genotoxic agents as well as interindividual variation in biotransformation capacity.

  8. Functional cooperativity between two TPA responsive elements in undifferentiated F9 embryonic stem cells.

    OpenAIRE

    Okuda, A; Imagawa, M; Sakai, M; Muramatsu, M

    1990-01-01

    We have recently identified an enhancer, termed GPEI, in the 5'-flanking region of the rat glutathione transferase P gene, that is composed of two imperfect TPA (phorbol 12-O-tetradecanoate 13-acetate) responsive elements (TREs). Unlike other TRE-containing enhancers, GPEI exhibits a strong transcriptional enhancing activity in F9 embryonic stem cells. Mutational analyses have revealed that the high activity of GPEI is mediated by two imperfect TREs. Each TRE-like sequence has no activity by ...

  9. Chemical and Molecular Biological Aspects of Alkylhydrazine-Induced Carcinogenesis in Human Cells in Vitro. Revised

    Science.gov (United States)

    1984-04-01

    DMH) and the metabolite methylazoxymethanol acetate ( MAMA ) have been shown to induce cancer in vivo in several species of rodents producing a variety of...The Pharmaceutical and Toxicological Research Institute (PTRI) I Co-Director, Developmental Chemotherapeutics, OSU Comprehensive Cancer Center...NNL[Methyl-l 4 C] I -dimethylhydrazine) of high specific activity Chapter III - Synthesis of (14 C] -labeled methylazoxymethanol 13 acetate ( MAMA ) of

  10. Tumor-promoting phorbol ester amplifies the inductions of tyrosine aminotransferase and ornithine decarboxylase by glucocorticoid

    International Nuclear Information System (INIS)

    Kido, H.; Fukusen, N.; Katunuma, N.

    1987-01-01

    In adrenalectomized rats, the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the inductions of tyrosine aminotransferase (TAT) and ornithine decarboxylase by glucocorticoids, even with sufficient concentration of glucocorticoids to have a maximal effect, whereas it had no effect on TAT activity and increased ornithine decarboxylase activity only slightly in the absence of glucocorticoids. Phorbol derivatives and components of TPA such as 4β-phorbol, phorbol 12-tetradecanoate, phorbol 13-acetate, and 4-O-methylphorbol 12-tetradecanoate 13-acetate, which have no tumor-promoting activity or ability to activate protein kinase C, did not have any effect on TAT induction by glucocorticoid. TPA enhanced the induction of TAT by various glucocorticoids but had no effect on induction of TAT by glucagon or insulin and did not enhance the induction of glucose-6-phosphate dehydrogenase by 17β-estradiol. These results suggest that TPA specifically enhances the induction of TAT and ornithine decarboxylase by glucocorticoids. Similar effects of TPA on TAT induction by glucocorticoid were observed in primary cultures of adult rat hepatocytes. Another activator of protein kinase C, rac-1,2-dioctanoylglycerol, was also found to have similar effects on the cells

  11. Moderate intake of myristic acid in sn-2 position has beneficial lipidic effects and enhances DHA of cholesteryl esters in an interventional study.

    Science.gov (United States)

    Dabadie, Henry; Peuchant, Evelyne; Bernard, Mireille; LeRuyet, Pascale; Mendy, François

    2005-06-01

    Among the saturated fatty acids (SFA), myristic acid is known to be one of the most atherogenic when consumed at high levels. Our purpose was to compare the effects of two moderate intakes of myristic acid on plasma lipids in an interventional study. Twenty-five male monks without dyslipidemia were given two isocaloric diets for 5 weeks each. In diet 1, 30% of the calories came from fat (8% SFA, 0.6% myristic acid) and provided 200 mg cholesterol/day. Calories of diet 2 were 34% fat (11% SFA, 1.2% myristic acid) with the same levels of oleate, linoleate, alpha-linolenate and cholesterol. A baseline diet was provided before each diet. In comparison with baseline, diets 1 and 2 induced a decrease in total cholesterol, LDL-cholesterol and triglycerides (Pdiet 2 than after diet 1 whereas HDL-cholesterol was higher (Pdiet 2 vs. baseline (Pdiet 1 (Pdiets were associated with an increase in alpha-linolenate of cholesteryl esters (Pdiet 2 was associated with an increase in DHA of cholesteryl esters (Pdiet 2, myristic acid intake was positively correlated with myristic acid of phospholipids, and alpha-linolenic acid intake was correlated with alpha-linolenic acid of cholesteryl esters. Moderate intake (1.2% of total calories) of myristic acid has beneficial lipidic effects and enhances DHA of cholesteryl esters.

  12. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

    OpenAIRE

    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-01-01

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lac...

  13. Antitumor-promoting activity of scopadulcic acid B, isolated from the medicinal plant Scoparia dulcis L.

    Science.gov (United States)

    Nishino, H; Hayashi, T; Arisawa, M; Satomi, Y; Iwashima, A

    1993-01-01

    Scopadulcic acid B (SDB), a tetracyclic diterpenoid isolated from a medicinal plant, Scoparia dulcis L., inhibited the effects of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro and in vivo; SDB inhibited TPA-enhanced phospholipid synthesis in cultured cells, and also suppressed the promoting effect of TPA on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene. The potency of SDB proved to be stronger than that of other natural antitumor-promoting terpenoids, such as glycyrrhetinic acid.

  14. Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Heikkilae, J.E.; Scott, J.G.; Suominen, L.A.; Akerman, K.E.O.

    1987-01-01

    Muscarinic receptor-linked increases in intracellular free Ca 2+ as measured with quin-2 and Ca 2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca 2+ and stimulation of Ca 2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3 H-NMS, by a non-labelled agonist was studied

  15. Inhibitory effect of Epstein-Barr virus activation by Citrus fruits, a cancer chemopreventor.

    Science.gov (United States)

    Iwase, Y; Takemura, Y; Ju-ichi, M; Kawaii, S; Yano, M; Okuda, Y; Mukainaka, T; Tsuruta, A; Okuda, M; Takayasu, J; Tokuda, H; Nishino, H

    1999-05-24

    To search useful compounds in Citrus fruit for cancer chemoprevention, we carried out a primary screening of extracts of fruit peels and seeds from 78 species of the genus Citrus and those from two Fortunella and one Poncirus species, which were closely related to the genus Citrus. These Citrus extracts inhibited the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) as a useful screening method for anti-tumor promoters. Our results indicated that Citrus containing substances may be inhibit susceptibility factors involved in the events leading to the development of cancer.

  16. Inhibitory effect of flavonoids from citrus plants on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors.

    Science.gov (United States)

    Iwase, Y; Takemura, Y; Ju-ichi, M; Ito, C; Furukawa, H; Kawaii, S; Yano, M; Mou, X Y; Takayasu, J; Tokuda, H; Nishino, H

    2000-06-01

    To search for possible anti-tumor promoters, thirteen flavones (1-13) obtained from the peel of Citrus plants were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation by a short-term in vitro assay. Of these flavones, 3,5,6,7,8,3',4'-heptamethoxyflavone (HPT) (13) exhibited significant inhibitory effects on the EBV-EA activation induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA). Further, compound 13 exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

  17. Glutarimide alkaloids and a terpenoid benzoquinone from Cordia globifera.

    Science.gov (United States)

    Parks, Joshua; Gyeltshen, Thinley; Prachyawarakorn, Vilailak; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

    2010-05-28

    Three new compounds, a meroterpene (2) having a cyclopropane moiety named globiferane and glutarimide alkaloids named cordiarimides A (3) and B (4), were isolated from the roots of Cordia globifera. Compounds 2-4 exhibited weak cytotoxic activity. Cordiarimide B (4) exhibited radical scavenging activity, as it inhibited superoxide anion radical formation in the xanthine/xanthine oxidase (XXO) assay, and also suppressed superoxide anion generation in differentiated HL-60 human promyelocytic leukemia cells when induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). This is the first report on the presence of glutarimide alkaloids in the genus Cordia.

  18. Effect of the activity of the Brazilian polyherbal formulation: Eucalyptus globulus Labill, Peltodon radicans Pohl and Schinus terebinthifolius Radd in inflammatory models

    Directory of Open Access Journals (Sweden)

    Karina C.P. Medeiros

    Full Text Available The Brazilian polyherbal formulation (BPF is composed by dyes of Eucalyptus globulus Labill, Peltodon radicans Pohl and Schinus terebinthifolius Raddi in alcohol at 13.3° GL. The formulation is popularly used in Paraíba state, Brazil since 1889 and it is used as an antiseptic and anti-inflammatory medicine. The aim of this study was to evaluate the anti-inflammatory property of the polyherbal formulation. For this purpose it was used the12-O-tetradecanoylphorbol 13-acetate (TPA and capsaicin-induced mouse ear edema and the carrageenan-induced rat paw edema. The BPF at dose of 26 mL/Kg inhibited both 12-O-tetradecanoylphorbol 13-acetate (TPA and capsaicin-induced ear edema by 49% (p < 0.05 and 24% (p < 0.01 respectively. Preliminary results on carrageenan-induced rat paw edema demonstrated that oral administration also inhibited the paw edema by approximately 29%. The results demonstrate that the Brazilian polyherbal formulation has anti-inflammatory activity and the better dose was the one used by the population.

  19. Retinoic acid modulation of ultraviolet light-induced epidermal ornithine decarboxylase activity

    International Nuclear Information System (INIS)

    Lowe, N.J.; Breeding, J.

    1982-01-01

    Irradiation of skin with ultraviolet light of sunburn range (UVB) leads to a large and rapid induction of the polyamine biosynthetic enzyme ornithine decarboxylase in the epidermis. Induction of epidermal ornithine decarboxylase also occurs following application of the tumor promoting agent 12-0-tetradecanoylphorbol-13 acetate and topical retinoic acid is able to block both this ornithine decarboxylase induction and skin tumor promotion. In the studies described below, topical application of retinoic acid to hairless mouse skin leads to a significant inhibition of UVB-induced epidermal ornithine decarboxylase activity. The degree of this inhibition was dependent on the dose, timing, and frequency of the application of retinoic acid. To show significant inhibition of UVB-induced ornithine decarboxylase the retinoic acid had to be applied within 5 hr of UVB irradiation. If retinoic acid treatment was delayed beyond 7 hr following UVB, then no inhibition of UVB-induced ornithine decarboxylase was observed. The quantities of retinoic acid used (1.7 nmol and 3.4 nmol) have been shown effective at inhibiting 12-0-tetradecanoyl phorbol-13 acetate induced ornithine decarboxylase. The results show that these concentrations of topical retinoic acid applied either before or immediately following UVB irradiation reduces the UVB induction of epidermal ornithine decarboxylase. The effect of retinoic acid in these regimens on UVB-induced skin carcinogenesis is currently under study

  20. Effect of tumor promoters on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kunz, B.A.; Hannan, M.A.; Haynes, R.H.

    1980-01-01

    Recently, it has been suggested that mitotic recombination is involved in tumor promotion. On this basis, one might expect tumor promoters to be recombinagenic. D7 is a diploid strain of yeast in which both mutation and mitotic recombination can be measured. We have used this strain to assay the known tumor promoters, iodacetate, anthralin, and 12-0-tetradecanoylphorbol-13-acetate, and the cocarcinogen, catechol, for mutagenicity, recombinagenicity, and the ability to enhance ultraviolet light (UV)-induced genetic events. In the absence of preirradiation with UV, iodoacetate was found to be recombinagenic whereas catechol was mutagenic; however, in both cases, the effects were small. Iodoacetate, anthralin, and catechol potentiated UV-induced mitotic crossing-over, aberrant colony formation, and mutation, while catechol also increased UV-induced gene conversion. We were unable to detect any mutagenic or recombinagenic effect of 12-0-tetradecanoyl-phorbol-13-acetate in either whole cells or spheroplasts. Our results do not indicate any consistent correlation between tumor-promoting activity and the ability of an agent to induce mitotic recombination in yeast. However, the ability to potentiate UV-induced mutation and mitotic recombination may reflect the cocarcinogenic activity of certain promoters

  1. Tumor promoter induced membrane-bound protein kinase C - its influence on hematogenous metastasis

    International Nuclear Information System (INIS)

    Gopalakrishna, R.; Barsky, S.H.

    1987-01-01

    A correlation between the amount of membrane-bound detergent-extractable protein kinase C activity in various B16 melanoma sublines (F10, F1, BL6) and their lung metastasizing abilities following intravenous injection was found. The F10 subline which exhibits higher metastasizing ability was found to have higher membrane-bound protein kinase C compared to the lower metastasizing subline, F1. Treatment of F1 cells with 100 nM 12-0 tetradecanoylphorbol-13-acetate (TPA) for 1h resulted in 90% decrease in protein kinase C activity in the cytosol with a concommitent increase in membrane-bound activity. These TPA-treated cells when injected intravenously in C57BL/6 mice produced 6-fold increase in pulmonary metastases compared to untreated F1 cells. However, biologically inactive analogues 4 α-phorbol 12,13-didecanoate and phorbol 13-acetate had no effect on either membrane-bound protein kinase C activity or pulmonary metastases. Treating F1 cells with the second-stage tumor promoter, mezerin, resulted in increase in both membrane association of protein kinase C and also lung metastases. Thus, these results strongly suggests that membrane associated protein kinase C activity influences hematogenous metastasis of these melanoma cells

  2. [Antiinflammatory activity of extracts and fractions obtained from Physalis peruviana L. calyces].

    Science.gov (United States)

    Franco, Luis A; Matiz, Germán E; Calle, Jairo; Pinzón, Roberto; Ospina, Luis F

    2007-03-01

    Cape gooseberry calyces (Physalis peruviana) have been used in folk medicine for their medicinal properties including anticancer, antimycobacterial, antipyretic, diuretic, immunomodulatory and antiinflammatory properties. The antiinflammatory effect was evaluated for extracts and fractions obtained from Physalis peruviana calyces in a mice model of acute inflammation. The fractions responsible for antiinflammatory activity were extracted for possible identification. The Physalis peruviana calyces were extracted by percolation with organic solvents. The primary hydroalcoholic fraction was purified by column chromatography. The antiinflammatory effect of extracts and fractions was evaluated using the 12-O-tetradecanoylphorbol-13-acetate-induced mouse model of ear edema. Thirty-eight secondary fractions were obtained by column chromatography of primary hydroalcoholic fraction. Six fractions, evaluated in 12-O-tetradecanoylphorbol-13-acetate-induced inflammation assay, showed significant antiinflammatory activity (pPhysalis peruviana calyces was confirmed and validated its use in folk medicine. Fractions responsible for the antiinflammatory action were identified and seem promising for phytomedicinal development. Further studies are needed to isolate and identify the active constituents of these fractions as well as to ascertain the mechanisms involved in the antiinflammatory effect.

  3. Time Dependent Production of Cytokines and Eicosanoids by Human Monocytic Leukaemia U937 Cells; Effects of Glucocorticosteroids

    Directory of Open Access Journals (Sweden)

    Ingrid M. Garrelds

    1999-01-01

    Full Text Available In the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cytokines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol-12 myristate acetate (PMA for 24h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 μg ml-1 and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10-11, 10-9 and 10-7 M for 96h. The production of interleukin- 1β (IL-1β, interleukin-6 (IL-6, prostaglandin E2 (PGE2 and thromboxane B2 (TxB2 gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-α reached its maximum between 6 and 12 h. Interferon-gamma (IFN-γ, interleukin-10 (IL-10 and leukotriene B4 (LTB4 were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone completely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells.

  4. Monoglycerides and fatty acids from Ibervillea sonorae root: isolation and hypoglycemic activity.

    Science.gov (United States)

    Hernández-Galicia, Erica; Calzada, Fernando; Roman-Ramos, Rubén; Alarcón-Aguilar, Francisco J

    2007-03-01

    Eleven monoglycerides (MG), 1-monopalmitin (1), glyceryl 1-monomargarate (2), 1-monostearin (3), glyceryl 1-monononadecylate ( 4), glyceryl 1-monoarachidate (5), glyceryl 1-monobehenate (6), glyceryl 1-monotricosanoate (7), glyceryl 1-monotetracosanoate (8), glyceryl 1-monopentacosanoate (9), glyceryl 1-monohexacosanoate (10) and glyceryl 1-monooctacosanoate (11), together with five fatty acids (FA), lauric acid (12), myristic acid (13), pentadecanoic acid (14), palmitic acid (15) and stearic acid (16) were isolated of the root of IBERVILLEA SONORAE Greene (Cucurbitaceae). Their structures were determined by spectroscopic and chemical methods as well as GC-MS analysis. The hypoglycemic activity of the dichloromethane (DCM) extract, of fractions (F1-F10 and SF1-SF5), of monoglycerides (MG) and of fatty acids (FA) mixtures obtained of the root from I. SONORAE was evaluated in normoglycemic and alloxan-induced diabetic mice. The results showed that by intraperitoneal administration the DCM extract (300 mg/kg), F9 (300 mg/kg) and SF1 (150 mg/kg) significantly reduced glucose levels in both models. For fraction SF1, the hypoglycemic activity was more pronounced than that of tolbutamide (150 mg/kg) used as control. However, neither MG (75 mg/kg) nor FA (75 mg/kg) mixtures isolated from SF1 exhibited a significant hypoglycemic effect. However, when MG and FA were combined in equal proportions (75 mg: 75 mg/kg), their effect was comparable to that of SF1. The observed activity for the DCM extract, F9, SF1 and the MG-FA mixture provides additional support for the popular use of this plant in the treatment of diabetes mellitus in Mexican traditional medicine.

  5. The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway

    Directory of Open Access Journals (Sweden)

    Roberts-Thomson Sarah J

    2006-07-01

    Full Text Available Abstract Background The vanilloid receptor 1 (TRPV1 is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described. Results In the present studies we investigated the hypothesis that the μ opioid receptor (MOP agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+ responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine. Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1. Conclusion The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.

  6. Chemopreventive effects of a curcumin-like diarylpentanoid [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] in cellular targets of rheumatoid arthritis in vitro.

    Science.gov (United States)

    Lee, Ka-Heng; Abas, Faridah; Mohamed Alitheen, Noorjahan Banu; Shaari, Khozirah; Lajis, Nordin Haji; Israf, Daud Ahmad; Syahida, Ahmad

    2015-07-01

    Synovial fibroblast has emerged as a potential cellular target in progressive joint destruction in rheumatoid arthritis development. In this study, BDMC33 (2,6-bis[2,5-dimethoxybenzylidene]cyclohexanone), a curcumin analogue with enhanced anti-inflammatory activity has been synthesized and the potency of BDMC33 on molecular and cellular basis of synovial fibroblasts (SF) were evaluated in vitro. Synovial fibroblast cells (HIG-82) were cultured in vitro and induced by phorbol-12-myristate acetate (PMA) to stimulate the expression of matrix metalloproteinase (MMPs) and pro-inflammatory cytokines. The protective effects of BDMC33 were evaluated toward MMP activities, pro-inflammatory cytokine expression and nuclear factor kappa-B (NF-κB) activation by using various bioassay methods, including zymography, Western blotting, reverse transcription polymerase chain reaction, immunofluorescense microscopy and electrophoretic mobility shift assay. The results showed that BDMC33 significantly inhibited the pro-gelatinase B (pro-MMP-9) and collagenase activities via suppression of MMP-1 in activated SF. In addition, BDMC33 strongly suppressed MMP-3 gene expression as well as inhibited COX-2 and IL-6 pro-inflammatory gene expression. We also demonstrated that BDMC33 abolished the p65 NF-κB nuclear translocation and NF-κB DNA binding activity in PMA-stimulated SF. BDMC33 represents an effective chemopreventive agent and could be used as a promising lead compound for further development of rheumatoid arthritis therapeutic intervention. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  7. Antitubercular activity and inhibitory effect on Epstein-Barr virus activation of sterols and polyisoprenepolyols from an edible mushroom, Hypsizigus marmoreus.

    Science.gov (United States)

    Akihisa, Toshihiro; Franzblau, Scott Gary; Tokuda, Harukuni; Tagata, Masaaki; Ukiya, Motohiko; Matsuzawa, Tsunetomo; Metori, Koichi; Kimura, Yumiko; Suzuki, Takashi; Yasukawa, Ken

    2005-06-01

    Seven sterols (1-7) and eight polyisoprenepolyols (8-15), isolated from the non-saponifiable lipid fraction of the dichloromethane extract of an edible mushroom, Hypsizigus marmoreus (Buna-shimeji), were tested for their antitubercular activity against Mycobacterium tuberculosis strain H37Rv using the Microplate Alamar Blue Assay (MABA). Six sterols (2-7) and two polyisoprenepolyols (8, 12) showed a minimum inhibitory concentration (MIC) in the range of 1-51 microg/ml, while the others (1, 9-11, 13-15) were inactive (MIC>128 microg/ml). The seven sterols (1-7) and three polyisoprenepolyols (8, 10, 12) were further evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Sterols 6 and 7 showed potent inhibitory effects while preserving the high viability of Raji cells.

  8. Codon 61 mutations in the c-Harvey-ras gene in mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene plus okadaic acid class tumor promoters.

    Science.gov (United States)

    Fujiki, H; Suganuma, M; Yoshizawa, S; Kanazawa, H; Sugimura, T; Manam, S; Kahn, S M; Jiang, W; Hoshina, S; Weinstein, I B

    1989-01-01

    Three okadaic acid class tumor promoters, okadaic acid, dinophysistoxin-1, and calyculin A, have potent tumor-promoting activity in two-stage carcinogenesis experiments on mouse skin. DNA isolated from tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and each of these tumor promoters revealed the same mutation at the second nucleotide of codon 61 (CAA----CTA) in the c-Ha-ras gene, determined by the polymerase chain reaction procedure and DNA sequencing. Three potent 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, TPA, teleocidin, and aplysiatoxin, showed the same effects. These results provide strong evidence that this mutation in the c-Ha-ras gene is due to a direct effect of DMBA rather than a selective effect of specific tumor promoters.

  9. Quantification of an Epstein-Barr virus-associated membrane antigen component

    International Nuclear Information System (INIS)

    North, J.R.; Morgan, A.J.; Thompson, J.L.; Epstein, M.A.

    1982-01-01

    A method is described for the preparation of a 125 I-labelled membrane antigen (MA) component (gp340) from B95-8 cell membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Good yields of antigenic material were obtained when renaturation of the [ 125 I]gp340 was carried out by removal of SDS in the presence of urea and subsequent removal of the urea. The availability of purified, radiolabelled gp340 has provided the essential basis for the development of a radioimmunoassay which, for the first time, permits quantification of this antigen. The assay has been used to demonstrate that cell membrane MA is a better source of gp340 for large-scale work than is the Epstein-Barr virus envelope and to measure the increase in expression of gp340 following treatment of cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). (Auth.)

  10. Topical anti-inflammatory activity of Calea prunifolia HBK (Asteraceae) in the TPA model of mouse ear inflammation

    International Nuclear Information System (INIS)

    Gomez, Milton; Gil, Juan F.

    2011-01-01

    Phytochemical study of Calea prunifolia HBK identified two compounds derived from p-hydroxyacetophenone, the 1-(2-hydroxy-5-(1-methoxyethyl)phenyl)-3-methylbut-2.en-1-one showed a satisfactory anti-inflammatory activity (58.33%), when considering that this is a natural product. Although the two derived compounds are structurally similar, the anti-inflammatory activity of 1-(2-hydroxy-5-methoxyphenyl)-3-methylbut-2-en-1-one was not significant (2.08%). The test was conducted in a model of inflammation induced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in the ear of mice. The positive control was tested with indomethacin and the negative control was done only with vehicle. These results allow the identification of a pharmacophore group that through molecular modeling studies and organic synthesis can result in compounds with improved anti-inflammatory activity. (author)

  11. Plasma application for detoxification of Jatropha phorbol esters

    International Nuclear Information System (INIS)

    Kongmany, S; Matsuura, H; Furuta, M; Okuda, S; Imamura, K; Maeda, Y

    2013-01-01

    Atmospheric pressure non-thermal dielectric barrier discharge (DBD) plasma generated by helium gas at high voltage and input power of about 50 W was first applied to detoxification of Jatropha curcas phorbol esters (J. PEs) as well as standard phorbol ester (4β-12-O-tetradecanoyl phorbol-13-acetate, TPA) in water and methanol. Plasma irradiation on the solution sample was conducted for 15 min. In aqueous solution, only 16% of TPA was degraded and complete degradation of J. PEs was observed. On the contrary, complete degradation of both TPA and J. PEs in methanol was achieved by the same plasma irradiation condition. Hydroxyl radical (.OH) generated by plasma irradiation of the solution is expected as the main radical inducing the degradation of PEs.

  12. Functional cooperativity between two TPA responsive elements in undifferentiated F9 embryonic stem cells.

    Science.gov (United States)

    Okuda, A; Imagawa, M; Sakai, M; Muramatsu, M

    1990-01-01

    We have recently identified an enhancer, termed GPEI, in the 5'-flanking region of the rat glutathione transferase P gene, that is composed of two imperfect TPA (phorbol 12-O-tetradecanoate 13-acetate) responsive elements (TREs). Unlike other TRE-containing enhancers, GPEI exhibits a strong transcriptional enhancing activity in F9 embryonic stem cells. Mutational analyses have revealed that the high activity of GPEI is mediated by two imperfect TREs. Each TRE-like sequence has no activity by itself but acts synergistically to form a strong enhancer which is active even in the very low level of AP-1 activity in F9 cells. Furthermore, we show that synthetic DNAs containing two perfect TREs in certain arrangements have strong transcriptional enhancing activities in F9 cells and the activity is greatly influenced by the relative orientation and the distance of two TREs. Images Fig. 1. Fig. 2. Fig. 3. PMID:2323334

  13. Effect of soy saponin on the growth of human colon cancer cells

    Science.gov (United States)

    Tsai, Cheng-Yu; Chen, Yue-Hwa; Chien, Yi-Wen; Huang, Wen-Hsuan; Lin, Shyh-Hsiang

    2010-01-01

    AIM: To investigate the effect of extracted soybean saponins on the growth of human colon cancer cells. METHODS: WiDr human colon cancer cells were treated with 150, 300, 600 or 1200 ppm of soy saponin to determine the effect on cell growth, cell morphology, alkaline phosphatase (AP) and protein kinase C (PKC) activities, and P53 protein, c-Fos and c-Jun gene expression. RESULTS: Soy saponin decreased the number of viable cells in a dose-dependent manner and suppressed 12-O-tetradecanol-phorbol-13-acetate-stimulated PKC activity (P saponins developed cytoplasmic vesicles and the cell membrane became rougher and more irregular in a dose-dependent manner, and eventually disassembled. At 600 and 1200 ppm, the activity of AP was increased (P saponin. CONCLUSION: Soy saponin may be effective in preventing colon cancer by affecting cell morphology, cell proliferation enzymes, and cell growth. PMID:20632438

  14. Porritoxins, metabolites of Alternaria porri, as anti-tumor-promoting active compounds.

    Science.gov (United States)

    Horiuchi, Masayuki; Tokuda, Harukuni; Ohnishi, Keiichiro; Yamashita, Masakazu; Nishino, Hoyoku; Maoka, Takashi

    2006-02-01

    To search for possible cancer chemopreventive agents from natural sources, we performed primary screening of metabolites of Alternaria porri by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. The ethyl acetate extract of A. porri showed the inhibitory effect on EBV-EA activation. Three porritoxins (1-3) were obtained as inhibitory active compounds for EBV-EA from ethyl acetate extract. 6-(3',3'-Dimethylallyloxy)-4-methoxy-5-methylphthalide (2) showed the strongest activity among them. Inhibitory effect of porritoxin (1) and (2) was superior to that of beta-carotene, a well-known anti-tumor promoter. Furthermore, the structure-activity correlation of porritoxins and their related compounds were discussed.

  15. Topical anti-inflammatory activity of Calea prunifolia HBK (Asteraceae) in the TPA model of mouse ear inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, Milton; Gil, Juan F., E-mail: miltongoba@uniquindio.edu.co [Grupo de Busqueda de Principios Bioactivos, Programa de Quimica, Universidad del Quindio, Armenia (Colombia)

    2011-09-15

    Phytochemical study of Calea prunifolia HBK identified two compounds derived from p-hydroxyacetophenone, the 1-(2-hydroxy-5-(1-methoxyethyl)phenyl)-3-methylbut-2.en-1-one showed a satisfactory anti-inflammatory activity (58.33%), when considering that this is a natural product. Although the two derived compounds are structurally similar, the anti-inflammatory activity of 1-(2-hydroxy-5-methoxyphenyl)-3-methylbut-2-en-1-one was not significant (2.08%). The test was conducted in a model of inflammation induced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in the ear of mice. The positive control was tested with indomethacin and the negative control was done only with vehicle. These results allow the identification of a pharmacophore group that through molecular modeling studies and organic synthesis can result in compounds with improved anti-inflammatory activity. (author)

  16. Modified eremophilanes and anti-inflammatory activity of Psacalium cirsiifolium

    International Nuclear Information System (INIS)

    Arciniegas, Amira; Perez-Castorena, Ana L.; Nieto-Camacho, Antonio; Vivar Alfonso Romo de; Villasenor, Jose Luis

    2013-01-01

    Four new modified eremophilanes, together with ten known cacalol derivatives, two caryophyllenes, one aromadendrene and one flavonoid were isolated from Psacalium cirsiifolium. The structures of these compounds were elucidated by spectroscopic analysis. The antiinflammatory activity of extracts and of seven of the isolated compounds was evaluated on 12-O-tetradecanoylphorbol-13-acetate (TPA) model of induced acute inflammation. The new compound 2α-hydroxyadenostin B (4) showed a dose dependent activity (IC 50 0.41 μmol per ear) and a neutrophil inhibition effect as measured by the myeloperoxidase (MPO) assay similar to that of indomethacin at 0.31 and 1.0 μmol per ear. (author)

  17. Microbial transformation of isosteviol and inhibitory effects on Epstein-Barr virus activation of the transformation products.

    Science.gov (United States)

    Akihisa, Toshihiro; Hamasaki, Yusuke; Tokuda, Harukuni; Ukiya, Motohiko; Kimura, Yumiko; Nishino, Hoyoku

    2004-03-01

    Microbial transformation of isosteviol (2), a beyerane-type diterpenoid obtained from stevioside (1) by acid hydrolysis, yielded 7beta-hydroxyisosteviol (3), 11beta-hydroxyisosteviol (5), and 12beta-hydroxyisosteviol (6) by the fungus Aspergillus niger, 17-hydroxyisosteviol (7) by the fungus Glomerella cingulata, and 3 and 7-oxoisosteviol (4) by the fungus Mortierella elongate. The five metabolites, 3-7, along with 1 and 2 were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells as a primary screening test for inhibitors of tumor promoters. All the diterpenes tested showed potent inhibitory effects, with the five metabolites 3-7 exhibiting more potent effects.

  18. Modified eremophilanes and anti-inflammatory activity of Psacalium cirsiifolium

    Energy Technology Data Exchange (ETDEWEB)

    Arciniegas, Amira; Perez-Castorena, Ana L.; Nieto-Camacho, Antonio; Vivar Alfonso Romo de, E-mail: alperezc@unam.mx [Instituto de Quimica, Universidad Nacional Autonoma de Mexico, Coyoacan, D.F. (Mexico); Villasenor, Jose Luis [Instituto de Biologia, Universidad Nacional Autonoma de Mexico, Coyoacan, D.F. (Mexico)

    2013-01-15

    Four new modified eremophilanes, together with ten known cacalol derivatives, two caryophyllenes, one aromadendrene and one flavonoid were isolated from Psacalium cirsiifolium. The structures of these compounds were elucidated by spectroscopic analysis. The antiinflammatory activity of extracts and of seven of the isolated compounds was evaluated on 12-O-tetradecanoylphorbol-13-acetate (TPA) model of induced acute inflammation. The new compound 2{alpha}-hydroxyadenostin B (4) showed a dose dependent activity (IC{sub 50} 0.41 {mu}mol per ear) and a neutrophil inhibition effect as measured by the myeloperoxidase (MPO) assay similar to that of indomethacin at 0.31 and 1.0 {mu}mol per ear. (author)

  19. Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein

    International Nuclear Information System (INIS)

    Sohn, Eun Jeong; Kim, Dae Won; Kim, Young Nam; Kim, So Mi; Lim, Soon Sung; Kang, Tae-Cheon; Kwon, Hyeok Yil; Kim, Duk-Soo; Cho, Sung-Woo; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Hwang, Hyun Sook; Choi, Soo Young

    2011-01-01

    Research highlights: → We studied effects of pergolide mesylate (PM) on in vitro and in vivo transduction of PEP-1-catalase. → PEP-1-catatase inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. → PM enhanced the transduction of PEP-1-catalase into HaCaT cells and skin tissue. → PM increased anti-inflammatory activity of PEP-1-catalase. → PM stimulated therapeutic action of anti-oxidant enzyme catalase in oxidative-related diseases. -- Abstract: The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor-α induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.

  20. Effect of iodide on glucose oxidation and 32P incorporation into phospholipids stimulated by different agents in dog thyroid slices

    International Nuclear Information System (INIS)

    Tseng, F.Y.; Rani, C.S.; Field, J.B.

    1989-01-01

    Since iodide (I-) inhibits TSH stimulation of cAMP formation, which mediates most of the effects of the hormone, it has been assumed that this accounts for the inhibitory action of iodide on the thyroid. However, TSH stimulation of 32P incorporation into phospholipids and stimulation of thyroid metabolism by other agonists, such as carbachol, phorbol esters, and ionophore A23187, is not cAMP mediated. The present studies examined the effect of iodide on stimulation of glucose oxidation and 32P incorporation into phospholipids by TSH and other agonists to determine if the inhibition of cAMP formation was responsible for the action of iodide. Preincubation of dog thyroid slices for 1 h with iodide (10(-4) M) inhibited TSH-, (Bu)2cAMP-, carbachol-, methylene blue-, 12-O-tetradecanoyl phorbol-13-acetate-, ionophore A23187-, prostaglandin E1-, and cholera toxin-stimulated glucose oxidation. I- also inhibited the stimulation by TSH, 12-O-tetradecanoyl phorbol-13-acetate, carbachol, and ionophore A23187 of 32P incorporation into phospholipids. The inhibition was similar whether iodide was added 2 h before or simultaneously with the agonist. I- itself sometimes stimulated basal glucose oxidation, but had no effect on basal 32P incorporation into phospholipids. The effects of iodide on basal and agonist-stimulated thyroid metabolism were blocked by methimazole (10(-3) M). When dog thyroid slices were preloaded with 32PO4 or [1-14C]glucose, the iodide inhibition of agonist stimulation disappeared, suggesting that the effect of iodide involves the transport process. In conclusion, I- inhibited stimulation of glucose oxidation and 32P incorporation into phospholipids by all agonists, indicating that the effect is independent of the cAMP system and that iodide autoregulation does not only involve this system. Oxidation and organification of iodide are necessary for the inhibition

  1. Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein

    Energy Technology Data Exchange (ETDEWEB)

    Sohn, Eun Jeong; Kim, Dae Won; Kim, Young Nam; Kim, So Mi [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Lim, Soon Sung [Department of Food Science and Nutrition and RIC Center, Hallym University, Chunchon 200-702 (Korea, Republic of); Kang, Tae-Cheon [Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chunchon 200-702 (Korea, Republic of); Kwon, Hyeok Yil [Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702 (Korea, Republic of); Kim, Duk-Soo [Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan-Si 330-090 (Korea, Republic of); Cho, Sung-Woo [Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736 (Korea, Republic of); Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Hwang, Hyun Sook, E-mail: wazzup@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Choi, Soo Young, E-mail: sychoi@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of)

    2011-03-18

    Research highlights: {yields} We studied effects of pergolide mesylate (PM) on in vitro and in vivo transduction of PEP-1-catalase. {yields} PEP-1-catatase inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. {yields} PM enhanced the transduction of PEP-1-catalase into HaCaT cells and skin tissue. {yields} PM increased anti-inflammatory activity of PEP-1-catalase. {yields} PM stimulated therapeutic action of anti-oxidant enzyme catalase in oxidative-related diseases. -- Abstract: The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1{beta}, and tumor necrosis factor-{alpha} induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.

  2. Cytometric evaluation of intracellular IFN-γ and IL-4 levels in thyroid follicular cells from patients with autoimmune thyroid diseases

    Directory of Open Access Journals (Sweden)

    Bossowski Artur

    2011-09-01

    Full Text Available Abstract Background In recent few years is underlined that altered balance of pro- and anti-inflammatory cytokines play an important role in the pathogenesis of AITD. The aim of this study was to estimate intracellular INF-γ and IL-4 levels in thyroid-infiltrating lymphocytes and thyrocytes isolated from thyroid tissues in 54 adolescent patients aged 8-21 years, with Graves' disease (GD; n = 18, Hashimoto's thyroiditis (HT; n = 18 and non-toxic multinodular goiter (NTMG; n = 18. Methods Fresh thyroid tissues were taken on culture medium RPMI -1640, it was mechanically prepared. In next step were added cell activators -12- myristate 13- the acetate (PMA and Ionomycin as well as the inhibitor of transportation of proteins - Breferdin A. They were cultured 24 hours in 50 ml flasks at 37°C in a 5-95% CO2-air water-saturated atmosphere. After that, thyrocytes were identified by mouse mAb directed against human TPO epitope 64 conjugated with rabbit anti-mouse antibodies IgG (Fab'2 labeled by FITC. After incubation at room temperature to each of samples added reagent A fixative the cellular membrane. In next step into the cell suspensions were added reagent B to permeabilization of cellular membrane and specific anti-IL-4-PE or anti-IFN-γ-PE mAbs. Identification of intracellular cytokines in T lymphocytes was performed in the same procedure with application of anti-CD4-PerCP and anti-CD8-PerCP mAbs specific for T lymphocytes. The cells were analyzed in a flow cytometry (Coulter EPICS XL. Results In examined group of patients with GD we observed statistically significant higher mean percentage of cells with phenotype CD4+IL-4 (p Conclusions We conclude that human thyrocytes in autoimmune thyroid disorders could be a source of cytokine production and that their activation influences local interaction with T lymphocytes inflowing to the thyroid gland.

  3. Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzymes. 15-deoxy-delta(12,14)-prostaglandin j2-induced expression of glutathione S-transferases.

    Science.gov (United States)

    Kawamoto, Y; Nakamura, Y; Naito, Y; Torii, Y; Kumagai, T; Osawa, T; Ohigashi, H; Satoh, K; Imagawa, M; Uchida, K

    2000-04-14

    Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes, such as glutathione S-transferases (GSTs). In the present study, we developed a cell culture system that potently responds to phenolic antioxidants and found that antitumor prostaglandins (PGs) are potential inducers of GSTs. We screened primary hepatocytes and multiple cell lines for inducing GST activity upon incubation with the phenolic antioxidant (tert-butylhydroquinone) and found that rat liver epithelial RL34 cells most potently responded. Based on an extensive screening of diverse chemical agents on the induction of GST activity in RL34 cells, the J2 series of PGs, 15-deoxy-Delta(12,14)-prostaglandin J2 (15-deoxy-Delta(12,14)-PGJ2) in particular, were found to be potential inducers of GST. Enhanced gene expression of Class pi GST isozyme (GSTP1) by 15-deoxy-Delta(12,14)-PGJ2 was evident as a drastic elevation of the mRNA level. Hence, we examined the molecular mechanism underlying the 15-deoxy-Delta(12, 14)-PGJ2-induced GSTP1 gene expression. From functional analysis of various deletion mutant genes, we found that the 15-deoxy-Delta(12, 14)-PGJ2 reponse element was localized in a region containing a GSTP1 enhancer I (GPEI) that consists of two imperfect phorbol 12-O-tetradecanoylphorbol-13-acetate response elements. When the GPEI was combined with the minimum GSTP1 promoter, the element indeed showed an enhancer activity in response to 15-deoxy-Delta(12, 14)-PGJ2. Point mutations of either of the two imperfect 12-O-tetradecanoylphorbol-13-acetate response elements in GPEI completely abolished the enhancer activity. Gel mobility shift assays demonstrated that 15-deoxy-Delta(12,14)-PGJ2 specifically stimulated the binding of nuclear proteins including the transcription factor c-Jun, but not Nrf2, to GPEI. These results

  4. Effects of ketamine on glucose uptake by glucose transporter type 3 expressed in Xenopus oocytes: The role of protein kinase C

    Energy Technology Data Exchange (ETDEWEB)

    Tomioka, Shigemasa, E-mail: tomioka@dent.tokushima-u.ac.jp [Department of Dental Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan); Kaneko, Miyuki [Department of Dental Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan); Satomura, Kazuhito [First Department of Oral and Maxillofacial Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan); Mikyu, Tomiko; Nakajo, Nobuyoshi [Department of Dental Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 18-15, Tokushima City, Tokushima 770-8504 (Japan)

    2009-10-09

    We investigated the effects of ketamine on the type 3 facilitative glucose transporter (GLUT3), which plays a major role in glucose transport across the plasma membrane of neurons. Human-cloned GLUT3 was expressed in Xenopus oocytes by injection of GLUT3 mRNA. GLUT3-mediated glucose uptake was examined by measuring oocyte radioactivity following incubation with 2-deoxy-D-[1,2-{sup 3}H]glucose. While ketamine and S(+)-ketamine significantly increased GLUT3-mediated glucose uptake, this effect was biphasic such that higher concentrations of ketamine inhibited glucose uptake. Ketamine (10 {mu}M) significantly increased V{sub max} but not K{sub m} of GLUT3 for 2-deoxy-D-glucose. Although staurosporine (a protein kinase C inhibitor) increased glucose uptake, no additive or synergistic interactions were observed between staurosporine and racemic ketamine or S(+)-ketamine. Treatment with ketamine or S(+)-ketamine partially prevented GLUT3 inhibition by the protein kinase C activator phorbol-12-myrisate-13-acetate. Our results indicate that ketamine increases GLUT3 activity at clinically relevant doses through a mechanism involving PKC inhibition.

  5. Interleukin 1 induces early protein phosphorylation and requires only a short exposure for late induced secretion of β-endorphin in a mouse pituitary cell line

    International Nuclear Information System (INIS)

    Fagarasan, M.O.; Axelrod, J.; Bishop, J.F.; Rinaudo, M.S.

    1990-01-01

    Previous work has shown that prolonged pretreatment of a mouse anterior pituitary cell line, AtT-20 cells, with the cytokine interleukin 1 (IL-1) stimulates β-endorphin release and potentiates the secretion induced by many secretagogues. Desensitization of protein kinase C (PKC) by pretreatment with phorbol ester [phorbol 12-tetradecanoate 13-acetate (TPA)] for 8 hr abolished the secretion induced by TPA as well as the enhancement of TPA-induced β-endorphin release produced by IL-1. Desensitization of PKC only partly abolished the potentiating effects of IL-1 on corticotropin-releasing factor-induced β-endorphin secretion. In contrast, IL-1-induced β-endorphin release was independent of PKC. The authors observed that treatment of AtT-20 cells with IL-1 markedly phosphorylated 19-, 20-, and 60-kDa proteins within minutes, presumably by early activation of protein kinases. Prolonged treatment with TPA, which was shown to desensitize an 87-kDa protein (a substrate for PKC), had no effect on IL-1-induced phosphorylation of 20-, 60-, and 87-kDa proteins, indicating that the phosphorylation of these proteins does not involve PKC. IL-1 does not generate cAMP in AtT-20 cells, suggesting that a cAMP-dependent protein kinase is also not involved. These observations indicate that once IL-1 generates an early signal, its presence is no longer necessary for the subsequent secretion of β-endorphin

  6. Anti-inflammatory activity of different agave plants and the compound cantalasaponin-1.

    Science.gov (United States)

    Monterrosas-Brisson, Nayeli; Ocampo, Martha L Arenas; Jiménez-Ferrer, Enrique; Jiménez-Aparicio, Antonio R; Zamilpa, Alejandro; Gonzalez-Cortazar, Manases; Tortoriello, Jaime; Herrera-Ruiz, Maribel

    2013-07-10

    Species of the agave genus, such as Agave tequilana, Agave angustifolia and Agave americana are used in Mexican traditional medicine to treat inflammation-associated conditions. These plants' leaves contain saponin compounds which show anti-inflammatory properties in different models. The goal of this investigation was to evaluate the anti-inflammatory capacity of these plants, identify which is the most active, and isolate the active compound by a bio-directed fractionation using the ear edema induced in mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) technique. A dose of 6 mg/ear of acetone extract from the three agave species induced anti-inflammatory effects, however, the one from A. americana proved to be the most active. Different fractions of this species showed biological activity. Finally the F5 fraction at 2.0 mg/ear induced an inhibition of 85.6%. We identified one compound in this fraction as (25R)-5α-spirostan-3β,6α,23α-triol-3,6-di-O-β-D-glucopyranoside (cantalasaponin-1) through 1H- and 13C-NMR spectral analysis and two dimensional experiments like DEPT NMR, COSY, HSQC and HMBC. This steroidal glycoside showed a dose dependent effect of up to 90% of ear edema inhibition at the highest dose of 1.5 mg/ear.

  7. Anti-Inflammatory Activity of Different Agave Plants and the Compound Cantalasaponin-1

    Directory of Open Access Journals (Sweden)

    Jaime Tortoriello

    2013-07-01

    Full Text Available Species of the agave genus, such as Agave tequilana, Agave angustifolia and Agave americana are used in Mexican traditional medicine to treat inflammation-associated conditions. These plants’ leaves contain saponin compounds which show anti-inflammatory properties in different models. The goal of this investigation was to evaluate the anti-inflammatory capacity of these plants, identify which is the most active, and isolate the active compound by a bio-directed fractionation using the ear edema induced in mice with 12-O-tetradecanoylphorbol-13-acetate (TPA technique. A dose of 6 mg/ear of acetone extract from the three agave species induced anti-inflammatory effects, however, the one from A. americana proved to be the most active. Different fractions of this species showed biological activity. Finally the F5 fraction at 2.0 mg/ear induced an inhibition of 85.6%. We identified one compound in this fraction as (25R-5α-spirostan-3β,6α,23α-triol-3,6-di-O-β-D-glucopyranoside (cantalasaponin-1 through 1H- and 13C-NMR spectral analysis and two dimensional experiments like DEPT NMR, COSY, HSQC and HMBC. This steroidal glycoside showed a dose dependent effect of up to 90% of ear edema inhibition at the highest dose of 1.5 mg/ear.

  8. Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus

    International Nuclear Information System (INIS)

    Schmitt, J.; Schlehofer, J.R.; Mergener, K.; Gissmann, L.; zur Hausen, H.

    1989-01-01

    Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur

  9. Some biological activities of pigments extracted from Micrococcus roseus (PTCC 1411) and Rhodotorula glutinis (PTCC 5257).

    Science.gov (United States)

    Rostami, Hossein; Hamedi, Hassan; Yolmeh, Mahmoud

    2016-12-01

    The importance of replacing synthetic pigments with natural types is increasing day by day in the food industry due to the harmful effects of some synthetic pigments. Microorganisms are a major source of natural pigments, which nowadays have attracted the attention of researchers. In this study, carotenoid pigments were produced by Micrococcus roseus and Rhodotorula glutinis, and some of their biological properties such as antimicrobial, antioxidant, anticancer, and anti-inflammatory activities were evaluated. Given the results, bacteria, especially gram-positive bacteria, had higher sensitivity to the pigments extracted from M. roseus (PEM) and R. glutinis (PER) compared to molds so that Bacillus cereus and Alternaria citri had the highest and the lowest sensitivity, respectively. PER showed a higher antioxidant activity compared with PEM in the various methods of measuring antioxidant activity. In vitro and in vivo anti-tumor-promoting activities of PER were measured significantly more than PEM (P <0.05). Both pigment extracts remarkably inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, so that ID 50 (50% inhibitory dose) of PEM and PER were 0.22 and 0.09 mg/ear, respectively. © The Author(s) 2016.

  10. Intense Pulsed Light: Friend or Foe? Molecular Evidence to Clarify Doubts.

    Science.gov (United States)

    Ferreira, Liliana; Vitorino, Rui; Neuparth, Maria João; Rodrigues, David; Gama, Adelina; Faustino-Rocha, Ana I; Ferreira, Rita; Oliveira, Paula A

    2018-02-01

    Intense pulsed light (IPL) has been extensively applied in the field of dermatology and aesthetics; however, the long-term consequences of its use are poorly unknown, and to the best of our knowledge there is no study on the effect of IPL in neoplastic lesions. In order to better understand the molecular mechanisms underlying IPL application in the skin, we used an animal model of carcinogenesis obtained by chemical induction with 12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Institute of Cancer Research (ICR) mice were administered DMBA and/or TPA and treated with IPL. Skin was evaluated by histopathology and 2DE-blot-MS/MS analysis. Our data evidenced an inflammatory response and a metabolic remodeling of skin towards a glycolytic phenotype after chronic exposure to IPL, which was accomplished by increased oxidative stress and susceptibility to apoptosis. These alterations induced by IPL were more notorious in the DMBA sensitized skin. Keratins and metabolic proteins seem to be the more susceptible to oxidative modifications that might result in loss of function, contributing for the histological changes observed in treated skin. Data highlight the deleterious impact of IPL on skin phenotype, which justifies the need for more experimental studies in order to increase our understanding of the IPL long-term safety. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  11. DNA damage-inducible transcripts in mammalian cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

    1988-01-01

    Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

  12. The synthesis of phosphatidylalcohols in HL-60 cells

    International Nuclear Information System (INIS)

    Tettenborn, C.S.

    1988-01-01

    This study focuses upon the phenomenon that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates a pathway for the synthesis of phosphatidylalcohols in HL-60 cells during the differentiation of these cells to macrophages. Using exogenous ethanol as a substrate for this pathway, the synthesis of phosphatidylethanol is induced by TPA well before full expression of differentiation. Experiments done in whole cell cultures demonstrated that this pathway could utilize a wide range of other alcohols as substrates, including glycerol, a potential endogenous substrate. Using radiolabeling and iodine-staining as criteria, this TPA-inducible pathway was shown to result in a dramatic alteration of phospholipid composition, depending on the availability of the alcohol headgroup. A cell-free system was developed to explore the enzymatic reactions involved in phosphatidylalcohol synthesis, a main goal of these studies. For this purpose, the lipid pools of HL-60 cells were prelabeled with [ 3 H]arachidonic acid in the absence of ethanol and total cell homogenates were prepared by sonication. The ability of the cell lysates to synthesize phosphatidylethanol was assayed after addition of ethanol to the system

  13. The effect of phorbols on metabolic cooperation between human fibroblasts

    International Nuclear Information System (INIS)

    Mosser, D.D.; Bols, N.C.

    1982-01-01

    Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of [ 3 H]hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total [ 3 H]hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of [ 3 H]nucleotides and is an indication of an interference with intercellular communication

  14. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

    International Nuclear Information System (INIS)

    Beretta, L.; Boutterin, M.C.; Sobel, A.

    1988-01-01

    We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and [ 32 P] phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins

  15. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    International Nuclear Information System (INIS)

    Deaciuc, I.V.; Spitzer, J.A.

    1989-01-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with [32P]H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis

  16. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

    Science.gov (United States)

    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-10-11

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.

  17. Cloning of the cDNA for human 12-lipoxygenase

    International Nuclear Information System (INIS)

    Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

    1990-01-01

    A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

  18. Cytogenetic investigations of chronic lymphocytic leukemia.

    Science.gov (United States)

    Wren, Catherine; Moriarty, Helen; Marsden, Katherine; Tegg, Elizabeth

    2010-04-15

    This study aimed to determine which culture method would yield the highest culture success rate, mitotic index, banding resolution, and abnormality rate in investigation of patients with chronic lymphocytic leukemia (CLL). A range of culture techniques for conventional cytogenetic (CC) analyses was compared: 24-hour unstimulated, 72 hours incubation with additional fetal calf serum, 72 hours stimulation with interleukin 4, 72 hours stimulation with lipopolysaccharide (LPS), 72 hours stimulation with TPA (12-O-tetradecanoylphorbol 13-acetate), and 72 hours stimulation with CpG-oligonucleotide DSP30 + Interleukin-2 (IL-2). CC abnormality rates were also compared to fluorescence in situ hybridization (FISH) results using probes for CLL (LSI D13S319/13q34/CEP 12: LSI ATM/p53). Forty-five samples from 24 patients (consisting of 11 newly diagnosed and 13 previously diagnosed patients) were included. For CC, a 100.0% culture success rate was achieved (n = 45) by means of an EDTA (ethylenediaminetetraacetic acid) peripheral blood sample with an associated 62.5% CC abnormality rate (n = 24). FISH detected an abnormality rate of 75.0% (n = 24). The combined CC and FISH abnormality rate was 87.5% (n = 24). This study demonstrates that CC that uses TPA and DSP30 + IL-2 on EDTA peripheral blood is effective in the investigation of CLL and may be used as a supplement to FISH studies. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Detection of chromosomal changes in chronic lymphocytic leukemia using classical cytogenetic methods and FISH: application of rich mitogen mixtures for lymphocyte cultures.

    Science.gov (United States)

    Koczkodaj, Dorota; Popek, Sylwia; Zmorzyński, Szymon; Wąsik-Szczepanek, Ewa; Filip, Agata A

    2016-04-01

    One of the research methods of prognostic value in chronic lymphocytic leukemia (CLL) is cytogenetic analysis. This method requires the presence of appropriate B-cell mitogens in cultures in order to obtain a high mitotic index. The aim of our research was to determine the most effective methods of in vitro B-cell stimulation to maximize the number of metaphases from peripheral blood cells of patients with CLL for classical cytogenetic examination, and then to correlate the results with those obtained using fluorescence in situ hybridization (FISH). The study group involved 50 consecutive patients with CLL. Cell cultures were maintained with the basic composition of culture medium and addition of respective stimulators. We used the following stimulators: Pokeweed Mitogen (PWM), 12-O-tetradecanoylphorbol 13-acetate (TPA), ionophore, lipopolysaccharide (LPS), and CpG-oligonucleotide DSP30. We received the highest mitotic index when using the mixture of PWM+TPA+I+DSP30. With classical cytogenetic tests using banding techniques, numerical and structural aberrations of chromosomes were detected in 46 patients, and no change was found in only four patients. Test results clearly confirmed the legitimacy of using cell cultures enriched with the mixture of cell stimulators and combining classical cytogenetic techniques with the FISH technique in later patient diagnosing. Copyright © 2016 American Federation for Medical Research.

  20. 'Water Structure' versus 'Radical Scavenger' theories as explanations for the suppressive effects of DMSO and related compounds on radiation-induced transformation in vitro

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Symons, M.C.R.

    1987-01-01

    We report here that dimethylsulfoxide (DMSO): (i) suppresses radiation-induced transformation in vitro, even when DMSO treatments begin as late as 10 days post-irradiation; (ii) inhibits the 12-O-tetradecanoylphorbol-13-acetate (TPA) enhancement of radiation-induced transformation in vitro; (iii) does not affect the expression of transformed cells as foci (when surrounded by non-transformed cells); and (iv) may be affecting radiation-induced transformation through its solvent properties (i.e. the 'Water Structure' theory), while its effects on the TPA enhancement of radiation transformation may be mediated by its free radical scavenging abilities. DMSO, dimethylformamide (DMF) and dimethylacetamide (DMA) are similar solvents which are all very effective in their ability to suppress radiation-induced transformation in vitro. As DMSO is known to be an extremely effective OH free-radical scavenging agent, while DMF and DMA are not as efficient at scavenging free radicals, our results suggest that properties other than free-radical scavenging ability may be important in the suppressive effects of these compounds on radiation-induced transformation in vitro. (author)

  1. Novel NSAID-Derived Drugs for the Potential Treatment of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Ivana Cacciatore

    2016-06-01

    Full Text Available Nonsteroidal anti-inflammatory drugs (NSAIDs have been suggested for the potential treatment of neurodegenerative diseases, such as Alzheimer’s disease (AD. Prolonged use of NSAIDs, however, produces gastrointestinal (GI toxicity. To overcome this serious limitation, the aim of this study was to develop novel NSAID-derived drug conjugates (Anti-inflammatory-Lipoyl derivatives, AL4–9 that preserve the beneficial effects of NSAIDS without causing GI problems. As such, we conjugated selected well-known NSAIDs, such as (S-naproxen and (R-flurbiprofen, with (R-α-lipoic acid (LA through alkylene diamine linkers. The selection of the antioxidant LA was based on the proposed role of oxidative stress in the development and/or progression of AD. Our exploratory studies revealed that AL7 containing the diaminoethylene linker between (R-flurbiprofen and LA had the most favorable chemical and in vitro enzymatic stability profiles among the synthesized compounds. Upon pretreatment, this compound exhibited excellent antioxidant activity in phorbol 12-miristate 13-acetate (PMA-stimulated U937 cells (lymphoblast lung from human and Aβ(25–35-treated THP-1 cells (leukemic monocytes. Furthermore, AL7 also modulated the expression of COX-2, IL-1β and TNF-α in these cell lines, suggesting anti-inflammatory activity. Taken together, AL7 has emerged as a potential lead worthy of further characterization and testing in suitable in vivo models of AD.

  2. Topical anti-inflammatory activity of a monofloral honey of Mimosa scabrella provided by Melipona marginata during winter in southern Brazil.

    Science.gov (United States)

    Borsato, Débora M; Prudente, Arthur S; Döll-Boscardin, Patrícia M; Borsato, Aurélio V; Luz, Cynthia F P; Maia, Beatriz H L N S; Cabrini, Daniela A; Otuki, Michel F; Miguel, Marilis D; Farago, Paulo V; Miguel, Obdulio G

    2014-07-01

    Melipona marginata is an endangered species of stingless bee from Brazil that produces honey with particular physicochemical features and a remarkable exotic flavor. To the best of our knowledge, this is the first report devoted to exploring the medicinal potential of this honey. Thus, the aim of this paper was to investigate the potential anti-inflammatory activity of honey extract from M. marginata on skin inflammation. The honey sample was classified as a monofloral honey of Mimosa scabrella. The presence of 11 phenolic compounds as kaempferol and caffeic acid was detected using the high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-UV-ESI-MS) method. The anti-inflammatory activity was measured using a 12-O-tetradecanoylphorbol-13-acetate-induced ear edema model of inflammation in mice. The topical application of the M. marginata honey extract (1.0 mg/ear) was able to reduce ear edema with an inhibitory effect of 54 ± 5%. This extract decreased the myeloperoxidase activity in 75 ± 3%, which suggests a lower leucocyte infiltration that was confirmed by histological analysis. This extract also provided a reduction of 55 ± 14% in the production of reactive oxygen species. This anti-inflammatory activity could be due to a synergic effect of the phenolic compounds identified in the honey sample. Taken together, these results open up new possibilities for the use of M. marginata honey extract in skin disorders.

  3. Role of arachidonic acid metabolism in transcriptional induction of tumor necrosis factor gene expression by phorbol ester

    Energy Technology Data Exchange (ETDEWEB)

    Horiguchi, J.; Spriggs, D.; Imamura, K.; Stone, R.; Luebbers, R.; Kufe, D.

    1989-01-01

    The treatment of human HL-60 promyelocytic leukemia cells with 12-0 tetradecanoylphorbol-13-acetate (TPA) is associated with induction of tumor necrosis factor (TNF) transcripts. The study reported here has examined TPA-induced signaling mechanisms responsible for the regulation of TNF gene expression in these cells. Run-on assays demonstrated that TPA increases TNS mRNA levels by transcriptional activation of this gene. The induction of TNF transcripts by TPA was inhibited by the isoquinolinesulfonamide derivative H7 but not by HA1004, suggesting that this effect of TPA is mediated by activation of protein kinase C. TPA treatment also resulted in increased arachidonic acid release. Moreover, inhibitors of phospholipase, A/sub 2/ blocked both the increase in arachidonic acid release and the induction of TNF transcripts. These findings suggest that TPA induces TNF gene expression through the formation of arachidonic acid metabolites. Although indomethacin had no detectable effect on this induction of TNF transcripts, ketoconazole, an inhibitor of 5-lipoxygenase, blocked TPA-induced increases in TNF mRNA levels. Moreover, TNF mRNA levels were increased by the 5-lipoxygenase metabolite leukotriene B/sub 4/. In contrast, the cyclooxygenase metabolite prostaglandin E/sub 2/ inhibited the induction of TNF transcripts by TPA. Taken together, these results suggest that TPA induces TNF gene expression through the arachidonic acid cascade and that the level of TNF transcripts is regulated by metabolites of the pathway, leukotriene B/sub 4/ and prostaglandin E/sub 2/.

  4. Enhanced incorporation of radioactive inorganic phosphate into phospholipids of HeLa cells by tumor promoters

    International Nuclear Information System (INIS)

    Nishino, H.; Fujiki, H.; Terada, M.; Sato, S.

    1983-01-01

    Teleocidin, a new tumor promoter, increased incorporation of radioactive inorganic phosphate ( 32 P/sub i/) into phospholipids in HeLa cells. This effect was detected within 1 h on incubation of the cells in medium containing teleocidin. The half-maximum effective dose of teleocidin was approximately 10 ng/ml. The main effect of teleocidin was on the incorporation of 32 P/sub i/ into the phosphatidylcholine fraction, with a lesser effect on 32 P/sub i/ incorporation into other phospholipid fractions. Increased incorporation of 32 P/sub i/ into phospholipids was also observed on incubation of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), dihydroteleocidin B, or lyngbyatoxin A, which are all complete tumor promoters, and also with mezerein, which is an incomplete and second stage promoter. On the other hand, at concentrations of up to 1 microgram/ml, 4-O-methyl TPA and C/sub a/ 2 + ionophore A23187, which are incomplete and first stage promoters, and phorbol, which has no promoting activity in skin carcinogenesis, did not cause any increased incorporation of 32 P/sub i/ into phospholipid fractions of HeLa cells

  5. Effects of a tumor promoter and an anti-promoter on spontaneous and UV-induced 6-thioguanine-resistant mutations and sister-chromatid exchanges in V79 Chinese hamster cells

    International Nuclear Information System (INIS)

    Fujiwara, Y.; Kano, Y.; Tatsumi, M.; Paul, P.

    1980-01-01

    The effects of a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or an anti-promoter antipain (protease inhibitor) on spontaneous and ultraviolet-induced sister-chromatid exchanges (SCEs) and 6-thioguanine-resistant (6TGsup(r)) recessive mutations were examined in V79 Chinese hamster cells in culture. TPA and/or antipain neither significantly altered base-line and UV-induced immediate SCE frequencies, nor decreased the level of delayed SCEs which persisted 6-7 days after irradiation. TPA and/or antipain appeared to enhance the recovery of UV-induced 6TGsup(r) colonies at the plateau expression phase despite non-mutagenicity by themselves and unaltered metabolic cooperation. Thus, the results conceivably imply that the 6TGsup(r)-recessive mutation expression, but not fixation, can be modulated at the cell level by TPA and/or antipain. Our results, together with the recent results of Loveday and Latt, may argue against the notion that TPA enhances the antipain-suppressible SCEs as an index of mitotic recombination in relevance with a tumor-promotion mechanism. (orig.)

  6. JWA deficiency suppresses dimethylbenz[a]anthracene-phorbol ester induced skin papillomas via inactivation of MAPK pathway in mice.

    Directory of Open Access Journals (Sweden)

    Zhenghua Gong

    Full Text Available Our previous studies indicated that JWA plays an important role in DNA damage repair, cell migration, and regulation of MAPKs. In this study, we investigated the role of JWA in chemical carcinogenesis using conditional JWA knockout (JWA(Δ2/Δ2 mice and two-stage model of skin carcinogenesis. Our results indicated that JWA(Δ2/Δ2 mice were resistant to the development of skin papillomas initiated by 7, 12-dimethylbenz(aanthracene (DMBA followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA. In JWA(Δ2/Δ2 mice, the induction of papilloma was delayed, and the tumor number and size were reduced. In primary keratinocytes from JWA(Δ2/Δ2 mice, DMBA exposure induced more intensive DNA damage, while TPA-promoted cell proliferation was reduced. The further mechanistic studies showed that JWA deficiency blocked TPA-induced activation of MAPKs and its downstream transcription factor Elk1 both in vitro and in vivo. JWA(Δ2/Δ2 mice are resistance to tumorigenesis induced by DMBA/TPA probably through inhibition of transcription factor Elk1 via MAPKs. These results highlight the importance of JWA in skin homeostasis and in the process of skin tumor development.

  7. Anti-Inflammatory Activity and Composition of Senecio salignus Kunth

    Science.gov (United States)

    Pérez González, Cuauhtemoc; Serrano Vega, Roberto; González-Chávez, Marco; Zavala Sánchez, Miguel Angel; Pérez Gutiérrez, Salud

    2013-01-01

    We investigated the anti-inflammatory activity of Senecio salignus. This medicinal plant is often used in Mexico for the treatment of fever and rheumatism. Chloroform and methanol extracts of the plant were tested on 12-O-tetradecanoylphorbol-13-acetate- (TPA-) induced edema in mice ears. The methanol extract of the plant inhibited edema by 36 ± 4.4% compared with the control, while the chloroform extract exhibited an even greater level of inhibition (64.1%). The chloroform extract was then fractionated, and the composition of the active fraction was determined by GC-MS. The anti-inflammatory activity of this fraction was then tested on TPA-induced ear edema in mice, and we found that the active fraction could inhibit edema by 46.9%. The anti-inflammatory effect of the fraction was also tested on carrageenan-induced paw edema in rats at doses of 100 mg/kg; a 58.9 ± 2.8% reduction of the edema was observed 4 h after administration of carrageenan, and the effect was maintained for 5 h. PMID:23691512

  8. Demonstration of the serum antibody to Epstein-Barr virus specific DNA polymerased (EBV-DP) from patients with nasopharyngeal carcinoma (NPC)

    Energy Technology Data Exchange (ETDEWEB)

    Tan, R.S.; Li, J.S.; Grill, S.; Nutter, L.M.; Cheng, Y.C.

    1986-03-05

    Raji cells, an EBV genome carrying and nonproducer cell line, treated with tetradecanoyl-phorbol-13-acetate (TPA) and n-butyrate could induce a special DNA polymerase which has properties that are similar to the EBV-DP induced by TPA in P/sub 3/HR-I cells, an EBV producer cell line. Since EBV was found to have a strong association with NPC, and antibodies against EBV proteins or enzymes were found in high levels in sera from these patients, the possible presence of serum antibody against EBV-DP was examined. The serum titer of antibody to EBV-DP was found to have 190 +/- 84 units/ml serum (mean +/- S.D.) in 48 sera from patients with NPC. The titer in 52 healthy donors was 31.4 +/- 28 unit/ml serum (p < 0.01). The antibody was found to be associated with the IgG but not the IgA fraction. The antibody titers against EBV-DP were not correlated with the titer against EBV-DNase or VCA-IgA. Whether the antibody observed is against an EBV-DP core protein or its stimulating protein, as demonstrated by this laboratory previously, is still being investigated. This study demonstrated the high frequency and high titer of antibody against EBV-DP in serum from patients with NPC, and suggested the potential of utilizing this antibody titer to compliment other methods for the early diagnosis or prognosis of NPC.

  9. Potentiation of phorbol ester-induced coronary vasoconstriction in dogs following endothelium disruption

    International Nuclear Information System (INIS)

    Roberts, R.B.; Ku, D.D.

    1986-01-01

    In the present study, the effect of phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA), activation of protein kinase C on coronary vascular reactivity was studied in isolated dog coronary arteries. Addition of TPA (10-100 nM) produced a slow, time- and dose-dependent contraction reaching a maximum at approx 2-3 hrs and was essentially irreversible upon washing. Disruption of the endothelium(EC) greatly accelerated the development as well as increase the magnitude of TPA contraction (50-100%). Prior treatment of vessels with phentolamine (1μM), cyproheptadine (1μH) and ibuprofen (1μg/ml) did not alter the TPA contraction. Furthermore, in contrast to previously reported calcium-dependence of TPA contraction in other vessels, complete removal of extracellular calcium (Ca 0 ) or addition of 1μM nimodipine after TPA(30nM) resulted in only 32 +/- 4% and 25 +/- 3% reversal of TPA contraction, respectively. Addition of amiloride (10μM to 1mM), however, resulted in a dose-dependent reversal of TPA contraction. The results of the present study indicate that a similar activation of protein kinase C by TPA leads to potent coronary vasoconstriction, which is not completely dependent on Ca 0 . More importantly, these results further support their hypothesis that EC also functions as an inhibitory barrier to prevent circulating vasoconstrictors from exerting their deleterious constrictory effects

  10. Metabolism of indole alkaloid tumor promoter, (-)-indolactam V, which has the fundamental structure of teleocidins, by rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Hagiwara, N.; Irie, K.; Tokuda, H.; Koshimizu, K.

    1987-07-01

    Metabolic activation and/or deactivation of indole alkaloid tumor promoter, (-)-indolactam V (ILV), was examined using rat liver microsomes. Reaction of ILV with the microsomes supplemented with NADPH and MgCl/sub 2/ gave three major metabolites, which were identified as (-)-N13-desmethylindolactam V and two diastereomers of (-)-2-oxyindolactam V at C-3. The tumor-promoting activities of these metabolites were evaluated by induction of Epstein-Barr virus early antigen and inhibition of specific binding of (/sup 3/H)-12-O-tetradecanoylphorbol-13-acetate to a mouse epidermal particulate fraction, and proved to be conspicuously lower than that of ILV. These results demonstrate that the metabolism of ILV results in detoxification, and that it itself is the tumor-promoting entity. Studies on the enzymes concerned with this metabolism suggested the involvement of cytochrome P-450-containing mixed-function oxidases. Similar deactivation seems to be possible by skin, where the mixed-function oxidases are known to exist.

  11. Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

    International Nuclear Information System (INIS)

    Sathish, Narayanan; Yuan Yan

    2010-01-01

    A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-Δ65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

  12. Phorbol ester tumor promoter induced the synthesis of two major cytoplasmic proteins: identity with two proteins induced under heat-shocked and glucose-starved conditions

    International Nuclear Information System (INIS)

    Zhang, H.; Chen, K.Y.; Liu, A.Y.C.

    1987-01-01

    The regulation of specific protein synthesis by the phorbol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was evaluated using the L-8 and C-2 myoblast and the 3T3-L1 fibroblast cell cultures. TPA increased, by 2-4 fold, the synthesis rates of two cytoplasmic proteins with apparent molecular weights of 89,000 and 74,000 as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. The concentration of TPA and the time of incubation needed to elicit this induction was determined to be 10 μg/ml and 20 hrs, respectively. Increasing the concentration of TPA to 100, 200, and 500 ng/ml did not result in a greater magnitude of induction. The possibility that these two TPA-induced proteins may be identical to proteins with similar molecular weights induced under heat-shocked or glucose-starved conditions was evaluated by 1-D and 2-D gel electrophoresis and autoradiography. Results provided evidence that the TPA-induced 89,000- and 74,000-dalton proteins were identical to hsp 89 and hsp 74, 2 out of a set of 8-9 proteins induced under heat shocked conditions. Furthermore, they are identical to two of the set of glucose-regulated proteins induced under a glucose-starved condition

  13. Suppression of skin inflammation in keratinocytes and acute/chronic disease models by caffeic acid phenethyl ester.

    Science.gov (United States)

    Lim, Kyung-Min; Bae, SeungJin; Koo, Jung Eun; Kim, Eun-Sun; Bae, Ok-Nam; Lee, Joo Young

    2015-04-01

    Skin inflammation plays a central role in the pathophysiology and symptoms of diverse chronic skin diseases including atopic dermatitis (AD). In this study, we examined if caffeic acid phenethyl ester (CAPE), a skin-permeable bioactive compound from propolis, was protective against skin inflammation using in vitro cell system and in vivo animal disease models. CAPE suppressed TNF-α-induced NF-κB activation and expression of inflammatory cytokines in human keratinocytes (HaCaT). The potency and efficacy of CAPE were superior to those of a non-phenethyl derivative, caffeic acid. Consistently, topical treatment of CAPE (0.5 %) attenuated 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced skin inflammation on mouse ear as CAPE reduced ear swelling and histologic inflammation scores. CAPE suppressed increased expression of pro-inflammatory molecules such as TNF-α, cyclooxygenase-2 and inducible NO synthase in TPA-stimulated skin. TPA-induced phosphorylation of IκB and ERK was blocked by CAPE suggesting that protective effects of CAPE on skin inflammation is attributed to inhibition of NF-κB activation. Most importantly, in an oxazolone-induced chronic dermatitis model, topical application of CAPE (0.5 and 1 %) was effective in alleviating AD-like symptoms such as increases of trans-epidermal water loss, skin thickening and serum IgE as well as histologic inflammation assessment. Collectively, our results propose CAPE as a promising candidate for a novel topical drug for skin inflammatory diseases.

  14. Pre-clinical efficacy assessment of Malva sylvestris on chronic skin inflammation.

    Science.gov (United States)

    Prudente, Arthur S; Sponchiado, Graziela; Mendes, Daniel A G B; Soley, Bruna S; Cabrini, Daniela A; Otuki, Michel F

    2017-09-01

    In the search for improved quality of life, the treatment of skin diseases like psoriasis (hyperproliferative disease) is valid, since it causes huge social discomfort to the patient. In this context, earlier studies showed that Malva sylvestris L. has anti-inflammatory activity demonstrated by acute animal models of skin inflammation, becoming a promising target for further studies. The present investigation aimed to verify the effect of hydroalcoholic extract of M. sylvestris (HEMS) on the chronic inflammatory and hyperproliferative response caused by multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) on mouse ears. Topical application of HEMS reduced oedema, leukocyte migration (mono- and polymorphonuclear cells) and keratinocyte hyperproliferation, confirmed by histology and proliferating cell nuclear antigen (PCNA) immunostaining. It was found that the anti-inflammatory effects of the extract did not involve the glucocorticoid system, and its incubation with HaCaT keratinocytes caused low toxicity and reduced cell proliferation by apoptosis. Thus, HEMS proved to be effective as an anti-psoriatic therapy, with the ability to prevent keratinocyte hyperproliferation and with low toxicity by topical application. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  15. Effects of α-particle radiation on rat tracheal epithelial cells

    International Nuclear Information System (INIS)

    Ford, J.R. Jr.

    1992-08-01

    By a combination of methods, which included flow cytometry and magnetic cell sorting, we have demonstrated that the cells of the rat tracheal epithelium which have the greatest proliferative capacity in culture and in vivo are the basal cells. Because of these findings it seems reasonable to suppose that the basal cells are the most likely target for the action of α-particle radiation in pseudostratified respiratory epithelium. This hypothesis is further supported by the finding that the basal cells are the cells which appear to respond to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The effects of 210 Po α-particles on the survival and oncogenic transformation of rat tracheal epithelial cells in suspension were investigated. Since these effects were assayed in culture, the results pertain to the reaction of only the basal cells to irradiation. The results indicate that α-particles are extremely cytotoxic in that a track segment of 4 μm, on average, is sufficient to cause the reproductive death of basal cells. This finding is supported by similar results obtained with two cell lines, Mv1Lu and CHO-K1 BH 4 . Production of proliferating epithelial foci by α-particles was not distinguishable from control and sham treatments. These results are in direct conflict with many of the results that have been obtained with C3H 1OT1/2 cells in similar transformation assays. Some possible reasons for these disparities are discussed and supporting evidence is provided

  16. The Effects of Low Dose Irradiation on Inflammatory Response Proteins in a 3D Reconstituted Human Skin Tissue Model

    Energy Technology Data Exchange (ETDEWEB)

    Varnum, Susan M.; Springer, David L.; Chaffee, Mary E.; Lien, Katie A.; Webb-Robertson, Bobbie-Jo M.; Waters, Katrina M.; Sacksteder, Colette A.

    2012-12-01

    Skin responses to moderate and high doses of ionizing radiation include the induction of DNA repair, apoptosis, and stress response pathways. Additionally, numerous studies indicate that radiation exposure leads to inflammatory responses in skin cells and tissue. However, the inflammatory response of skin tissue to low dose radiation (<10 cGy) is poorly understood. In order to address this, we have utilized a reconstituted human skin tissue model (MatTek EpiDerm FT) and assessed changes in 23 cytokines twenty-four and forty eight hours following treatment of skin with either 3 or 10 cGy low-dose of radiation. Three cytokines, IFN-γ, IL-2, MIP-1α, were significantly altered in response to low dose radiation. In contrast, seven cytokines were significantly altered in response to a high radiation dose of 200 cGy (IL-2, IL-10, IL-13, IFN-γ, MIP-1α, TNF α, and VEGF) or the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (G-CSF, GM-CSF, IL-1α, IL-8, MIP-1α, MIP-1β, RANTES). Additionally, radiation induced inflammation appears to have a distinct cytokine response relative to the non-radiation induced stressor, TPA. Overall, these results indicate that there are subtle changes in the inflammatory protein levels following exposure to low dose radiation and this response is a sub-set of what is seen following a high dose in a human skin tissue model.

  17. Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

    Science.gov (United States)

    Li, Yafan; Wheeler, Deric L; Ananthaswamy, Honnavara N; Verma, Ajit K; Oberley, Terry D

    2007-12-01

    Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

  18. Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Myung-Hoon; Kim, Do-Hee [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Na, Hye-Kyung [Department of Food and Nutrition, Sungshin Women' s University, Seoul (Korea, Republic of); Kim, Jung-Hwan; Kim, Ha-Na [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Haegeman, Guy [LEGEST, University of Gent (Belgium); Surh, Young-Joon, E-mail: surh@snu.ac.kr [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2014-10-15

    Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

  19. Cancer-promoting effect of capsaicin on DMBA/TPA-induced skin tumorigenesis by modulating inflammation, Erk and p38 in mice.

    Science.gov (United States)

    Liu, Zhaoguo; Zhu, Pingting; Tao, Yu; Shen, Cunsi; Wang, Siliang; Zhao, Lingang; Wu, Hongyan; Fan, Fangtian; Lin, Chao; Chen, Chen; Zhu, Zhijie; Wei, Zhonghong; Sun, Lihua; Liu, Yuping; Wang, Aiyun; Lu, Yin

    2015-07-01

    Epidemiologic and animal studies revealed that capsaicin (8-methyl-N-vanillyl-6-noneamide) can act as a carcinogen or cocarcinogen. However, the influence of consumption of capsaicin-containing foods or vegetables on skin cancer patients remains largely unknown. In the present study, we demonstrated that capsaicin has a cocarcinogenic effect on 9, 10-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumorigenesis. Our results showed that topical application of capsaicin on the dorsal skin of DMBA-initiated and TPA-promoted mice could significantly accelerate tumor formation and growth and induce more and larger skin tumors than the model group (DMBA + TPA). Moreover, capsaicin could promote TPA-induced skin hyperplasia and tumor proliferation. Mechanistic study found that inflammation-related factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were highly elevated by pretreatment with capsaicin, suggesting an inflammation-dependent mechanism. Furthermore, mice that were administered capsaicin exhibited significant up-regulation of phosphorylation of nuclear factor kappaB (NF-κB), Erk and p38 but had no effect on JNK. Thus, our results indicated that inflammation, Erk and P38 collectively played a crucial role in cancer-promoting effect of capsaicin on carcinogen-induced skin cancer in mice. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Depression- and anxiety-like behaviour is related to BDNF/TrkB signalling in a mouse model of psoriasis.

    Science.gov (United States)

    JiaWen, W; Hong, S; ShengXiang, X; Jing, L

    2018-04-01

    The prevalence of anxiety and depression is significantly higher in individuals with psoriasis than in the general population. Clinical data also show that anti-anxiety and antidepression drugs can reduce skin lesions in patients with psoriasis, but the actual mechanism is still poorly understood. To investigate whether brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrKB) signalling plays a role in the mechanism underlying psoriasis with depression and anxiety behaviours. Expression of BDNF and tropomyosin receptor kinase B (TrKB) in the K5.Stat3C mouse, an animal model of psoriasis, were investigated by reverse transcription PCR and Western blotting. Anxiety-like behaviours in the elevated-plus maze test and changes in BDNF/TrkB that have been implicated in depression and anxiety behaviours were measured. Skin lesions induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) were also measured when the mice were administered fluoxetine and K252a, an antagonist of TrkB. The antidepression and anti-anxiety drug fluoxetine reduced TPA-induced skin lesions and increased expression of BDNF and TrkB in K5.Stat3C mice. More importantly, the effects of fluoxetine were reversed by the TrkB antagonist K252a. BDNF/TrkB signalling participates in the pathological mechanism of depression and anxiety behaviours in psoriasis. Our findings provide a new therapeutic strategy for the treatment of skin lesions in psoriasis. © 2018 British Association of Dermatologists.

  1. Cellular Dichotomy Between Anchorage-Independent Growth Responses to bFGF and TA Reflects Molecular Switch in Commitment to Carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Waters, Katrina M.; Tan, Ruimin; Opresko, Lee K.; Quesenberry, Ryan D.; Bandyopadhyay, Somnath; Chrisler, William B.; Weber, Thomas J.

    2009-11-01

    We have investigated gene expression patterns underlying reversible and irreversible anchorage-independent growth (AIG) phenotypes to identify more sensitive markers of cell transformation for studies directed at interrogating carcinogenesis responses. In JB6 mouse epidermal cells, basic fibroblast growth factor (bFGF) induces an unusually efficient and reversible AIG response, relative to 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced AIG which is irreversible. The reversible and irreversible AIG phenotypes are characterized by largely non-overlapping global gene expression profiles. However, a subset of differentially expressed genes were identified as common to reversible and irreversible AIG phenotypes, including genes regulated in a reciprocal fashion. Hepatic leukemia factor (HLF) and D-site albumin promoter-binding protein (DBP) were increased in both bFGF and TPA soft agar colonies and selected for functional validation. Ectopic expression of human HLF and DBP in JB6 cells resulted in a marked increase in TPA- and bFGF-regulated AIG responses. HLF and DBP expression were increased in soft agar colonies arising from JB6 cells exposed to gamma radiation and in a human basal cell carcinoma tumor tissue, relative to paired non-tumor tissue. Subsequent biological network analysis suggests that many of the differentially expressed genes that are common to bFGF- and TPA-dependent AIG are regulated by c-Myc, SP-1 and HNF-4 transcription factors. Collectively, we have identified a potential molecular switch that mediates the transition from reversible to irreversible AIG.

  2. Inhibitory effects of curcumin and capsaicin on phorbol ester-induced activation of eukaryotic transcription factors, NF-kappaB and AP-1.

    Science.gov (United States)

    Surh, Y J; Han, S S; Keum, Y S; Seo, H J; Lee, S S

    2000-01-01

    Recently, considerable attention has been focused on identifying dietary and medicinal phytochemicals that can inhibit, retard or reverse the multi-stage carcinogenesis. Spices and herbs contain phenolic substances with potent antioxidative and chemopreventive properties. Curcumin, a yellow colouring agent from turmeric and capsaicin, a pungent principle of red pepper exhibit profound anticarcinogenic and antimutagenic activities. Two well-defined eukaryotic transcription factors, nuclear factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) have been implicated in pathogenesis of many human diseases including cancer. These transcription factors are known to be activated by a wide array of external stimuli, such as tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor, reactive oxygen species, bacterial lipopolysaccharide, and ultraviolet. In the present study, we found that topical application of TPA onto dorsal skin of female ICR mice resulted in marked activation of epidermal NF-kappaB and AP-1. Curcumin and capsaicin, when topically applied prior to TPA, significantly attenuated TPA-induced activation of each transcription factor in mouse skin. Likewise, both compounds inhibited NF-kappaB and AP-1 activation in cultured human promyelocytic leukemia (HL-60) cells stimulated with TPA. Based on these findings, it is likely that curcumin and capsaicin exert anti-tumor promotional effects through suppression of the tumor promoter-induced activation of transcription factors, NF-kappaB and AP-1.

  3. Radiation induction of cancer of the skin

    International Nuclear Information System (INIS)

    Fry, R.J.M.; Storer, J.B.; Burns, F.J.

    1985-01-01

    The induction of epidermal tumors was studied using exposures to 25 kV x-rays with or without subsequent exposures to 12-0-tetradeconyl phorbol-13 acetate (TPA) or ultraviolet radiation (uvr) 280-400 nm. Fractionation regimens and total exposure up to 4000R produced no squamous cell carcinomas. When these regimes were followed by TPA an incidence of about 80% was obtained, and incidence of 60% when uvr exposures followed the x-irradiation. A dose-dependent increase in fibrosarcomas was found when x-irradiation was followed by 24 weeks of topical treatment with TPA. These results support the contention that uvr can enhance the expression of cells initiated by x-rays. The experimental evidence is compared with the data from the tinea capitis patients treated with x-rays. In C3HF/He male mice exposed to 50, 100, 150 and 200 rads 137 Cs gamma rays the induction rate for fibrosarcomas was 2.9 x 10 -4 per cGy/per mouse. This result compares with 2.5 x 10 -6 transformations per surviving cell per cGy with 10T1/2 cells that are fibroblasts derived from C3H mice. 16 refs., 1 fig., 1 tab

  4. Cancer chemoprevention by ginseng in mouse liver and other organs.

    Science.gov (United States)

    Nishino, H; Tokuda, H; Ii, T; Takemura, M; Kuchide, M; Kanazawa, M; Mou, X Y; Bu, P; Takayasu, J; Onozuka, M; Masuda, M; Satomi, Y; Konoshima, T; Kishi, N; Baba, M; Okada, Y; Okuyama, T

    2001-01-01

    Oral administration of red ginseng extracts (1% in diet for 40 weeks) resulted in the significant suppression of spontaneous liver tumor formation in C3H/He male mice. Average number of tumors per mouse in control group was 1.06, while that in red ginseng extracts-treated group was 0.33 (p<0.05). Incidence of liver tumor development was also lower in red ginseng extracts-treated group, although the difference from control group was not statistically significant. Anti-carcinogenic activity of white ginseng extracts, besides red ginseng extracts, was also investigated. In the present study, the administration of white ginseng extracts was proven to suppress tumor promoter-induced phenomena in vitro and in vivo. It is of interest that oral administration of the extracts of Ren-Shen-Yang- Rong-Tang, a white ginseng-containing Chinese medicinal prescription, resulted in the suppression of skin tumor promotion by 12-o-tetradecanoylphorbol-13-acetate in 7,12-dimethylbenz[a]anthracene-initiated CD-1 mice. These results suggest the usefulness of ginseng in the field of cancer prevention. PMID:11748379

  5. Inflammation, oxidative DNA damage, and carcinogenesis

    International Nuclear Information System (INIS)

    Lewis, J.G.; Adams, D.O.

    1987-01-01

    Inflammation has long been associated with carcinogenesis, especially in the promotion phase. The mechanism of action of the potent inflammatory agent and skin promoter 12-tetradecanoyl phorbol-13-acetate (TPA) is unknown. It is though that TPA selectively enhances the growth of initiated cells, and during this process, initiated cells progress to the preneoplastic state and eventually to the malignant phenotype. The authors and others have proposed that TPA may work, in part, by inciting inflammation and stimulating inflammatory cells to release powerful oxidants which then induce DNA damage in epidermal cells. Macrophages cocultured with target cells and TPA induce oxidized thymine bases in the target cells. This process is inhibited by both catalase and inhibitors of lipoxygenases, suggesting the involvement of both H 2 O 2 and oxidized lipid products. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin that C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H 2 O 2 and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice. These data support the hypothesis that inflammation and the release of genotoxic oxidants may be one mechanism whereby initiated cells receive further genetic insults. They also further complicate risk assessment by suggesting that some environmental agents may work indirectly by subverting host systems to induce damage rather than maintaining homeostasis

  6. Essential oil from waste leaves of Curcuma longa L. alleviates skin inflammation.

    Science.gov (United States)

    Kumar, Anant; Agarwal, Karishma; Singh, Monika; Saxena, Archana; Yadav, Pankaj; Maurya, Anil Kumar; Yadav, Anju; Tandon, Sudeep; Chanda, Debabrata; Bawankule, Dnyaneshwar U

    2018-02-10

    Curcuma longa L. is an important industrial crop used by medicinal and cosmetic industries in the world. Its leaves are a waste material after harvesting rhizomes. The aim of the study was to evaluate the chemical and pharmacological profile of essential oil from waste leaves of Curcuma longa (EOCl) against skin inflammation. EOCl was subjected to gas chromatography (GC) analysis for identification of essential oil constituents and its anti-inflammatory evaluation through in vitro and in vivo models. Chemical fingerprinting using GC and GC-MS analysis of EOCl revealed the presence of 11 compounds, representing 90.29% of the oil, in which terpinolene (52.88%) and α-phellandrene (21.13%) are the major components. In the in vitro testing EOCl inhibited the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the human keratinocyte cell line (HaCaT). Topical application of EOCl produced anti-inflammatory effects by reducing ear thickness, ear weight and ameliorating the level of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) at protein and mRNA levels as well as regulating the overproduction of oxidative markers and restoring the histopathological damage in a TPA-induced mouse model of inflammation. These findings of topical anti-inflammatory properties of EOCl provide a scientific basis for medicinal use of this plant material against inflammatory disorders.

  7. Poly-γ-Glutamic Acid Induces Apoptosis via Reduction of COX-2 Expression in TPA-Induced HT-29 Human Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Eun Ju Shin

    2015-04-01

    Full Text Available Poly-γ-glutamic acid (PGA is one of the bioactive compounds found in cheonggukjang, a fast-fermented soybean paste widely utilized in Korean cooking. PGA is reported to have a number of beneficial health effects, and interestingly, it has been identified as a possible anti-cancer compound through its ability to promote apoptosis in cancer cells, although the precise molecular mechanisms remain unclear. Our findings demonstrate that PGA inhibits the pro-proliferative functions of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, a known chemical carcinogen in HT-29 human colorectal cancer cells. This inhibition was accompanied by hallmark apoptotic phenotypes, including DNA fragmentation and the cleavage of poly (ADP-ribose polymerase (PARP and caspase 3. In addition, PGA treatment reduced the expression of genes known to be overexpressed in colorectal cancer cells, including cyclooxygenase 2 (COX-2 and inducible nitric oxide synthase (iNOS. Lastly, PGA promoted activation of 5' adenosine monophosphate-activated protein (AMPK in HT-29 cells. Taken together, our results suggest that PGA treatment enhances apoptosis in colorectal cancer cells, in part by modulating the activity of the COX-2 and AMPK signaling pathways. These anti-cancer functions of PGA make it a promising compound for future study.

  8. Salvia miltiorrhiza extract inhibits TPA-induced MMP-9 expression and invasion through the MAPK/AP-1 signaling pathway in human breast cancer MCF-7 cells.

    Science.gov (United States)

    Kim, Jeong-Mi; Noh, Eun-Mi; Song, Hyun-Kyung; Lee, Minok; Lee, Soo Ho; Park, Sueng Hyuk; Ahn, Chan-Keun; Lee, Guem-San; Byun, Eui-Baek; Jang, Beom-Su; Kwon, Kang-Beom; Lee, Young-Rae

    2017-09-01

    Cancer cell invasion is crucial for metastasis. A major factor in the capacity of cancer cell invasion is the activation of matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix. Salvia miltiorrhiza has been used as a promotion for blood circulation to remove blood stasis. Numerous previous studies have demonstrated that S. miltiorrhiza extracts (SME) decrease lipid levels and inhibit inflammation. However, the mechanism behind the effect of SME on breast cancer invasion has not been identified. The inhibitory effects of SME on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression were assessed using western blotting, reverse transcription-quantitative polymerase chain reaction and zymography assays. MMP-9 upstream signal proteins, including mitogen-activated protein kinases and activator protein 1 (AP-1) were also investigated. Cell invasion was assessed using a matrigel invasion assay. The present study demonstrated the inhibitory effects of the SME ethanol solution on MMP-9 expression and cell invasion in TPA-treated MCF-7 breast cancer cells. SME suppressed TPA-induced MMP-9 expression and MCF-7 cell invasion by blocking the transcriptional activation of AP-1. SME may possess therapeutic potential for inhibiting breast cancer cell invasiveness.

  9. Cell model for the study of receptor and regulatory functions of human proHB-EGF

    Directory of Open Access Journals (Sweden)

    N. V. Korotkevych

    2014-08-01

    Full Text Available Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation of transfected cells with TPA (12-O-Tetradecanoylphorbol-13-acetate, proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule – CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

  10. Docosahexaenoic Acid Inhibits Tumor Promoter-Induced Urokinase-Type Plasminogen Activator Receptor by Suppressing PKCδ- and MAPKs-Mediated Pathways in ECV304 Human Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Sen Lian

    Full Text Available The overexpression of urokinase-type plasminogen activator receptor (uPAR is associated with inflammation and virtually all human cancers. Despite the fact that docosahexaenoic acid (DHA has been reported to possess anti-inflammatory and anti-tumor properties, the negative regulation of uPAR by DHA is still undefined. Here, we investigated the effect of DHA on 12-O-tetradecanoylphorbol-13-acetate (TPA-induced uPAR expression and the underlying molecular mechanisms in ECV304 human endothelial cells. DHA concentration-dependently inhibited TPA-induced uPAR. Specific inhibitors and mutagenesis studies showed that PKCδ, JNK1/2, Erk1/2, NF-κB, and AP-1 were critical for TPA-induced uPAR expression. Application of DHA suppressed TPA-induced translocation of PKCδ, activation of the JNK1/2 and Erk1/2 signaling pathways, and subsequent AP-1 and NF-κB transactivation. In conclusion, these observations suggest a novel role for DHA in reducing uPAR expression and cell invasion by inhibition of PKCδ, JNK1/2, and Erk1/2, and the reduction of AP-1 and NF-κB activation in ECV304 human endothelial cells.

  11. Effects of ketamine on glucose uptake by glucose transporter type 3 expressed in Xenopus oocytes: The role of protein kinase C

    International Nuclear Information System (INIS)

    Tomioka, Shigemasa; Kaneko, Miyuki; Satomura, Kazuhito; Mikyu, Tomiko; Nakajo, Nobuyoshi

    2009-01-01

    We investigated the effects of ketamine on the type 3 facilitative glucose transporter (GLUT3), which plays a major role in glucose transport across the plasma membrane of neurons. Human-cloned GLUT3 was expressed in Xenopus oocytes by injection of GLUT3 mRNA. GLUT3-mediated glucose uptake was examined by measuring oocyte radioactivity following incubation with 2-deoxy-D-[1,2- 3 H]glucose. While ketamine and S(+)-ketamine significantly increased GLUT3-mediated glucose uptake, this effect was biphasic such that higher concentrations of ketamine inhibited glucose uptake. Ketamine (10 μM) significantly increased V max but not K m of GLUT3 for 2-deoxy-D-glucose. Although staurosporine (a protein kinase C inhibitor) increased glucose uptake, no additive or synergistic interactions were observed between staurosporine and racemic ketamine or S(+)-ketamine. Treatment with ketamine or S(+)-ketamine partially prevented GLUT3 inhibition by the protein kinase C activator phorbol-12-myrisate-13-acetate. Our results indicate that ketamine increases GLUT3 activity at clinically relevant doses through a mechanism involving PKC inhibition.

  12. Dihydro-β-agarofuran sesquiterpenes from celastraceae species as anti-tumour-promoting agents: Structure-activity relationship.

    Science.gov (United States)

    Núñez, Marvin J; Jiménez, Ignacio A; Mendoza, Cristina R; Chavez-Sifontes, Marvin; Martinez, Morena L; Ichiishi, Eiichiro; Tokuda, Ryo; Tokuda, Harukuni; Bazzocchi, Isabel L

    2016-03-23

    Inhibition of tumour promotion in multistage chemical carcinogenesis is considered a promising strategy for cancer chemoprevention. In an ongoing investigation of bioactive secondary metabolites from Celastraceae species, five new dihydro-β-agarofuran sesquiterpenes (1-5), named Chiapens A-E, and seventeen known ones, were isolated from Maytenus chiapensis. Their structures were elucidated by extensive NMR spectroscopic and mass spectrometric techniques, and their absolute configurations were determined by circular dichroism studies, chemical correlations and biogenic means. The isolated compounds, along with twenty known sesquiterpenes, previously isolated from Zinowiewia costaricensis, have been tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorpol-13-acetate (TPA). Thirty three compounds from this series showed stronger effects than that of β-carotene, the reference inhibitor. The structure-activity relationship (SAR) analysis revealed that the type of substituent, in particular at the C-1 position of the sesquiterpene scaffold, was able to modulate the anti-tumour promoting activity. Compounds 3, 6, and 33 showed significant effects in an in vivo two-stage mouse-skin carcinogenesis model. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Quantification of tumour initiating effect of jute batching oil and its distillates over mouse skin.

    Science.gov (United States)

    Agarwal, R; Kumar, S; Shukla, Y; Antony, M; Mehrotra, N K

    1985-09-30

    In order to identify the tumour initiating constituent(s) of a mineral oil, jute batching oil (JBO), used in the processing of jute fibres, it was fractionally distilled in various boiling range fractions. The latter were then subjected to in vivo assessment of their aryl hydrocarbon hydroxylase (AHH) inducing potential in mouse epidermis. Fractions with almost similar AHH inducing potential were regrouped and studied for their tumour initiating potential over mouse skin following two-stage initiation-promotion protocol and using 12-O-tetradecanoyl phorbol-13-acetate (TPA) as tumour promoter. It was noticed that: (1) JBO as initiator, provoked local development of benign skin tumours over mouse back; (2) fractions of JBO boiling below 335 degrees C and above 399 degrees C accounted for most of the tumour initiating potential of the oil; (3) the histological features of the tumours (i.e. benign papillomas and keratoacanthomas) initiated by these fractions were similar to those developed after being initiated with unfractionated or reconstituted JBO; (4) removal of these fractions from JBO may be attempted which could decontaminate the batch oil from most of its tumorigenic components and make it safer for industrial use.

  14. Broccoli and watercress suppress matrix metalloproteinase-9 activity and invasiveness of human MDA-MB-231 breast cancer cells

    International Nuclear Information System (INIS)

    Rose, Peter; Huang, Qing; Ong, Choon Nam; Whiteman, Matt

    2005-01-01

    A high dietary intake of cruciferous vegetables has been associated with a reduction in numerous human pathologies particularly cancer. In the current study, we examined the inhibitory effects of broccoli (Brassica oleracea var. italica) and watercress (Rorripa nasturtium aquaticum) extracts on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cancer cell invasion and matrix metalloproteinase-9 activity using human MDA-MB-231 breast cancer cells. Aberrant overexpression of matrix metalloproteinases, including metalloproteinase-9, is associated with increased invasive potential in cancer cell lines. Our results demonstrate that extracts of broccoli and Rorripa suppressed TPA-induced MMP-9 activity and invasiveness in a concentration dependant manner as determined by zymographic analysis. Furthermore, fractionation of individual extracts followed by liquid chromatography mass spectroscopy analysis (LC-MS) revealed that the inhibitory effects of each vegetable were associated with the presence of 4-methysulfinylbutyl (sulforaphane) and 7-methylsulphinylheptyl isothiocyanates. Taken together, our data indicate that isothiocyanates derived form broccoli and Rorripa inhibit metalloproteinase 9 activities and also suppress the invasive potential of human MDA-MB-231 breast cancer cells in vitro. The inhibitory effects observed in the current study may contribute to the suppression of carcinogenesis by diets high in cruciferous vegetables

  15. Role of the Slug Transcription Factor in Chemically-Induced Skin Cancer

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    Kristine von Maltzan

    2016-02-01

    Full Text Available The Slug transcription factor plays an important role in ultraviolet radiation (UVR-induced skin carcinogenesis, particularly in the epithelial-mesenchymal transition (EMT occurring during tumor progression. In the present studies, we investigated the role of Slug in two-stage chemical skin carcinogenesis. Slug and the related transcription factor Snail were expressed at high levels in skin tumors induced by 7,12-dimethylbenz[α]anthracene application followed by 12-O-tetradecanoylphorbol-13-acetate (TPA treatment. TPA-induced transient elevation of Slug and Snail proteins in normal mouse epidermis and studies in Slug transgenic mice indicated that Slug modulates TPA-induced epidermal hyperplasia and cutaneous inflammation. Although Snail family factors have been linked to inflammation via interactions with the cyclooxygenase-2 (COX-2 pathway, a pathway that also plays an important role in skin carcinogenesis, transient TPA induction of Slug and Snail appeared unrelated to COX-2 expression. In cultured human keratinocytes, TPA induced Snail mRNA expression while suppressing Slug expression, and this differential regulation was due specifically to activation of the TPA receptor. These studies show that Slug and Snail exhibit similar patterns of expression during both UVR and chemical skin carcinogenesis, that Slug and Snail can be differentially regulated under some conditions and that in vitro findings may not recapitulate in vivo results.

  16. Photodynamic therapy using a novel irradiation source, LED lamp, is similarly effective to photodynamic therapy using diode laser or metal-halide lamp on DMBA- and TPA-induced mouse skin papillomas.

    Science.gov (United States)

    Takahashi, Hidetoshi; Nakajima, Susumu; Ogasawara, Koji; Asano, Ryuji; Nakae, Yoshinori; Sakata, Isao; Iizuka, Hajime

    2014-08-01

    Photodynamic therapy (PDT) is useful for superficial skin tumors such as actinic keratosis and Bowen disease. Although PDT is non-surgical and easily-performed treatment modality, irradiation apparatus is large and expensive. Using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-ο-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin papilloma model, we compared the efficacy of TONS501- and ALA-PDT with a LED lamp, a diode laser lamp or a metal-halide lamp on the skin tumor regression. TONS501-PDT using 660 nm LED lamp showed anti-tumor effect at 1 day following the irradiation and the maximal anti-tumor effect was observed at 3 days following the irradiation. There was no significant difference in the anti-tumor effects among TONS501-PDT using LED, TONS501-PDT using diode laser, and 5-aminolevulinic acid hydrochloride (ALA)-PDT using metal-halide lamp. Potent anti-tumor effect on DMBA- and TPA-induced mouse skin papilloma was observed by TONS501-PDT using 660 nm LED, which might be more useful for clinical applications. © 2014 Japanese Dermatological Association.

  17. Endoplasmic reticulum stress causes EBV lytic replication.

    Science.gov (United States)

    Taylor, Gwen Marie; Raghuwanshi, Sandeep K; Rowe, David T; Wadowsky, Robert M; Rosendorff, Adam

    2011-11-17

    Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

  18. Anti-allergic effects of a nonameric peptide isolated from the intestine gastrointestinal digests of abalone (Haliotis discus hannai) in activated HMC-1 human mast cells.

    Science.gov (United States)

    Ko, Seok-Chun; Lee, Dae-Sung; Park, Won Sun; Yoo, Jong Su; Yim, Mi-Jin; Qian, Zhong-Ji; Lee, Chang-Min; Oh, Junghwan; Jung, Won-Kyo; Choi, Il-Whan

    2016-01-01

    The aim of the present study was to examine whether the intestine gastrointestinal (GI) digests of abalone [Haliotis discus hannai (H. discus hannai)] modulate inflammatory responses and to elucidate the mechanisms involved. The GI digests of the abalone intestines were fractionated into fractions I (>10 kDa), II (5-10 kDa) and Ⅲ (abalone intestine GI digests (AIGIDs), fraction Ⅲ inhibited the passive cutaneous anaphylaxis (PCA) reaction in mice. Subsequently, a bioactive peptide [abalone intestine GI digest peptide (AIGIDP)] isolated from fraction Ⅲ was determined to be 1175.2 Da, and the amino acid sequence was found to be PFNQGTFAS. We noted that the purified nonameric peptide (AIGIDP) attenuated the phorbol‑12‑myristate 13-acetate plus calcium ionophore A23187 (PMACI)-induced histamine release and the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in human mast cells (HMC-1 cells). In addition, we also noted that AIGIDP inhibited the PMACI‑induced activation of nuclear factor‑κB (NF-κB) by suppressing IκBα phosphorylation and that it suppressed the production of cytokines by decreasing the phosphorylation of JNK. The findings of our study indicate that AIGIDP exerts a modulatory, anti-allergic effect on mast cell-mediated inflammatory diseases.

  19. Calcium permeability of the T lymphocyte plasma membrane: counteraction of phorbol ester and A23187

    Energy Technology Data Exchange (ETDEWEB)

    Csermely, P.; Szamel, M.; Somogyi, J.

    1986-01-01

    The intracellular calcium concentration (Ca/sub i/) of T lymphocytes was measured using the fluorescent indicator quin2. Different ionophores effectively enhanced the Ca permeability of the plasma membrane. The effective concentration of the ionophores required for permeabilization increased in the order of ionomycin, A23187 and X537-A (lasalocid-A). 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in submicromolar concentrations did not change Ca/sub i/. The addition of TPA immediately before the A23187-permeabilization did not alter the Ca ionophoretic effect of A23187. However, prolonged incubation with TPA decreased the efficiency of A23187 permeabilizing the plasma membrane for calcium ions. This effect was concentration and time dependent, being maximal at TPA concentrations higher than 10 nM with a preincubation time of 1.5 hours. TPA induced relative A23187 insensitivity is most probably not due to a direct effect of TPA on the ionophore as it is concentration and time dependent. Moreover the fluorescence and fluorescence polarization of A23187 as well as the energy transfer between the tryptophan groups of the membrane proteins and A23187 showed no significant change during incubation with TPA. These results indicate that membrane fluidity changes or A23187 immobilization also do not play a prominent role in the explanation of the phenomenon. However the supposed intracellular heavy metal content of T lymphocyte might be a possible source of the TPA induced relative insensitization towards A23187.

  20. Hair follicle stem cell proliferation, Akt and Wnt signaling activation in TPA-induced hair regeneration.

    Science.gov (United States)

    Qiu, Weiming; Lei, Mingxing; Zhou, Ling; Bai, Xiufeng; Lai, Xiangdong; Yu, Yu; Yang, Tian; Lian, Xiaohua

    2017-06-01

    Regeneration of hair follicles relies on activation of hair follicle stem cells during telogen to anagen transition process in hair cycle. This process is rigorously controlled by intrinsic and environmental factors. 12-o-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, accelerates reentry of hair follicles into anagen phase. However, it is unclear that how TPA promotes the hair regeneration. In the present study, we topically applied TPA onto the dorsal skin of 2-month-old C57BL/6 female mice to examine the activity of hair follicle stem cells and alteration of signaling pathways during hair regeneration. We found that refractory telogen hair follicles entered anagen prematurely after TPA treatment, with the enhanced proliferation of CD34-positive hair follicle stem cells. Meanwhile, we observed Akt signaling was activated in epidermis, hair infundibulum, bulge and hair bulb, and Wnt signaling was also activated after hair follicle stem cells proliferation. Importantly, after overexpression of DKK1, a specific Wnt signaling inhibitor, the accelerated reentry of hair follicles into anagen induced by TPA was abolished. Our data indicated that TPA-induced hair follicle regeneration is associated with activation of Akt and Wnt/β-catenin signaling.

  1. EFFECT OF SOME PLANT OILS ON THE FATTY ACIDS OF HUMP BEETLE GIBBIUM PSYLLOIDES

    International Nuclear Information System (INIS)

    ABBASY, S.A.; ALY, W.M.; REZK, S.A.

    2008-01-01

    The efficiency of some plant oils (sesame oil, camphor oil, castor oil and camomile oil) as insecticides were studied on newly emerged males and females Gibbium psylloids and also the effect of LC50 of each plant oils on fatty acids composition and relative concentration were determined. The obtained results showed that sesame oil had proved to be more effective with it's advantage over other tested oils of being used at lower concentration and it followed by chamomile oil then camphor oil and finally castor oil. The susceptibility of the males and females to plant oils was the same. There were differences in the number and relative concentration of fatty acids between males and females. Nine fatty acids were detected by the analysis of the whole body of untreated males , these fatty acids are capric (C10) , lauric (C12) , myristic (C14), palmiloleic (C16), margic (C17), stearic (C18) , oleic (C18:1) , linoleic (C18:2) and linolenic (C18:3) . Treatments of males with LC50 sesame oil lead to the appearance of ten fatty acids with increase of caprylic acid (C8) while LC50 chamomile oil lead to the appearance of six fatty acids and fatty acid with C10 , C18:1 and C18:2 were disappeared as compared to control. LC50 of camphor oil lead to the appearance of ten fatty acids and castor oil lead to the appearance of six fatty acids. In untreated females, five fatty acids with C12 , C16 , C17 , C18 and C18:1 were detected. Treatments with sesame oil lead to the appearance of new fatty acid with C18:2 and chamomile oil lead to the appearance of nine fatty acids with increase of C8 , C10 , C14 and C18:2 as compared with untreated females. Camphor oil treatment lead to the appearance of nine fatty acids with increase of four fatty acids with C6 , C8 , C10 and C18:2 as compared to untreated females and fatty acid with C18:1 was disappeared in the treatments. Castor oil leads to the appearance of seven fatty acids. The relative concentration of C12 was the highest fatty acid in

  2. AKTIFITAS ANTIINFLAMASI TOPIKAL MINYAK ATSIRI DAN EKSTRAK ETER TUMBUHAN TENGGULUN, PROTIUM JAVANICUM, BURM TERHADAP MODEL INFLAMASI KULIT PADA TIKUS

    Directory of Open Access Journals (Sweden)

    I Wayan Suirta

    2016-06-01

    Full Text Available ABSTRAK: Tenggulun (Protium javanicum, Burm secara tradisional telah dimanfaatkan masyarakat Bali sebagai tanaman obat untuk mengobati bengkak. Penelitian ini bertujuan untuk menentukan aktivitas antiinflamasi ekstrak eter kulit batang dan minyak atsiri daun tengulun pada udem (inflamasi telinga tikus yang diinduksi TPA (12-O-tetradecanoylphorbol-13-acetate secara topikal. Ekstraksi dilakukan dengan maserasi dan isolasi minyak atsiri dengan distilasi uap. Pada penelitian ini digunakan rancangan acak lengkap dengan sembilan kelompok perlakuan dengan masing-masing perlakuan terdiri dari lima ekor tikus. Inflamasi diukur 6 jam setelah perlakuan. Hasil triturasi 180g ekstrak etanol kulit batang tenggulun diperoleh 6,30 g ekstrak dietil eter dan hasil distilasi uap 4,5 kg daun segar diperoleh 2,5 g minyak atsiri. Hasil uji fitokimia ekstrak kulit batang tenggulun mengandung senyawa golongan terpenoid dan minyak atsiri mengandung senyawa flavonoid, terpenoid, dan steroid. Hasil uji aktifitas antiinflamasi menunjukkan bahwa persentase hambatan peradangan dari ekstrak tenggulun didapatkan : KEI (58,34% , KEII (54,17% , KEIII (45,85% dan KAI (50,02% yang menunjukkan hambatan yang tidak berbeda secara bermakna dengan hambatan yang diberikan oleh kontrol positip  KP (62,50%. Ekstrak eter 12 mg dan 20 mg masih memberikan hambatan peradangan yang lebih besar dari minyak atsiri 20 mg. Hasil uji histologi menunjukkan ekstrak eter dan minyak atsiri dapat menghambat migrasi sel radang lebih dari 50%.  Jumlah sel radang yang bermigrasi ke daerah peradangan sebanyak 17 sel untuk ekstrak eter dosis 20 mg/telinga  dan 20 sel radang untuk minyak atsiri dosis 20 mg/ telinga. Dengan induksi TPA jumlah sel radang yang bermigrasi ke daerah peradangan sebanyak 64 sel ABSTRACT: Protium javanicum, Burm (Tenggulun has been used traditionally in Bali as medicines to treat inflamation. This research aimed to evaluate topical anti-inflammatory activity of stem bark ether

  3. Pancreatic acini possess endothelin receptors whose internalization is regulated by PLC-activating agents.

    Science.gov (United States)

    Hildebrand, P; Mrozinski, J E; Mantey, S A; Patto, R J; Jensen, R T

    1993-05-01

    Endothelin-1 (ET-1) and ET-3 mRNA have been found in the pancreas. We investigated the ability of ET-1, ET-2, and ET-3 to interact with and alter dispersed rat pancreatic acinar cell function. Radiolabeled ETs bound in a time- and temperature-dependent fashion, which was specific and saturable. Analysis demonstrated two classes of receptors, one class (ETA receptor) had a high affinity for ET-1 but a low affinity for ET-3, and the other class (ETB receptor) had equally high affinities for ET-1 and ET-3. No specific receptor for ET-2 was identified. Pancreatic secretagogues that activate phospholipase C (PLC) inhibited binding of 125I-labeled ET-1 (125I-ET-1) or 125I-ET-3, whereas agents that act through adenosine 3',5'-cyclic monophosphate (cAMP) did not. A23187 had no effect on 125I-ET-1 or 125I-ET-3 binding, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate reduced binding. The effect of cholecystokinin octapeptide (CCK-8) was mediated through its own receptor. Stripping of surface bound ligand studies demonstrated that both 125I-labeled ET-1 and 125I-labeled ET-3 were rapidly internalized. CCK-8 decreased the internalization but did not change the amount of surface bound ligand. Endothelins neither stimulate nor alter changes in enzyme secretion, intracellular calcium, cAMP, or [3H]inositol trisphosphate (IP3). This study demonstrates the presence of ETA and ETB receptors on rat pancreatic acini; occupation of both receptors resulted in rapid internalization, which is regulated by PLC-activating secretagogues. Occupation of either ET receptor did not alter intracellular calcium, cAMP, IP3, or stimulate amylase release.

  4. Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

    Science.gov (United States)

    Metz, D C; Patto, R J; Mrozinski, J E; Jensen, R T; Turner, R J; Gardner, J D

    1992-10-15

    In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.

  5. Lactobacillus salivarius LA307 and Lactobacillus rhamnosus LA305 attenuate skin inflammation in mice.

    Science.gov (United States)

    Holowacz, S; Blondeau, C; Guinobert, I; Guilbot, A; Hidalgo, S; Bisson, J F

    2018-02-27

    Oral probiotics potential for the management of dermatological diseases is vast. However, results of available studies in skin diseases, such as atopic dermatitis (AD), are inconsistent, partly because probiotic effects are strain specific. Careful selection of probiotic strains is therefore indispensable to ensure efficacy of treatment. In this study, Lactobacillus salivarius LA307, Lactobacillus rhamnosus LA305 and Bifidobacterium bifidum PI22, three strains that were previously identified for their interesting immunomodulatory properties in allergy and/or colitis models, were assessed in the prevention of chronic skin inflammation induced by repeated applications of 12-O-tetradecanoylphorbol-13-acetate in hairless SKH-1 mice. Macroscopic and microscopic evaluation of skin lesions was performed together with measurements of serum levels of interleukin (IL)-1β, IL-6, tumour necrosis factor alpha (TNF-α), IL-17, IL-22, IL-10 and IL-4. Daily oral treatment with the three strains at the dose of 1×10 9 cfu/day for 3 weeks limited the development of chronic skin inflammation, the effects being strain dependent. Indeed the two Lactobacillus strains significantly limited the intensity of skin inflammation both at the macroscopic and microscopic levels. Macroscopic observations were correlated to the histological observations and the resulting microscopic score. This limitation of the development of AD-like skin lesions involved the modulation of cytokine production. Treatment with the two Lactobacillus strains induced a decrease in the serum levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, IL-17, IL-22 and at the opposite an increase in the production of the anti-inflammatory cytokine IL-10 and also of IL-4. Globally, B. bifidum PI22 had lower benefits. These results obtained in mice suggest that L. salivarius LA307 and L. rhamnosus LA305 could be good candidates for preserving skin integrity and homeostasis via the modulation of the gut microbiota and that

  6. Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

    International Nuclear Information System (INIS)

    Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

    2009-01-01

    Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

  7. Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells.

    Science.gov (United States)

    Hirose, H; Suhara, T; Kaji, N; Sakuma, N; Sekijima, M; Nojima, T; Miyakoshi, J

    2008-01-01

    A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation. (c) 2007 Wiley-Liss, Inc.

  8. Transformation assay in Bhas 42 cells: a model using initiated cells to study mechanisms of carcinogenesis and predict carcinogenic potential of chemicals.

    Science.gov (United States)

    Sasaki, Kiyoshi; Umeda, Makoto; Sakai, Ayako; Yamazaki, Shojiro; Tanaka, Noriho

    2015-01-01

    Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

  9. Effects of Intermediates between Vitamins K2 and K3 on Mammalian DNA Polymerase Inhibition and Anti-Inflammatory Activity

    Directory of Open Access Journals (Sweden)

    Takeshi Azuma

    2011-02-01

    Full Text Available Previously, we reported that vitamin K3 (VK3, but not VK1 or VK2 (=MK-4, inhibits the activity of human DNA polymerase γ (pol γ. In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF-α induced by lipopolysaccharide (LPS. Moreover, MK-2 was found to inhibit the action of nuclear factor (NF-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.

  10. Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation

    International Nuclear Information System (INIS)

    Kupper, T.S.; Chua, A.O.; Flood, P.; McGuire, J.; Gubler, U.

    1987-01-01

    Interleukin 1 (IL-1) is a family of polypeptides initially found to be produced by activated monocytes and macrophages that mediate a wide variety of cellular responses to injury and infection. Epidermal epithelial cells (keratinocytes) produce ''epidermal cell-derived thymocyte activating factor'' or ETAF, which has been recently shown to be identical to IL-1. Human epidermis is normally exposed to significant amounts of solar ultraviolet radiation. Certain ultraviolet wavelengths (UVB, 290-320 nm) are thought to be responsible for most of the immediate and long-term pathological consequences of excessive exposure to sunlight. In this study, we asked whether exposure to UVB irradiation induced IL-1 gene expression in cultured human keratinocytes. Cultured human keratinocytes contain detectable amounts of IL-1 alpha and beta mRNA and protein in the absence of apparent stimulation; these levels could be significantly enhanced 6 h after exposure to 10 ng/ml of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Exposure to UVB irradiation with an emission spectrum comparable to that of sunlight (as opposed to that of an unfiltered artificial UV light source) significantly increased the steady state levels IL-1 alpha and beta mRNA in identical populations of human keratinocytes. This was reflected in the production of increased IL-1 activity by these cultures in vitro. In the same cell population, exposures to UVB irradiation did not alter the level of actin mRNA; therefore, the effect of UV irradiation on IL-1 represents a specific enhancement of IL-1 gene expression. Local increases of IL-1 may mediate the inflammation and vasodilation characteristic of acute UVB-injured skin, and systemic release of this epidermal IL-1 may account for fever, leukocytosis, and the acute phase response seen after excessive sun exposure

  11. Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA

    International Nuclear Information System (INIS)

    Friedmann, P.S.; Wren, F.E.; Matthews, J.N.

    1990-01-01

    Epidermal melanocytes (MC) synthesize melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV-induced activation of melanogenesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E1, E2, D2, F2 alpha, and di-acyl glycerol upon the UV-induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradiated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di-acyl glycerol, 1-oleyl 2-acetyl glycerol (OAG), caused a significant (P less than 0.0001), dose-related augmentation of melanin content both in human MC (seven-fold) and S91 cells (three-fold). UVR caused a significant augmentation of the OAG-induced melanogenesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di-octanoyl glycerol, another di-acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12-0 tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV-induced melanin synthesis in either cell type. These data suggest that the UV-induced signal activating melanogenesis could be mediated by di-acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C

  12. Enhanced caveolin-1 expression increases migration, anchorage-independent growth and invasion of endometrial adenocarcinoma cells

    International Nuclear Information System (INIS)

    Diaz-Valdivia, Natalia; Bravo, Denisse; Huerta, Hernán; Henriquez, Soledad; Gabler, Fernando; Vega, Margarita; Romero, Carmen; Calderon, Claudia; Owen, Gareth I.; Leyton, Lisette; Quest, Andrew F. G.

    2015-01-01

    Caveolin-1 (CAV1) has been implicated both in tumor suppression and progression, whereby the specific role appears to be context dependent. Endometrial cancer is one of the most common malignancies of the female genital tract; however, little is known about the role of CAV1 in this disease. Here, we first determined by immunohistochemistry CAV1 protein levels in normal proliferative human endometrium and endometrial tumor samples. Then using two endometrial cancer cell lines (ECC: Ishikawa and Hec-1A) we evaluated mRNA and protein levels of CAV1 by real time qPCR and Western blot analysis, respectively. The role of CAV1 expression in ECC malignancy was further studied by either inducing its expression in endometrial cancer cells with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (4β-TPA) or decreasing expression using short-hairpin RNA constructs, and then evaluating the effects of these changes on ECC proliferation, transmigration, matrigel invasion, and colony formation in soft agar. Immunohistochemical analysis of endometrial epithelia revealed that substantially higher levels of CAV1 were present in endometrial tumors than the normal proliferative epithelium. Also, in Ishikawa and Hec-1A endometrial cancer cells CAV1 expression was readily detectable. Upon treatment with 4β-TPA CAV1 levels increased and coincided with augmented cell transmigration, matrigel invasion, as well as colony formation in soft agar. Reduction of CAV1 expression using short-hairpin RNA constructs ablated these effects in both cell types whether treated or not with 4β-TPA. Alternatively, CAV1 expression appeared not to modulate significantly proliferation of these cells. Our study shows that elevated CAV1, observed in patients with endometrial cancer, is linked to enhanced malignancy of endometrial cancer cells, as evidenced by increased migration, invasion and anchorage-independent growth. The online version of this article (doi:10.1186/s12885-015-1477-5) contains

  13. Topical anti-inflammatory activity of Polygonum cuspidatum extract in the TPA model of mouse ear inflammation

    Directory of Open Access Journals (Sweden)

    Wicker Louise

    2008-02-01

    Full Text Available Abstract Background This study tested the ability of a characterized extract of Polygonum cuspidatum (PCE to inhibit mouse ear inflammation in response to topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA. Methods A 50% (wt:vol ethanolic solution of commercial 200:1 PCE was applied to both ears of female Swiss mice (n = 8 at 0.075, 0.15, 0.3, 1.25 and 2.5 mg/ear 30 min after TPA administration (2 μg/ear. For comparison, 3 other groups were treated with TPA and either 1 the vehicle (50% ethanol alone, 2 indomethacin (0.5 mg/ear, or 3 trans-resveratrol (0.62 mg/ear. Ear thickness was measured before TPA and at 4 and 24 h post-TPA administration to assess ear edema. Ear punch biopsies were collected at 24 h and weighed as a second index of edema. Myeloperoxidase activity was measured in each ear punch biopsy to assess neutrophil infiltration. Results PCE treatment at all doses significantly reduced ear edema compared to the TPA control. The PCE response was dose-dependent and 2.5 mg PCE significantly inhibited all markers of inflammation to a greater extent than indomethacin (0.5 mg. MPO activity was inhibited at PCE doses ≥ 1.25 mg/ear. Trans-resveratrol inhibited inflammation at comparable doses. Conclusion PCE inhibits development of edema and neutrophil infiltration in the TPA-treated mouse ear model of topical inflammation.

  14. Anti-Inflammatory Effects and Mechanisms of Fatsia polycarpa Hayata and Its Constituents

    Directory of Open Access Journals (Sweden)

    Hsueh-Ling Cheng

    2013-01-01

    Full Text Available Fatsia polycarpa, a plant endemic to Taiwan, is an herbal medicine known for treating several inflammation-related diseases, but its biological function needs scientific support. Thus, the anti-inflammatory effects and mechanisms of the methanolic crude extract (MCE of F. polycarpa and its feature constituents, that is, brassicasterol (a phytosterol, triterpenoids 3α-hydroxyolean-11,13(18-dien-28-oic acid (HODA, 3α-hydroxyolean-11-en-28,13β-olide (HOEO, fatsicarpain D, and fatsicarpain F, were investigated. MCE and HOEO, but not brassicasterol, dose-dependently inhibited lipopolysaccharide- (LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophage line, whereas HODA, fatsicarpain D and fatsicarpain F were toxic to RAW cells. Additionally, MCE and HOEO suppressed LPS-induced production of nitric oxide, prostaglandin E2, and interleukin-1β and interfered with LPS-promoted activation of the inhibitor kappa B kinase (IKK/nuclear factor-κB (NF-κB pathway, and that of the mitogen-activated protein kinases (MAPKs extracellular signal regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38. In animal tests, MCE and HOEO effectively ameliorated 12-O-tetradecanoylphorobol-13 acetate- (TPA-induced ear edema of mice. Thus, MCE of F. polycarpa exhibited an obvious anti-inflammatory activity in vivo and in vitro that likely involved the inhibition of the IKK/NF-κB pathway and the MAPKs, which may be attributed by triterpenoids such as HOEO.

  15. Anti-inflammatory effect of transduced PEP-1-heme oxygenase-1 in Raw 264.7 cells and a mouse edema model

    International Nuclear Information System (INIS)

    Kwon, Soon Won; Sohn, Eun Jeong; Kim, Dae Won; Jeong, Hoon Jae; Kim, Mi Jin; Ahn, Eun Hee; Kim, Young Nam; Dutta, Suman; Kim, Duk-Soo; Park, Jinseu; Eum, Won Sik; Hwang, Hyun Sook; Choi, Soo Young

    2011-01-01

    Highlights: → Recombinant PEP-1 heme oxygenase-1 expression vector was constructed and overexpressed. → We investigated transduction efficiency of PEP-1-HO-1 protein in Raw 264.7 cells. → PEP-1-HO-1 was efficiently transduced into Raw 264.7 cells in a dose and time dependent manner. → PEP-1-HO-1 exerted anti-inflammatory activity in Raw 264.7 cells and in a mice edema model. → PEP-1-HO-1 could be used as a therapeutic drug against inflammatory diseases. -- Abstract: Heme oxygenase-1 (HO-1), which catalyzes the degradation of free heme to biliverdin, carbon monoxide (CO), and free iron (Fe 2+ ), is up-regulated by several cellular stress and cell injuries, including inflammation, ischemia and hypoxia. In this study, we examined whether fusion of HO-1 with PEP-1, a protein transduction domain that is able to deliver exogenous molecules to living cells or tissues, would facilitate HO-1 delivery to target cells and tissues, and thereby effectively exert a therapeutically useful response against inflammation. Western blot analysis demonstrated that PEP-1-HO-1 fusion proteins were transduced into Raw 264.7 cells in time- and dose-dependent manners, and were stably maintained in the cells for about 60 h. In addition, fluorescence analysis revealed that only PEP-1-HO-1 fusion proteins were significantly transduced into the cytoplasm of cells, while HO-1 proteins failed to be transduced. In lipopolysaccharide (LPS)-stimulated Raw 264.7 cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse edema model, transduced PEP-1-HO-1 fusion proteins effectively inhibited the overexpression of pro-inflammatory mediators and cytokines. Also, histological analysis demonstrated that PEP-1-HO-1 remarkably suppressed ear edema. The results suggest that the PEP-1-HO-1 fusion protein can be used as a therapeutic molecule against reactive oxygen species-related inflammatory diseases.

  16. Macrophage migration inhibitory factor triggers chemotaxis of CD74+CXCR2+ NKT cells in chemically induced IFN-γ-mediated skin inflammation.

    Science.gov (United States)

    Hsieh, Chia-Yuan; Chen, Chia-Ling; Lin, Yee-Shin; Yeh, Trai-Ming; Tsai, Tsung-Ting; Hong, Ming-Yuan; Lin, Chiou-Feng

    2014-10-01

    IFN-γ mediates chemically induced skin inflammation; however, the mechanism by which IFN-γ-producing cells are recruited to the sites of inflammation remains undefined. Secretion of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, from damaged cells may promote immune cell recruitment. We hypothesized that MIF triggers an initial step in the chemotaxis of IFN-γ-producing cells in chemically induced skin inflammation. Using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears, MIF expression was examined, and its role in this process was investigated pharmacologically. The cell populations targeted by MIF, their receptor expression patterns, and the effects of MIF on cell migration were examined. TPA directly caused cytotoxicity accompanied by MIF release in mouse ear epidermal keratinocytes, as well as in human keratinocytic HaCaT cells. Treatment with the MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester considerably attenuated TPA-induced ear swelling, leukocyte infiltration, epidermal cell proliferation, and dermal angiogenesis. Inhibition of MIF greatly diminished the dermal infiltration of IFN-γ(+) NKT cells, whereas the addition of exogenous TPA and MIF to NKT cells promoted their IFN-γ production and migration, respectively. MIF specifically triggered the chemotaxis of NKT cells via CD74 and CXCR2, and the resulting depletion of NKT cells abolished TPA-induced skin inflammation. In TPA-induced skin inflammation, MIF is released from damaged keratinocytes and then triggers the chemotaxis of CD74(+)CXCR2(+) NKT cells for IFN-γ production. Copyright © 2014 by The American Association of Immunologists, Inc.

  17. The effect of chemical carcinogenesis on rat glutathione S-transferase P1 gene transcriptional regulation.

    Science.gov (United States)

    Liu, D; Liao, M; Zuo, J; Henner, W D; Fan, F

    2001-03-01

    To investigate mechanisms of rat glutathione S-transferase P1 gene (rGSTP1) expression regulation during chemical carcinogenesis. we studied enhancer elements located in the region between -2.5 kb to -2.2 kb. The region was upstream from the start site of transcription and was divided into two major fragments, GPEI and GPEII. The GPEII fragment was further divided into two smaller fragments, GPEII- I and GPEII-2. Using a luciferase reporter system, we identified a strong enhancer of GPEI and a weak enhancer of GPEII in HeLa and a rat hepatoma cell line CBRH79 19 cell. The enhancer of GPEII was located within the GPEII-I region. Chemical stimulation by glycidyl methatylate (GMA) and phorbol 12-o-tetradecanoate 13-acetate (TPA) analysis revealed that induction of rGSTP1 expression was mainly through GPEI. Although H2O2 could enhance GPEII enhancer activity, the enhancement is not mediated by the NF-kappaB factor that bound the NF-kappaB site in GPEII. Using electrophoretic mobility shift assays (EMSA) and the UV cross-linking assays, we found that HeLa and CBRH7919 cells had proteins that specifically bound GPEI core sequence and a 64 kDa protein that interacted with GPEII-1. The cells from normal rat liver did not express the binding proteins. Therefore, the trans-acting factors seem to be closely related to GPEI, GPEII enhancer activities and may play an important role in high expression of rGSTPI gene.

  18. Novel function of the chromosome 7 open reading frame 41 gene to promote leukemic megakaryocyte differentiation by modulating TPA-induced signaling.

    Science.gov (United States)

    Sun, X; Lu, B; Hu, B; Xiao, W; Li, W; Huang, Z

    2014-03-28

    12-O-tetradecanoylphorbol-13-acetate (TPA) activates multiple signaling pathways, alters gene expression and causes leukemic cell differentiation. How TPA-induced genes contribute to leukemic cell differentiation remains elusive. We noticed that chromosome 7 open reading frame 41 (C7ORF41) was a TPA-responsive gene and its upregulation concurred with human megakaryocyte differentiation. In K562 cells, ectopic expression of C7ORF41 significantly increased CD61 expression, enhanced ERK and JNK signaling, and upregulated RUNX1 and FLI1, whereas C7ORF41 knockdown caused an opposite phenotype. These observations suggest that C7ORF41 may promote megakaryocyte differentiation partially through modulating ERK and JNK signaling that leads to upregulation of RUNX1 and FLI1. In supporting this, C7ORF41 overexpression rescued megakaryocyte differentiation blocked by ERK inhibition while JNK inhibition abrogated the upregulation of FLI1 by C7ORF41. Furthermore, we found that Y34F mutant C7ORF41 inhibited megakaryocyte differentiation. nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was the major activator of C7ORF41 that in turn repressed NF-κB activity by inhibiting its phosphorylation at serine 536, while MAPK/ERK was the potent repressor of C7ORF41. Finally, we showed that C7ORF41 knockdown in mouse fetal liver cells impaired megakaryocyte differentiation. Taken together, we have identified the function of a novel gene C7ORF41 that forms interplaying regulatory network in TPA-induced signaling and promotes leukemic and normal megakaryocyte differentiation.

  19. Novel function of the chromosome 7 open reading frame 41 gene to promote leukemic megakaryocyte differentiation by modulating TPA-induced signaling

    International Nuclear Information System (INIS)

    Sun, X; Lu, B; Hu, B; Xiao, W; Li, W; Huang, Z

    2014-01-01

    12-O-tetradecanoylphorbol-13-acetate (TPA) activates multiple signaling pathways, alters gene expression and causes leukemic cell differentiation. How TPA-induced genes contribute to leukemic cell differentiation remains elusive. We noticed that chromosome 7 open reading frame 41 (C7ORF41) was a TPA-responsive gene and its upregulation concurred with human megakaryocyte differentiation. In K562 cells, ectopic expression of C7ORF41 significantly increased CD61 expression, enhanced ERK and JNK signaling, and upregulated RUNX1 and FLI1, whereas C7ORF41 knockdown caused an opposite phenotype. These observations suggest that C7ORF41 may promote megakaryocyte differentiation partially through modulating ERK and JNK signaling that leads to upregulation of RUNX1 and FLI1. In supporting this, C7ORF41 overexpression rescued megakaryocyte differentiation blocked by ERK inhibition while JNK inhibition abrogated the upregulation of FLI1 by C7ORF41. Furthermore, we found that Y34F mutant C7ORF41 inhibited megakaryocyte differentiation. nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was the major activator of C7ORF41 that in turn repressed NF-κB activity by inhibiting its phosphorylation at serine 536, while MAPK/ERK was the potent repressor of C7ORF41. Finally, we showed that C7ORF41 knockdown in mouse fetal liver cells impaired megakaryocyte differentiation. Taken together, we have identified the function of a novel gene C7ORF41 that forms interplaying regulatory network in TPA-induced signaling and promotes leukemic and normal megakaryocyte differentiation

  20. Efficacy of DSP30-IL2/TPA for detection of cytogenetic abnormalities in chronic lymphocytic leukaemia/small lymphocytic lymphoma.

    Science.gov (United States)

    Holmes, P J; Peiper, S C; Uppal, G K; Gong, J Z; Wang, Z-X; Bajaj, R

    2016-10-01

    Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia in the Western Hemisphere. Cytogenetic abnormalities in CLL are used for diagnosis, prognosis and treatment. However, detecting these is difficult because mature B cells do not readily divide in culture. Here, we present data on two mitogen cocktails: CpG-oligonucleotide DSP30/Interleukin-2 (IL-2) and DSP30/IL-2 in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA). We analysed 165 cases of CLL with FISH and cytogenetics from January 2011 to June 2013. In 2011, three cultures were set-up: unstimulated, DSP30/IL-2-stimulated and TPA-stimulated. In 2012-2013, two cultures were set-up: unstimulated and stimulated with TPA/DSP30/IL-2. In 2011, FISH had a detection rate of 91% and cytogenetics using DSP30/IL2 had a detection rate of 91% (n = 22). In 2012-2013, FISH had a detection rate of 79% and cytogenetics using TPA/DSP30/IL-2 had a detection rate of 98% (n = 40). The percentage of cases with normal FISH but abnormal cytogenetics increased from 9% in 2011 to 21% in 2012-2013. The TPA/DSP30/IL-2 cultures in 2012-2013 detected more novel abnormalities (n = 5) as compared to DSP30/IL-2 alone (n = 3). TPA/DSP30/IL2 was as good as or better than DSP30/IL2 alone. TPA/DSP30/IL-2 offers a high detection rate for CLL abnormalities with a single stimulated culture and may increase detection of clinically significant abnormalities. © 2016 John Wiley & Sons Ltd.

  1. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    International Nuclear Information System (INIS)

    Watkins, D.T.

    1991-01-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules

  2. Water structure versus radical scavenger theories as explanations for the suppressive effects of DMSO and related compounds on radiation-induced transformation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, A.R.; Symons, M.C.

    1987-05-01

    We report here that dimethylsulfoxide (DMSO): suppresses radiation-induced transformation in vitro, even when DMSO treatments begin as late as 10 days post-irradiation (when cells are in the confluent, stationary phase of growth); inhibits the 12-O-tetradecanoylphorbol-13-acetate (TPA) enhancement of radiation-induced transformation in vitro; does not affect the expression of transformed cells as foci (when surrounded by non-transformed cells); and may be affecting radiation-induced transformation through its solvent properties (i.e. the Water Structure theory), while its effects on the TPA enhancement of radiation transformation may be mediated by its free radical scavenging abilities. DMSO, dimethylformamide (DMF) and dimethylacetamide (DMA) are similar solvents which are all very effective in their ability to suppress radiation-induced transformation in vitro (at concentrations in the cellular media down to 0.01%). As DMSO is known to be an extremely effective OH. free-radical scavenging agent, while DMF and DMA are not as efficient at scavenging free radicals, our results suggest that properties other than free-radical scavenging ability may be important in the suppressive effects of these compounds on radiation-induced transformation in vitro. It is known that low concentrations of such basic aprotic solvents modify water structure so as to suppress the protic (H-bond donor) reactivity of water and enhance its basic (H-bond receptor) reactivity. These reactivity changes may well be responsible for the effects noted above. DMSO, DMF and DMA are also capable of suppressing the TPA enhancement of radiation transformation (at concentrations of the compounds of 0.1% or higher). For this effect, the ability of these compounds to scavenge OH. shows a general correlation with their ability to suppress the TPA enhancement of transformation, suggesting that the Radical Scavenger theory may explain the ability of DMSO to suppress promotion in vitro.

  3. 2,3,7,8-tetrachlorodibenzo-p-dioxin: examination of biochemical effects involved in the proliferation and differentiation of XB cells

    International Nuclear Information System (INIS)

    Knutson, J.C.; Poland, A.

    1984-01-01

    XB, a cell line derived form a mouse teratoma, differentiates into stratified squamous epithelium when incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To examine the mediators of this response the effects produced by TCDD and those elicited by other compounds which stimulated epidermal proliferation and/or differentiation in mice were compared, XB/3T3 cultures keratinize when incubated with cholera toxin, epidermal growth factor (EGF), or TCDD , but not 12-0-tetradecanoylphorbol-13-acetate (TPA). Incubation of XB cells with TCDD for 48 hours produces an increase in thymidine incorporation, a response which is neither as large nor as rapid as that produced by cholera toxin, TPA, or EGF. Although both cholera toxin and TCDD stimulate differentiation and thymidine incorporation in XB/3T3 cultures, cholera toxin increases cAMP 30-fold in these cells, while TCDD does not affect cAMP accumulation. Inhibitors of arachidonic acid metabolism, which block epidermal proliferative responses to TPA in vivo, do not prevent the differentiation of XB cells in response to TCDD. In XB/3T3 cultures, TPA stimulates arachidonic acid release at all times tested (1,6, and 24 hours) and increases the incorporation of 32 P/sub i/ into total phospholipids and phosphatidyl-choline after 3 hours. In contrast, D affects neither arachidonic acid release nor the turnover of phosphatidylinositol, or phosphatidylcholine at any of the times tested. Although biochemical effects which have been suggested as part of the mechanism of TCDD and produced by other epiderma