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Sample records for 12-myristate 13-acetate tpa

  1. Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells.

    PubMed

    Noé, V; Alemany, C; Nicolás, M; Ciudad, C J

    2001-06-01

    4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.

  2. Effects of phorbol 12-myristate 13-acetate and cortisol interaction on steroid-binding capacity in the rat.

    PubMed Central

    Janssens, J P; de Loecker, W

    1979-01-01

    The specificity of the cortisol-receptor protein is examined in plasma and liver cytosol of rats. Phorbol 12-myristate 13-acetate does not inhibit the binding of cortisol to transcortin, nor does it affect the binding capacity of dexamethasone to the intracellular glucocorticoid receptor, but, by interacting with the cortisol molecule, it interferes with hormone-mediated processes in the cell. PMID:534535

  3. Contraction of rat thoracic aorta strips induced by phorbol 12-myristate 13-acetate

    SciTech Connect

    Itoh, H.; Lederis, K.

    1987-02-01

    Phorbol 12-myristate 13-acetate (PMA) induced a slow and progressive increase in tension of rat thoracic aorta strips in the presence of extracellular CaS . Complete relaxation could not be obtained in CaS -free buffer containing 1 mM ethyleneglycol-bis(US -aminoethylether)-N,N'-tetraacetic acid (EGTA) and 10 X M PMA. In the absence of extracellular CaS , PMA (10 X M) induced a small but sustained contraction which was not altered by the addition of another 2 mM EGTA and 3 x 10 V M verapamil. Papaverine (10 U M) relaxed the PMA-induced contraction to the base line, but phentolamine (10 V M), cyproheptadine (10 V M), atropine (10 V M) and tetrodotoxine (10 W M) did not change the contraction. CaS -depleted muscle strips, prepared by four repeated applications of 10 X M norepinephrine in CaS -free buffer, were contracted by 10 X M PMA, but at a lower maximum tension than nontreated strips. The action of PMA on rat aorta strips in CaS -free buffer did not require the presence of the adventitial layer or endothelial cells. These results suggest that PMA may induce activation of protein kinase C and smooth muscle contraction in the absence of extracellular CaS , without an increase in myoplasmic CaS .

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

    PubMed Central

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

    2016-01-01

    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. PMID:27994457

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

    PubMed

    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.

  6. Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate.

    PubMed

    Feyen, J H; Petersen, D N; Kream, B E

    1988-04-01

    We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

  7. The effect of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) on whole blood oxidative response as assessed by luminol-amplified chemiluminescence in dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The differences between lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) on whole blood oxidative response using luminol-amplified chemiluminescence (CL) are currently unknown in cattle. Luminol-dependent CL measures the amount of reactive oxygen species released from leukocytes a...

  8. Phorbol 12-myristate 13-acetate prevents isoproterenol-induced morphological change in cultured vascular smooth muscle cells

    SciTech Connect

    Nabika, Toru; Chaldakov, G.N.; Nara, Yasuo; Endo, Jiro; Yamori, Yukio )

    1988-10-01

    The effect of phorbol 12-myristate 13-acetate (PMA) on isoproterenol (ISO)- and dibutyryl cAMP (dBcAMP)-induced morphological change and cytoskeletal reorganization was studied in cultured vascular smooth muscle cells (VSMC) using the fluorescence staining of actin and microtubules. The treatment of VSMC with 1.0 {mu}M of ISO or with 1.0 mM of dBcAMP for 90 min induced the disruption of actin-containing stress fibers followed by cytoplasmic arborization. The addition of 100 nM of PMA prevented both the destruction of actin fibers and cell arborization induced either by ISO or by dBcAMP. These results indicated that the inhibition of arborization by PMA was mediated through the activation of protein kinase C. Colchicine at 5.0 {mu}M also had an inhibitory effect on ISO- and dBcAMP-induced cell arborization. However, immunofluorescence studies revealed that colchicine but not PMA elicited the reorganization of microtubules, suggesting that the effect of PMA was mediated through a mechanism different from that of colchicine. The observations indicated that the morphology of VSMC was regulated through the alteration of cytoskeletal organization induced by cAMP-mediated and by protein kinase C-dependent systems.

  9. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells*

    PubMed Central

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N. K.

    2015-01-01

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation. PMID:26269597

  10. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells.

    PubMed

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N K

    2015-09-25

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

  11. Expression of the human B-cell surface protein CD20: alteration by phorbol 12-myristate 13-acetate

    SciTech Connect

    Valentine, M.A.; Cotner, T.; Gaur, L.; Torres, R.; Clark, E.A.

    1987-11-01

    The monoclonal antibody 1F5 recognizes human B-cell surface protein CD20 and can activate resting B cells; with this antibody the authors found CD20 to be a 35/37-kDa non-disulfide-linked protein. The protein has a pI of 7.5-8.0 and is phosphorylated in B-cell lines, tonsillar B cells, and peripheral blood B cells. Both CD20 surface expression and phosphorylation are increased on buoyant tonsillar B cells activated in vivo. Because phorbol 12-myristate 13-acetate (PMA) supports the activation signal initiated by monoclonal antibody 1F5, they studied the effect of PMA on CD20 expression. After brief incubation with mitogenic levels of PMA, the number of dense tonsillar B cells positive for CD20 protein transiently decreased. Paradoxically, the cells remaining positive had more surface CD20 than did control cells, and these remaining surface CD20 molecules were hyperphosphorylated. Furthermore, PMA not only induced phosphorylation of CD20 protein on Raji cells but also increased the internalization of CD20 molecules; both phosphorylation and internalization of CD20 molecules were decreased with the protein kinase C inhibitor palmitoyl carnitine. Conditions that increase CD20 phosphorylation are shown also to increase surface mobility of the molecule, suggesting that CD20 protein internalization may be a critical early event for B-cell entry into the G/sub 1/ phase of the cell cycle.

  12. Control of macrophage cell differentiation in human promyelocytic HL-60 leukemia cells by 1,25-dihydroxyvitamin D/sub 3/ and phorbol-12-myristate-13-acetate

    SciTech Connect

    Murao, S.; Gemmell, M.A.; Callaham, M.F.; Anderson, N.L.; Huberman, E.

    1983-10-01

    Human promyelocytic leukemia cells (HL-60) were induced to differentiate into macrophage-like cells in a dose (3 x 10/sup -10/ to 10/sup -7/ M) and time (1 to 6 days)-dependent manner by 1,25-dihydroxyvitamin D/sub 3/ and the tumor promoter, phorbol-12-myristate-13-acetate. Differentiation was determined by an increase in the percentage of morphologically mature cells, in lysozyme and nonspecific esterase activities, and in reactivity with the murine OKM1 monoclonal antibody. Two HL-60 cell variants, designated as R-80 and B-II, were also examined. R-80 cells, which are resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate, also exhibited resistance, although to a lesser degree, to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/. Te resistance to the action of the two compounds is presumably not due to similar binding sites for the two inducers, since 1,25-dihydroxyvitamin D/sub 3/ was unable to compete for the phorbol diester binding sites as measured by (/sup 3/H)phorbol-12,13-dibutyrate binding. B-II cells were resistant to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide. Two-dimensional electrophoretic analysis of HL-60 cell protein patterns indicated that treatment of the HL-60 cells with 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide caused the cells to express various monocyte-macrophage and granulocyte marker proteins. These results indicate that 1,25-dihydroxyvitamin D/sub 3/ induces in the HL-60 cells a phenotype that resembles, but is not identical to, that of peripheral monocytes-macrophages. 40 references, 3 figures, 1 table.

  13. Double minute chromatin bodies and other chromosome alterations in human myeloid HL-60 leukemia cells susceptible or resistant to induction of differentiation by phorbol-12-myristate-13-acetate

    SciTech Connect

    Au, W.W.; Callaham, M.F.; Workman, M.L.; Huberman, E.

    1983-12-01

    An analysis of the chromosomal karyotype of the human promyelocytic HL-60 leukemia cell line and of a number of its sublines that exhibit varying degrees of resistance to induction of differentiation by phorbol-12-myristate-13-acetate was conducted. The HL-60 cell line and the derived sublines contained two consistent marker chromosomes (9p- and t(10;13)), which suggested that they have a common and possibly clonal origin. HL-60 cells that are susceptible to phorbol-12-myristate-13-acetate-induced cell differentiation contained double minute chromatine bodies. The sublines with different degrees of resistance showed a corresponding sequential reduction of double minute chromatin bodies in metaphase cells. This loss of double minute chromatin bodies was not associated with an appearance of homogeneously staining chromosomal regions. Resistant and susceptible HL-60 cell differed also in a number of other chromosomal alteration, including gains or losses involving chromosomes 5, 8, 11, 13, 16, and 17. Thus, it is suggested that acquisition of resistance to phorbol-12-myristate-13-acetate-induced cell differentiation in the HL-60 cells may involve one or more of the above chromosomal changes.

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

    PubMed

    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.

  15. Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2

    SciTech Connect

    Jorgensen, Kjersti; Skrede, Martina; Cruciani, Veronique; Mikalsen, Svein-Ole; Slipicevic, Ana; Florenes, Vivi Ann . E-mail: v.a.florenes@labmed.uio.no

    2005-04-01

    The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotides against PKC-{alpha}, or the PKC inhibitor Goe6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.

  16. A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation*

    PubMed Central

    Azevedo, Estefania P.; Rochael, Natalia C.; Guimarães-Costa, Anderson B.; de Souza-Vieira, Thiago S.; Ganilho, Juliana; Saraiva, Elvira M.; Palhano, Fernando L.; Foguel, Debora

    2015-01-01

    Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

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

    SciTech Connect

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

    2011-10-01

    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.

  18. Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1.

    PubMed Central

    Tözeren, A; Mackie, L H; Lawrence, M B; Chan, P Y; Dustin, M L; Springer, T A

    1992-01-01

    This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton. Images FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 8 FIGURE 9 PMID:1358239

  19. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus.

    PubMed

    Lund, Maria E; To, Joyce; O'Brien, Bronwyn A; Donnelly, Sheila

    2016-03-01

    The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/macrophages. This is because, following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics, in many respects, primary human macrophages. Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to inflammatory stimuli, as well as the development and screening of potential therapeutics, there is currently no standardised protocol for the reliable differentiation of THP-1 monocytes to a macrophage phenotype using PMA. Consequently, reports using THP-1 cells have demonstrated significant phenotypic and functional differences between resultant THP-1 macrophage populations, which are largely attributable to the varying PMA differentiation methods used. Thus, to guarantee consistency and reproducibility between studies, and to ensure the relevance of THP-1 cells as an appropriate model for primary human macrophages, it is crucial to develop a standardised protocol for the differentiation of THP-1 macrophages. Accordingly, we compared the function and phenotype of THP-1 macrophages generated using the range of published PMA differentiation protocols, specifically in response to the pro-inflammatory stimulus, lipopolysaccharide (LPS). Our results demonstrated that the function of the resultant THP-1 macrophage populations, as determined by tumour necrosis factor (TNF) secretion in response to LPS stimulation, varied significantly, and was dependent upon the concentration of PMA used to stimulate the differentiation of monocytes, and the period of rest following PMA exposure. These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells.

  20. Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs.

    PubMed

    Ciapa, B; Crossley, I; De Renzis, G

    1988-07-01

    We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.

  1. Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

    SciTech Connect

    Ciapa, B.; Payan, P. ); Allemand, D. )

    1989-12-01

    The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive {sup 86}Rb uptake and amiloride-sensitive {sup 24}Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na{sup +}/H{sup +} and Na{sup +}/K{sup +} exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na{sup +}/H{sup +} activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na{sup +}/H{sup +} exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 minutes. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na{sup +} stat, pH stat).

  2. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is anti-tumorigenic in liver cancer cells via inhibiting YAP through AMOT

    PubMed Central

    Zhu, Guoqing; Chen, Yan; Zhang, Xiao; Wu, Qi; Zhao, Yinghui; Chen, Yuxin; Sun, Fenyong; Qiao, Yongxia; Wang, Jiayi

    2017-01-01

    TPA stimulates carcinogenesis in various types of cancers. However, we found that TPA inhibits transformative phenotypes in liver cancer cells via the translocation of YAP from the nucleus, where it functions as a transcriptional co-factor, to the cytoplasm. Such effects led to a separation of YAP from its dependent transcription factors. The inhibitory effects of TPA on YAP were AMOT dependent. Without AMOT, TPA was unable to alter YAP activity. Importantly, the depletion of YAP and AMOT blocked the TPA-reduced transformative phenotypes. In sum, TPA has been established as an anti-tumorigenic drug in liver cancer cells via YAP and AMOT. PMID:28322318

  3. Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phosphatidylethanolamine metabolism in HeLa cells

    SciTech Connect

    Mueller, H.W.; Vance, D.E.

    1986-05-01

    The potent tumor promoter, TPA, exerts its earliest effects at the plasma membrane. Recent findings have shown that TPA stimulates a phospholipase C-mediated turnover of phosphatidyl-choline in several different cell types. The present study was undertaken to investigate whether TPA elicits a similar effect on the phosphatidylethanolamine (PE) pool of HeLa cells. Three different series of experiments were performed. First, in HeLa cells pulse-labeled with (/sup 3/H)ethanolamine, TPA stimulated a 5-fold release of aqueous radiolabeled products into the extra-cellular medium after a 1-hour incubation. Second, when (/sup 3/H)ethanolamine and TPA were added simultaneously to the cells, TPA stimulated a 2-fold incorporation of radiolabel into the cellular PE pool. In both the release and incorporation of (/sup 3/H)ethanolamine, TPA had no significant effect on PE mass. Finally, when HeLa cells were incubated with exogenous 1-radyl-2-acyl-sn-glycero-3-phospho-(/sup 3/H)ethanolamine, TPA stimulated the formation of an aqueous radiolabeled product in the medium, which was identified as phosphoethanolamine. These results provide evidence that TPA stimulates a phospholipase C-mediated turnover of PE.

  4. Stimulation of progesterone production by phorbol-12-myristate 13-acetate (PMA) in cultured Leydig tumor cells

    SciTech Connect

    Chaudhary, L.R.; Raju, V.S.; Stocco, D.M.

    1987-05-01

    It has been shown that addition of hCG or c-AMP to cultured Leydig tumor cells (MA-10) increases synthesis of progesterone as the major steroid. To investigate the possible involvement of protein kinase C (PK-C) in the regulation of steroid synthesis, the authors have studied the effect of PMA, an activator of PK-C, on progesterone production in MA-10 cells. The addition of PMA (100 ng/ml) stimulated steroid production whereas 4 -phorbol-12,13-didecanoate, an inactive phorbol ester, did not have any effects. Like hCG and c-AMP, PMA-stimulated progesterone production was inhibited by cycloheximide. hCG-stimulated steroid synthesis was inhibited by PMA. The addition of PMA to MA-10 Leydig cells further increased the c-AMP-stimulated progesterone production. To determine whether c-AMP has a obligatory role in the regulation of steroid production, the effect of adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (TFA), was studied on progesterone production in the presence of hCG. At lower dose (17 ng/ml) hCG-stimulated intracellular c-AMP levels and steroid production were inhibited by TFA (300 M). At higher dose of hCG (34 ng/ml) TFA did not inhibit the hCG-stimulated intracellular c-AMP levels, however, progesterone production was inhibited. Results suggest that the action of hCG, c-AMP and PMA in controlling steroidogenesis might be regulated by similar but different mechanisms.

  5. Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

    PubMed

    Hussaini, I M; Karns, L R; Vinton, G; Carpenter, J E; Redpath, G T; Sando, J J; VandenBerg, S R

    2000-07-21

    Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.

  6. Studies on the induction of Epstein-Barr virus (EBV) DNA polymerase (POL) and deoxyribonuclease (DNase) by the combined action of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and N-butyrate (SB in EBV-carrying cells

    SciTech Connect

    Nutter, L.M.; Tan, R.S.; Grill, S.; Li, J.S.; Cheng, Y.C.

    1986-03-05

    TPA and SB were found to induce EBV early antigen in EBV-carrying Raji cells, a Burkitt's Lymphoma-derived human cell line. The mode of interaction of these agents was unclear. They have examined the induction of EBV-POL and DNase activities by TPA and SB. It was found that neither agent alone could induce EBV-POL and DNase activities, even though the virus DNA could be induced by either compound alone. Induction of virus enzymes could only occur when cells were exposed to both compounds. A 2h exposure to TPA followed by 46h to SB resulted in levels of induction of EBV-POL and DNase activities comparable to those induced with simultaneous exposure to both agents for 48h. No induction of the enzymes will occur if the sequence of exposure to these agents is reversed. Phospholipase C, which increases intracellular diacylglycerol (and subsequently the activation of Protein Kinase C), and 5-Aza-deoxycytidine, a DNA hypomethylating agent, were able to partially substitute for TPA and SB, respectively. These results suggest that the mechanism of induction of EBV enzyme activities by TPA and SB could involve both Protein Kinase C activation and DNA hypomethylation. Furthermore, the synthesis of EBV DNA is not sufficient for induction of these virus enzyme activities.

  7. Metabolic conversion of 12-O-tetradecanoylphorbol-13-acetate in adult and newborn mouse skin and mouse liver microsomes.

    PubMed

    Berry, D L; Bracken, W M; Fischer, S M; Viaje, A; Slaga, T J

    1978-08-01

    Tritiated 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to adult mouse skin; at specified time intervals the mice were killed, and the labeled phorbol was extracted and subjected to separation and quantitation by high-pressure liquid chromatography. After 24 hr, TPA comprised greater than 96% of the recovered label from the skin, and its apparent half-life was 17.8 hr. Pretreatment of adult skin with TPA for 4 weeks before treatment with labeled TPA resulted in an increase in the clearance rate of TPA from the skin. Skin from newborn mice was capable of converting TPA into monoesters and phorbol, but the clearance rate in the adult was about 12 times more rapid than it was in the newborn. Epidermal homogenates converted TPA into 12-O-tetradecanoylphorbol, phorbol-13-acetate, and phorbol. Hepatic homogenates were able to convert TPA to monoesters and phorbol at rates 14 to 15 times faster than were epidermal homogenates. Attempts to isolate any previously undescribed metabolites of TPA by use of liver homogenates were unsuccessful, and mixed-function oxidation did not contribute to the metabolism of TPA. From inhibitor studies it was judged that esterases were implicated in the conversion of TPA to monoesters and phorbol. The results support the hypothesis that the tumor-promoting activity of TPA is directly related to its concentration in a specific tissue and that conversion of TPA to an active metabolite probably does not occur.

  8. 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

    PubMed Central

    Tsumbu, Cesar N.; Deby-Dupont, Ginette; Tits, Monique; Angenot, Luc; Franck, Thierry; Serteyn, Didier; Mouithys-Mickalad, Ange

    2011-01-01

    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. PMID:22254126

  9. Anti-edema effects of brown seaweed (Undaria pinnatifida) extract on phorbol 12-myristate 13-acetate-induced mouse ear inflammation.

    PubMed

    Khan, Mohammed Nurul Absar; Yoon, Seung-Je; Choi, Jae-Suk; Park, Nam Gyu; Lee, Hyung-Ho; Cho, Ji-Young; Hong, Yong-Ki

    2009-01-01

    The brown seaweed Undaria pinnatifida (Harvey) Suringar is used in traditional medicine to treat fever, urination problems, lumps and swelling, and as a dietary supplement for post-childbirth women. We examined the anti-inflammatory activities of the seaweed. The methanol extract of the seaweed was active against mouse ear edema induced by phorbol myristate acetate (PMA), with an IC(50) of 10.3 mg/ml. The extract reduced the edema to a half-maximal level when applied at the concentration of 40 mg/ml within 3 hours before or 2 hours after application of PMA. Extract taken from the blade section of the seaweed demonstrated the highest activity. The Northern form of U. pinnatifida was more active than the Southern form. In the analgesic test, the methanol extract suppressed the acetic acid-induced writhing response, with an IC(50) of 0.48 g/kg body weight. The extract also demonstrated antipyretic activity in yeast-induced hyperthermic mice. Activity-related constituents were arachidonic, eicosapentaenoic, and stearidonic acids.

  10. Modulation of phospholipid metabolism in murine keratinocytes by tumor promoter, 12-O-tetradecanoylphorbol-13-acetate

    SciTech Connect

    Galey, C.I.; Ziboh, V.A.; Marcelo, C.L.; Voorhees, J.J.

    1985-10-01

    The possibility that phospholipid deacylation may be a critical event in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-associated effects on mouse skin prompted us to examine in vitro the effects of TPA on arachidonic acid metabolism in neonatal mouse keratinocytes. Three-day old neonatal keratinocytes were prelabeled with ( UC)arachidonic acid (( UC)AA) and ( UC) stearic acid (( UC)ST) and used to characterize the lipases that were activated when these cells were treated with TPA in culture. Data from these studies demonstrate that phosphatidylcholine (PC) and phosphatidylinositol (PI) are the major phospholipids that undergo early hydrolysis to release arachidonic acid when challenged by TPA. Of particular interest was the novel observation of the hydrolysis of UC-labeled PI in these keratinocytes, the accumulation of ( UC)1,2-diacylglyceride and the lack of the ( UC)diacylglyceride phosphorylation to form ( UC)phosphatidic acid. This lack of ( UC) phosphatidic accumulation implied that although TPA enhanced the hydrolysis of ( UC)PI resulting in increased ( UC)diacylglyceride it did not enhance the resynthesis of the ( UC)PI via the phosphorylation of the ( UC)diacylglyceride. Therefore, TPA probably is not involved in the turnover of PI in these cells but is involved in the activation of PC hydrolyzing phospholipase A2 and PI hydrolyzing phospholipase C in these keratinocytes releasing arachidonic acid which then undergoes oxygenation reactions to provide biologically active eicosanoids.

  11. Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TTPA) upon membrane ionic exchanges in sea urchin eggs.

    PubMed

    Ciapa, B; Allemand, D; Payan, P

    1989-12-01

    The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive 86Rb uptake and amiloride-sensitive 24Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na+/H+ and Na+/K+ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na+/H+ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na+/H+ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na+ pump, Na+/H+ exchange) without eliciting corresponding regulatory mechanisms (Na+ stat, pH stat).

  12. Some lupane-type triterpenes inhibit tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin.

    PubMed

    Yasukawa, K; Yu, S; Yamanouchi, S; Takido, M; Akihisa, T; Tamura, T

    1995-04-01

    We have found that several lupane-type triterpenes, including lupeol, its acetate, betulin and betulinic acid, inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, and that betulinic acid inhibits tumor promotion in two-stage carcinogenesis in mice. Among seven lupane-type triterpenes assayed, these compounds inhibited the inflammatory activity induced by TPA in mice. The 50 % inhibitory dose of these compounds for TPA-induced inflammation was 0.4-4.0 μmol. Furthermore, topical application of lupeol, lupeol 3-acetate and betulin markedly suppressed the tumor-promoting effect of TPA (1 μg/mouse) in mouse skin initiated with 7,12-dimethyl-benz[a]anthracene (50 μg/mouse), at a grade corresponding to that of betulinic acid.

  13. 12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

    SciTech Connect

    Shiba, Yoshiki; Sasaki, Yasuto; Kanno, Yoshinobu )

    1988-10-01

    The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodomine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.

  14. Tumor promoter 12-O-tetradecanoylphorbol 13-acetate stimulates simian virus 40 induction by DNA-damaging agents and tumor initiators

    SciTech Connect

    Nomura, S.; Shobu, N.; Oishi, M.

    1983-05-01

    Simian virus 40 (SV40)-transformed Syrian hamster kidney cells produce infectious SV40 virus particles after treatments which damage DNA, such as UV irradiation or mitomycin C treatment. We have found that the induction of SV40 by DNA-damaging agents is greatly stimulated when a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), is present in the medium. Phorbol, which has a molecular structure similar to TPA but does not have any tumor-promoting activity, showed no such stimulatory effect on SV40 induction. This apparent synergistic effect of DNA-damaging agents and tumor promoter (TPA) was more pronounced when a tumor initiator, benzo (a)pyrene or 2-acetamido-fluorene, was combined with TPA. The effect of TPA on UV-triggered SV40 induction was greatly influenced by the timing of TPA addition to the culture medium, which was most efficient when addition of TPA was 5 to 20 h before UV irradiation. The effect of TPA, however, was not observed in SV40 rescue from hamster cells by cell fusion with permissive monkey (C7) cells.

  15. SCF/c-kit signaling is required in 12-O-tetradecanoylphorbol-13-acetate-induced migration and differentiation of hair follicle melanocytes for epidermal pigmentation.

    PubMed

    Qiu, Weiming; Yang, Ke; Lei, Mingxing; Yan, Hongtao; Tang, Hui; Bai, Xiufeng; Yang, Guihong; Lian, Xiaohua; Wu, Jinjin

    2015-05-01

    Hair follicle melanocyte stem cells (McSCs) are responsible for hair pigmentation and also function as a major melanocyte reservoir for epidermal pigmentation. However, the molecular mechanism promoting McSCs for epidermal pigmentation remains elusive. 12-O-tetradecanoylphorbol-13-acetate (TPA) mimics key signaling involved in melanocyte growth, migration and differentiation. We therefore investigated the molecular basis for the contribution of hair follicle McSCs to epidermal pigmentation using the TPA induction model. We found that repetitive TPA treatment of female C57BL/6 mouse dorsal skin induced epidermal pigmentation by increasing the number of epidermal melanocytes. Particularly, TPA treatment induced McSCs to initiate proliferation, exit the stem cell niche and differentiate. We also demonstrated that TPA promotes melanoblast migration and differentiation in vitro. At the molecular level, TPA treatment induced robust expression of stem cell factor (SCF) in keratinocytes and c-kit in melanoblasts and melanocytes. Administration of ACK2, a neutralizing antibody against the Kit receptor, suppressed mouse epidermal pigmentation, decreased the number of epidermal melanocytes, and inhibited melanoblast migration. Taken together, our data demonstrate that TPA promotes the expansion, migration and differentiation of hair follicle McSCs for mouse epidermal pigmentation. SCF/c-kit signaling was required for TPA-induced migration and differentiation of hair follicle melanocytes. Our findings may provide an excellent model to investigate the signaling mechanisms regulating epidermal pigmentation from mouse hair follicle McSCs, and a potential therapeutic option for skin pigmentation disorders.

  16. Suppression of Transglutaminase-2 is Involved in Anti-Inflammatory Actions of Glucosamine in 12-O-Tetradecanoylphorbol-13-Acetate-Induced Skin Inflammation

    PubMed Central

    Cho, Sun A; Lee, Hye Ja; Lee, Eun Ji; Kang, June Hee; Kim, You Lee; Kim, Hyun Ji; Oh, Seung Hyun; Choi, Changsun; Lee, Ho; Kim, Soo Youl

    2012-01-01

    Glucosamine (GS) is well known for the treatment of inflam-mation. However, the mechanism and efficacy of GS for skin inflammation are unclear. The aim of this study was to evaluate the effects and mechanism of GS in the mouse 12-O-tetradecanoyl 13-acetate (TPA)-induced ear edema model. TPA-induced ear edema was evoked in ICR or transglutaminase 2 (Tgase-2) (-/-) mice. GS was administered orally (10-100 mg/kg) or topically (0.5-2.0 w/v %) prior to TPA treatment. Orally administered GS at 10 mg/kg showed a 76 or 57% reduction in ear weight or myeloperoxidase, respectively, and a decreased expression of cyclooxy-genase-2 (COX-2), NF-κB and Tgase-2 in TPA-induced ear edema by western blot and immunohistochemistry. Role of Tgase-2 in TPA ear edema is examined using Tgase-2 (-/-) mice and TPA did not induce COX-2 expression in ear of Tgase-2 (-/-) mice. These observations suggested that Tgase-2 is involved in TPA-induced COX-2 expression in the inflamed ear of mice and anti-inflammatory effects of glucosamine is mediated through suppression of Tgase-2 in TPA ear edema. PMID:24009824

  17. Effect of Combined Treatment with Ursolic Acid and Resveratrol on Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate.

    PubMed

    Cho, Jiyoon; Rho, Okkyung; Junco, Jacob; Carbajal, Steve; Siegel, Dionicio; Slaga, Thomas J; DiGiovanni, John

    2015-09-01

    In this study, the effects of combining ursolic acid + resveratrol, for possible combined inhibitory effects on skin tumor promotion, were evaluated. Ursolic acid, resveratrol, and the combination of ursolic acid + resveratrol were applied topically prior to 12-O-tetracanoylphorbol-13-acetate (TPA) treatment on mouse skin to examine their effect on TPA-induced signaling pathways, epidermal hyperproliferation, skin inflammation, inflammatory gene expression, and skin tumor promotion. The combination of ursolic acid + resveratrol produced a greater inhibition of TPA-induced epidermal hyperproliferation. The combination of ursolic acid + resveratrol inhibited TPA-induced signaling pathways, including EGFR, STAT3, Src, Akt, Cox-2, Fas, NF-κB, p38 MAPK, c-Jun, and JNK1/2 while increasing levels of tumor suppressors, such as p21 and PDCD4, to a greater extent compared with the groups treated with the individual compounds. Ursolic acid + resveratrol also induced a dramatic increase of p-AMPK-α(Thr172). Combined treatment with ursolic acid + resveratrol resulted in a greater inhibition of expression of proinflammatory cytokines, including Il1a, Il1b, and Il22. Furthermore, NF-κB, Egr-1, and AP-1 DNA binding activities after TPA treatment were dramatically decreased by the combination of ursolic acid + resveratrol. Treatment with ursolic acid + resveratrol during skin tumor promotion with TPA produced greater inhibition of tumor multiplicity and tumor size than with either agent alone. Collectively, the greater ability of the combination of ursolic acid + resveratrol to inhibit skin tumor promotion was due to the greater inhibitory effects on growth factor and inflammatory signaling, skin inflammation, and epidermal hyperproliferation induced by TPA treatment.

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

    PubMed Central

    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, or procaine (0.1-1 mM), induction of the decarboxylase by TPA was inhibited by more than 90%. In vivo, lidocaine essentially abolishes the decarboxylase response of mouse epidermis when applied shortly after TPA. In contrast, local anesthetics have no effect on the enzyme's activity when added directly to the assay mixture and, in concert with TPA, have only a minimal effect on overall protein synthesis relative to controls. However, lidocaine has no effect on TPA-stimulated DNA synthesis in vitro (12-fold with or without lidocaine). Local anesthetics also markedly inhibit induction of the decarboxylase by ultraviolet light, which is probably not membrane mediated. Furthermore, in culture, lidocaine has only a small inhibitory effect on ornithine decarboxylase when given before TPA but is an effective inhibitor even when given up to 4-5 hr after the promoter, a time when decarboxylase activity has already increased. These findings suggest that local anesthetics, which are tertiary amines, do not act at the site of interaction of TPA and its putative receptor but may be acting specifically on polyamine biosynthesis. These drugs could be useful agents to determine the role of the polyamine pathway in tumor promotion. PMID:6933562

  19. Effect of Combined Treatment with Ursolic Acid and Resveratrol on Skin Tumor Promotion by 12-O-tetradecanoylphorbol-13-acetate

    PubMed Central

    Cho, Jiyoon; Rho, Okkyung; Junco, Jacob; Carbajal1, Steve; Siegel, Dionicio; Slaga, Thomas J.; DiGiovanni, John

    2015-01-01

    In this study, the effects of combining ursolic acid (UA) + resveratrol (Res), for possible combined inhibitory effects on skin tumor promotion were evaluated. UA, Res and the combination of UA + Res were applied topically prior to TPA treatment on mouse skin to examine their effect on TPA-induced signaling pathways, epidermal hyperproliferation, skin inflammation, inflammatory gene expression and skin tumor promotion. The combination of UA + Res produced a greater inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal hyperproliferation. The combination of UA + Res inhibited TPA-induced signaling pathways, including EGFR, STAT3, Src, Akt, Cox-2, Fas, NF-κB, p38 MAPK, c-Jun, and JNK1/2 while increasing levels of tumor suppressors such as p21 and PDCD4 to a greater extent compared to the groups treated with the individual compounds. UA + Res also induced a dramatic increase of p-AMPK-αThr172. Combined treatment with UA + Res resulted in a greater inhibition of expression of proinflammatory cytokines including IL-1α, IL-1β, and IL-22. Furthermore, NF-κB, Egr-1, and AP-1 DNA binding activities after TPA treatment were dramatically decreased by the combination of UA + Res. Treatment with UA + Res during skin tumor promotion with TPA produced greater inhibition of tumor multiplicity and tumor size than with either agent alone. Collectively, the greater ability of the combination of UA + Res to inhibit skin tumor promotion was due to the greater inhibitory effects on growth factor and inflammatory signaling, skin inflammation and epidermal hyperproliferation induced by TPA treatment. PMID:26100520

  20. Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate

    SciTech Connect

    Stith, B.J.; Maller, J.L.

    1987-04-01

    Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate are involved in occyte maturation was investigated. Microinjection of IP/sub 3/ into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP/sub 3/ concentration of 1 ..mu..M. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. These results indicate that protein kinase C is capable of regulating oocyte maturation of Xenopus.

  1. Inhibitory effect of some triterpenes from cacti on 32Pi-incorporation into phospholipids of HeLa cells promoted by 12-O-tetradecanoylphorbol-13-acetate.

    PubMed

    Kinoshita, K; Yang, Y; Koyama, K; Takahashi, K; Nishino, H

    1999-05-01

    Seventeen triterpenes isolated from cacti and the 10 derivatives were examined for the inhibition of tumor promoter-induced effects in vitro, such as stimulation of 32Pi-incorporation into phospholipids of cultured cells. Betulinic acid (1), cochalic acid (15), erythrodiol (16), oleanolic acid (21) and queretaroic acid (24) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated 32Pi-incorporation into phospholipids of the cultured cells.

  2. 4-Methylumbelliferone inhibits the phosphorylation of hyaluronan synthase 2 induced by 12-O-tetradecanoyl-phorbol-13-acetate.

    PubMed

    Kuroda, Yoshiyuki; Kasai, Kosuke; Nanashima, Naoki; Nozaka, Hiroyuki; Nakano, Manabu; Chiba, Mitsuru; Yoneda, Masahiko; Nakamura, Toshiya

    2013-04-01

    The effect of 4-methylumbelliferone (MU), a hyaluronan synthase-suppressor, on O-linked β-Nacetylglucosaminylation (O-GlcNAcylation) was investigated in cultured human skin fibroblasts, and we found that MU stimulated O-GlcNAcylation of the cellular proteins. Since O-GlcNAcylation affects protein phosphorylation via Ser/Thr kinases, we examined the effect of MU on both the phosphorylation of hyaluronan synthase 2 (HAS2) and hyaluronan production. The cells were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and MU independently or in combination. The protein fraction of each cell culture was extracted and divided into 2 parts-phosphorylated and non-phosphorylated fractions-by immobilized metal-affinity chromatography. The hyaluronan level in the medium was determined by an ELISA-like assay. Addition of MU decreased the level of hyaluronan in the medium and that of HAS2 in the phosphorylated protein fraction. On the contrary, the addition of TPA increased the levels of both of them. Interestingly, the combination of TPA and MU lowered the levels of them in treated cells as compared to those in untreated control cells. These results suggest that TPA activated protein kinase C (PKC), which stimulates the phosphorylation of HAS2, and increased hyaluronan production. Further, MU may inhibit the phosphorylation of HAS2 by PKC through the stimulation of O-GlcNAcylation.

  3. Nordihydroguaiaretic Acid from Creosote Bush (Larrea tridentata) Mitigates 12-O-Tetradecanoylphorbol-13-Acetate-Induced Inflammatory and Oxidative Stress Responses of Tumor Promotion Cascade in Mouse Skin

    PubMed Central

    Rahman, Shakilur; Ansari, Rizwan Ahmed; Rehman, Hasibur; Parvez, Suhel; Raisuddin, Sheikh

    2011-01-01

    Nordihydroguaiaretic acid (NDGA) is a phenolic antioxidant found in the leaves and twigs of the evergreen desert shrub, Larrea tridentata (Sesse and Moc. ex DC) Coville (creosote bush). It has a long history of traditional medicinal use by the Native Americans and Mexicans. The modulatory effects of topically applied NDGA was studied on acute inflammatory and oxidative stress responses in mouse skin induced by stage I tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). Double TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion cascade. Pretreatment of NDGA in TPA-treated mice mitigated cutaneous lipid peroxidation and inhibited production of hydrogen peroxide. NDGA treatment also restored reduced glutathione level and activities of antioxidant enzymes. Elevated activities of myeloperoxidase, xanthine oxidase and skin edema formation in TPA-treated mice were also lowered by NDGA indicating a restrained inflammatory response. Furthermore, results of histological study demonstrated inhibitory effect of NDGA on cellular inflammatory responses. This study provides a direct evidence of antioxidative and anti-inflammatory properties of NDGA against TPA-induced cutaneous inflammation and oxidative stress corroborating its chemopreventive potential against skin cancer. PMID:19861506

  4. Evaluation of pentacyclic triterpenes found in Perilla frutescens for inhibition of skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

    PubMed Central

    Cho, Jiyoon; Tremmel, Lisa; Rho, Okkyung; Camelio, Andrew M.; Siegel, Dionicio; Slaga, Thomas J.; DiGiovanni, John

    2015-01-01

    A series of pentacyclic tritperpenes found in Perilla frutescens (P. frutescens), including ursolic acid (UA), oleanolic acid (OA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) were evaluated for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and compared to UA as the prototype compound. All compounds were given topically 30 min prior to each TPA application and significantly inhibited skin tumor promotion. 3-epiCA and MA were significantly more effective than UA at inhibiting tumor development. All of these compounds significantly inhibited epidermal proliferation induced by TPA, however, CA, 3-epiCA and MA were more effective than UA. All compounds also reduced skin inflammation (assessed by infiltration of mast cells and T-cells) and inflammatory gene expression induced by TPA, however, 3-epiCA and MA were again more effective than UA. The greater ability of 3-epiCA and MA to inhibit skin tumor promotion was associated with greater reduction of Cox-2 and Twist1 proteins and inhibition of activation (i.e., phosphorylation) of IGF-1R, STAT3 and Src. Further study of these compounds, especially 3-epiCA and MA, for chemopreventive activity in other cancer model systems is warranted. PMID:26513295

  5. Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in murine skin by systemic effects of ultraviolet irradiation.

    PubMed

    Gensler, H L; Simpson, P J; Powell, M B

    1992-07-01

    Systemic effects of UVB irradiation (280-320 nm) have been shown to prevent subsequent chemical tumorigenesis induced by an initiation-promotion protocol. The present investigation was designed to determine whether initiation or promotion is prevented by UV irradiation. Groups of 25 B6D2F1/J mice received 12 weeks of intermittent dorsal UVB radiation treatments administered before, or 3 weeks after, initiation with a single application of 7,12-dimethylbenz[a]anthracene on the ventral skin. All mice were promoted ventrally with 5 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) applied three times weekly throughout the experiment. UV irradiation consisted of five 30-min exposures per week to a bank of 6 Westinghouse FS40 sunlamps. UV irradiation applied before or after initiation resulted in a decrease of 18-16 tumors per group of 25 mice, for a reduction of 61 and 50%, respectively, at 24 weeks after the first TPA treatment. Thus, prevention of tumor development was similar whether the UV influence was present or not during initiation. This finding suggests that the UV prevention of promotion could account for UV inhibition of skin tumors induced by an initiation-promotion regimen. Consistent with this concept, pretreatment of mice with dorsal UVB radiation was found to reduce DNA synthesis after exposure to TPA by 46%, although it did not decrease tritiated benzo[a]pyrene binding to DNA, in ventral epidermis. Thus, UVB irradiation systemically reduced TPA-induced tumor promotion in murine skin.

  6. Transplacental arsenic plus postnatal 12-O-teradecanoyl phorbol-13-acetate exposures associated with hepatocarcinogenesis induce similar aberrant gene expression patterns in male and female mouse liver

    SciTech Connect

    Liu Jie . E-mail: Liu6@niehs.nih.gov; Xie Yaxiong; Merrick, B. Alex; Shen Jun; Ducharme, Danica M.K.; Collins, Jennifer; Diwan, Bhalchandra A.; Logsdon, Daniel; Waalkes, Michael P.

    2006-06-15

    Our prior work shows that in utero arsenic exposure alone is a complete transplacental carcinogen, producing hepatocellular carcinoma in adult male offspring but not in females. In a follow-up study to potentially promote arsenic-initiated tumors, mice were exposed to arsenic (85 ppm) from gestation day 8 to 18 and then exposed to 12-O-teradecanoyl phorbol-13-acetate (TPA), a well-known tumor promoter after weaning. The dermal application of TPA (2 {mu}g/0.1 ml acetone, twice/week for 21 weeks) after transplacental arsenic did not further increase arsenic-induced liver tumor formation in adult males but significantly increased liver tumor formation in adult females. Thus, for comparison, liver tumors and normal liver samples taken from adult male and female mice at necropsy were analyzed for aberrant gene/protein expression by microarray, real-time RT-PCR and Western blot analysis. Arsenic/TPA treatment resulted in increased expression of {alpha}-fetoprotein, k-ras, c-myc, estrogen receptor-{alpha}, cyclin D1, cdk2na, plasminogen activator inhibitor-1, cytokeratin-8, cytokeratin-18, glutathione S-transferases and insulin-like growth factor binding proteins in liver and liver tumors from both male and female mice. Arsenic/TPA also decreased the expression of BRCA1, betaine-homocysteine methyltransferase, CYP7B1, CYP2F2 and insulin-like growth factor-1 in normal and cancerous livers. Alterations in these gene products were associated with arsenic/TPA-induced liver tumors, regardless of sex. Thus, transplacental arsenic plus postnatal TPA exposure induced similar aberrant gene expression patterns in male and female mouse liver, which are persistent and potentially important to the mechanism of arsenic initiation of hepatocarcinogenesis.

  7. Protection against 12-O-tetradecanoylphorbol-13-acetate-caused inflammation in SENCAR mouse ear skin by polyphenolic fraction isolated from green tea.

    PubMed

    Katiyar, S K; Agarwal, R; Ekker, S; Wood, G S; Mukhtar, H

    1993-03-01

    Earlier studies conducted in our laboratory have shown that a polyphenolic fraction isolated from green tea (GTP) possesses anti-skin tumor initiating and anti-skin tumor promoting activity in the two-stage skin tumorigenesis protocol in SENCAR mouse. We have also shown that topical application of GTP inhibits tumor promoter-caused induction of epidermal ornithine decarboxylase activity in SENCAR mice in a dose-dependent manner, and that its oral feeding in drinking water to SKH-1 hairless mice enhances antioxidant and phase II enzyme activity in liver, lung, small bowel and skin. In this study, we show that single or multiple applications of GTP on SENCAR mouse ear prior to or after the application of 12-O-tetradecanoylphorbol-13-acetate (TPA) afford significant protection (P < 0.05) against TPA-induced edema. Pre-application of GTP also afforded significant protection against TPA-induced hyperplasia in the ear skin. The percentage protection by GTP both in terms of epidermal thickness and vertical cell layers was 75 and 90% respectively (P < 0.005). In further studies, we assessed the protective effect of GTP against TPA-caused infiltration of neutrophils in the ear skin of SENCAR mouse, by determining a naturally occurring constituent of neutrophils, myeloperoxidase, as a quantitative marker of tissue neutrophil content. Prior application of GTP resulted in significant protection against TPA-caused infiltration of neutrophils (P < 0.005). These results suggest that GTP possesses potential as a cancer chemopreventive agent against stage I tumor promotion.

  8. Involvement of retrotransposition of long interspersed nucleotide element-1 in skin tumorigenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate.

    PubMed

    Okudaira, Noriyuki; Goto, Motohito; Yanobu-Takanashi, Rieko; Tamura, Masato; An, Akihiro; Abe, Yukiko; Kano, Shigeyuki; Hagiwara, Shotaro; Ishizaka, Yukihito; Okamura, Tadashi

    2011-11-01

    Tumor development induced by 7,12-dimethylbenz[a]anthracene (DMBA) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) is a well-characterized model of multistep carcinogenesis. DMBA mutates the Ha-ras gene, whereas TPA promotes the growth of transformed cells by activating cellular signaling molecules. It remains to be clarified how repeated TPA treatment endows transformed cells with autonomous cell growth. Long interspersed nucleotide element-1 (L1) is an endogenous retroelement, and 80-100 copies of L1 function as autonomous mobile elements. Although the L1 retrotransposition (RTP) has been found in various human tumors, implying the possible mobility of L1 during carcinogenesis, little is known about how L1-RTP arises in tumor cells, owing to a lack of experimental models. To dissect the mechanism of L1-RTP during carcinogenesis, we established a line of transgenic mice carrying human L1 and enhanced green fluorescent protein (hL1-EGFP mice) and subjected them to DMBA/TPA-induced skin tumorigenesis. Of 15 skin tumors examined, 13 were positive for L1-RTP; L1-RTP was not detected in normal skin tissues adjacent to the tumors. Moreover, nine L1-RTP-positive tumors were positive for activated Ha-ras, and immunohistochemical analysis revealed cells positive for both L1-RTP and phosphorylated Stat3, a marker of tumor cells. Additional in vivo experiments suggested that L1-RTP occurred during tumor promotion by TPA. This is the first report on the involvement of L1-RTP in chemical carcinogenesis. We propose hL1-EGFP mice as a versatile system for investigating the mode of L1-RTP in tumor development and discuss the possible role of L1-RTP in tumorigenesis.

  9. Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

    PubMed Central

    Chen, Jia-Jing; Chen, Haw-Wen; Liu, Kai-Li; Yeh, Shu-Lan; Wang, Tsu-Shing; Liu, Shu-Hui; Tsai, Chia-Han; Li, Chien-Chun

    2016-01-01

    Fascin-1, an actin-bundling protein, plays an important role in cancer cell migration and invasion; however, the underlying mechanism remains unclear. On the basis of a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell migration model, it was shown that TPA increased fascin-1 mRNA and protein expression and fascin-1-dependent cell migration. TPA dose- and time-dependently increased PKCδ and STAT3α activation and GSK3β phosphorylation; up-regulated Wnt-1, β-catenin, and STAT3α expression; and increased the nuclear translocation of β-catenin and STAT3α. Rottlerin, a PKCδ inhibitor, abrogated the increases in STAT3α activation and β-catenin and fascin-1 expression. WP1066, a STAT3 inhibitor, suppressed TPA-induced STAT3α DNA binding activity and β-catenin expression. Knockdown of β-catenin attenuated TPA-induced fascin-1 and STAT3α expression as well as cell migration. In addition to MCF-7, migration of Hs578T breast cancer cells was inhibited by silencing fascin-1, β-catenin, and STAT3α expression as well. TPA also induced Wnt-1 expression and secretion, and blocking Wnt-1 signaling abrogated β-catenin induction. DHA pretreatment attenuated TPA-induced cell migration, PKCδ and STAT3α activation, GSK3β phosphorylation, and Wnt-1, β-catenin, STAT3α, and fascin-1 expression. Our results demonstrated that TPA-induced migration is likely associated with the PKCδ and Wnt-1 pathways, which lead to STAT3α activation, GSK3β inactivation, and β-catenin increase and up-regulation of fascin-1 expression. Moreover, the anti-metastatic potential of DHA is partly attributed to its suppression of TPA-activated PKCδ and Wnt-1 signaling. PMID:27036017

  10. Effects of TPA on short-circuit current across frog skin

    SciTech Connect

    Mauro, T.; O'Brien, T.G.; Civan, M.M.

    1987-02-01

    TPA (12-O-tetradecanoylphorbol-13-acetate) is an effective tumor promoter that affects a variety of ion transport processes. To examine the relationship between effects on transport and growth and differentiation, the authors have been studying the actions of TPA on frog skin, a particularly well-characterized epithelium. They have reported that high concentrations of TPA stimulate base-line short-circuit current (I/sub SC/) and inhibit the subsequent natriferic action of vasopressin. The current study of 89 preparations extends those findings. The K/sub m/ of the stimulatory effect of TPA is approx. 3 nM; this high affinity indicates that the transport phenomenon does not simply reflect a nonspecific interaction of phorbol ester with the plasma membranes. TPA acts largely or entirely at the mucosal surface of both split and whole skins; thus the sidedness of the effect does not arise from adsorption onto the underlying connective tissue when TPA is applied to the serosal surface of whole skin. Amiloride, an inhibitor of apical Na entry, abolishes I/sub SC/ across frog skins pretreated with TPA. The phorbol ester also increases I/sub SC/ across split skins, preparations which do not produce net Cl transport. The present results indicate that frog skin is highly responsive to TPA at concentrations known to activate protein kinase C in broken-cell preparations. The actions on I/sub SC/ appear to reflect changes in transepithelial Na transport modulated at the apical membranes. The full biochemical events triggered by TPA remain to be clarified; in part, TPA's actions may be mediated by leukotrienes produced by activation of the lipoxygenase pathway of arachidonic acid metabolism.

  11. c-fos sequence necessary for basal expression and induction by epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore.

    PubMed Central

    Fisch, T M; Prywes, R; Roeder, R G

    1987-01-01

    We have investigated the sequence requirements for induction of the human c-fos gene by epidermal growth factor (EGF), 12-O-tetradecanoyl-13-acetate (TPA), and the calcium ionophore A23187 by transfecting c-fos promoter mutants into HeLa and A431 cells. Induction by both EGF and TPA in HeLa cells required the presence of the c-fos enhancer located at -317 to -298 relative to the mRNA cap site. A23187, however, did not induce expression of the transfected gene, even though it strongly induced expression of the endogenous gene, suggesting that it has different requirements for induction than do EGF and TPA. We have also investigated the role of promoter sequences downstream of the enhancer in general expression and induction of c-fos. A sequence between -97 and -76, which includes an 8-base-pair perfect direct repeat, was needed for efficient general expression but not for induction of the gene. A factor in nuclear extracts that bound specifically to this sequence was detected by a gel mobility shift assay. A 7-base-pair sequence, located between -63 and -57 relative to the mRNA cap site and previously shown to be important for general expression of mouse c-fos, was also important for general expression of the human gene. In addition, this element was important for inducibility by EGF and TPA, since induction was significantly reduced when internal deletion mutants that retained the enhancer but lacked the -63 to -57 sequence element were analyzed in transfecting assays. Images PMID:3119989

  12. Epidermal proliferation of nude mouse skin, pig skin, and pig skin grafts. Failure of nude mouse skin to respond to the tumor promoter 12- O-tetradecanoyl phorbol 13-acetate

    PubMed Central

    1980-01-01

    Human skin transplanted to nude mice offers a possible experimental system for the study of normal epidermal proliferation and differentiation, and for their pathological counterparts. Crucial to the development of such a system is the demonstration that such grafts retain the responsive features of donor skin. To document that donor proliferative characteristics are maintained in the grafts, a comparative analysis of agents that induce proliferation was made on skin of mice homozygous and heterozygous for nude, on pig skin, and on pig skin transplanted onto nude mice. A wave of epidermal proliferation could be induced in pig skin and pig skin grafted onto nude mice, but not in nude mouse skin after the topical application of 10 ng 12-O- tetradecanoyl phorbol 13-acetate (TPA). A 10-fold greater concentration of TPA or 5% croton oil induced proliferation in all species of epidermis studied. Mice, heterozygous for nude, showed a normal response to 10 ng TPA, suggesting that the ability to respond to TPA may be related, in part, to a recessive genetic trait. Nude mouse skin transplanted to a heterozygous littermate capable of responding to 10 ng TPA does not respond. These observations argue that: the graft retains its donor proliferative characteristics when transplanted to the nude, and the inability of the nude mouse to respond to lower doses of TPA may be related to absorption, the nude gene(s), or an inherent threshold to response. The lack of response to the promoter TPA provides a plausible explanation for the decreased incidence of tumors arising in nude mice during two-stage carcinogenesis experiments. PMID:7000965

  13. 12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-M/sub r/ form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in Middle T function

    SciTech Connect

    Matthews, J.T.; Benjamin, T.L.

    1986-05-01

    The 58,000-M/sub r/ form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes. The authors report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60/sup c-src/.

  14. Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation.

    PubMed

    Chien, Chih-Chiang; Wu, Ming-Shun; Shen, Shing-Chuan; Yang, Liang-Yo; Wu, Wen-Shin; Chen, Yen-Chou

    2013-04-01

    The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.

  15. Modulation by glycyrrhetinic acid derivatives of TPA-induced mouse ear oedema.

    PubMed Central

    Inoue, H.; Mori, T.; Shibata, S.; Koshihara, Y.

    1989-01-01

    1. The anti-inflammatory effects of glycyrrhetinic acid and its derivatives on TPA (12-O-tetradecanoylphorbol-13-acetate)-induced mouse ear oedema were studied. The mechanisms of TPA-induced ear oedema were first investigated with respect to the chemical mediators. 2. The formation of ear oedema reached a maximum 5 h after TPA application (2 micrograms per ear) and the prostaglandin E2 (PGE2) production of mouse ear increased with the oedema formation. 3. TPA-induced ear oedema was prevented by actinomycin D and cycloheximide (0.1 mg per ear, respectively) when applied during 60 min after TPA treatment. 4. Of glycyrrhetinic acid derivatives examined, dihemiphthalate derivatives (IIe, IIe', IIIa, IIIa', IVa, IVa') most strongly inhibited ear oedema on both topical (ID50, 1.6 mg per ear for IIe, 2.0 mg per ear for IIIa and 1.6 mg per ear for IVa) and oral (ID50, 88 mg kg-1 for IIe', 130 mg kg-1 for IIIa' and 92 mg kg-1 for IVa') administration. 5. Glycyrrhetinic acid (Ia) and its derivatives applied 30 min before TPA treatment were much more effective in inhibiting oedema than when applied 30 min after TPA. A dihemiphthalate of triterpenoid compound IVa completely inhibited oedema, even when applied 3 h before TPA treatment. 6. Glycyrrhetinic acid (Ia) and deoxoglycyrrhetol (IIa), the parent compounds, produced little inhibition by oral administration at less than 200 mg kg-1. 7. These results suggest that the dihemiphthalate derivatives of triterpenes derived from glycyrrhetinic acid by chemical modification are useful for the treatment of skin inflammation by both topical and oral application. PMID:2924072

  16. Large scale production and purification of human IL-2 from buffy coat lymphocytes stimulated with 12-O-tetradecanoylphorbol 13-acetate and calcium ionophore A23187.

    PubMed

    Grote, W; Klaar, J; Mühlradt, P F; Monner, D A

    1987-10-23

    Methods for the production of high titers of interleukin-2 (IL-2) from human buffy coat lymphocytes, and subsequent purification of the IL-2 are described. 50 buffy coats containing 1 X 10(11) leukocytes were first depleted of erythrocytes by batchwise leukapheresis using a Haemonetics model 15 blood wash centrifuge. Further lymphocyte enrichment was achieved using a one-step sedimentation in the presence of hydroxyethyl starch, which produced suspensions of more than 90% lymphocytes. This degree of lymphocyte purity was important since phagocytes were inhibitory to 12-O-tetradecanoylphorbol 13-acetate/calcium ionophore (TPA/A23187)-induced IL-2 production when their concentration exceeded 15% of the total cells. Cell culture was performed in stirred fermenters. Using TPA/A23187 induction, up to 500 micrograms of IL-2 per liter were produced. The IL-2 was purified by absorption from the supernatants onto controlled pore glass and elution with 50% ethylene glycol, followed by Fractogel chromatography, and then preparative high-performance liquid chromatography (HPLC) using an RP-6 column and elution with a gradient of n-propanol. A final HPLC rechromatography step using an analytical RP-6 column gave a homogeneous preparation with specific activity of 1.2 X 10(7) U/mg and a recovery from the starting supernatant of 22%.

  17. Inhibitory effect of herbal remedies on 12-O-tetradecanoylphorbol-13-acetate-promoted Epstein-Barr virus early antigen activation.

    PubMed

    Kapadia, Govind J; Azuine, Magnus A; Tokuda, Harukuni; Hang, Eric; Mukainaka, T; Nishino, Hoyoku; Sridhar, Rajagopalan

    2002-03-01

    For the past several years we have been evaluating natural products as potential cancer chemopreventive agents in a short term in vitro assay involving Epstein--Barr virus early antigen (EBV-EA) activation in Raji cells promoted by phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Because of the current interest in the use of herbal remedies, we considered examining them for their cancer chemopreventive activities, using their extracts with a view to uncovering such benefits (if any) these remedies might possess. Thirty-six extracts of 32 herbs belonging to 27 families in use as herbal remedies including those of gingko, black cohosh, echinacea, kava-kava, saw palmetto, turmeric, angelica, wild yam, cat's claw, passion flower, muira puama, feverfew, blueberry, chasteberry, licorice, nettle, golden seal, pygeum, ginger, valerian and hops were prepared and evaluated. Turmeric at a concentration of 10 microg x ml (-1)exhibited the most potent anti-EBV-EA activity, which is ten times more than passionflower, that is next in the order of activity. At the concentration level of 100 microg ml (-1), several of the herbal remedies tested inhibited the EBV-EA in Raji cells exposed to the tumor promoter TPA (32 pM) by more than 90%. We also report for the first time the activities of 16 new medicinal plants as potential cancer chemopreventive agents. Since inhibitors of EBV-EA promoted by TPA in vitro have been shown to be effective anti-tumor promoting agents in laboratory animal models, our results indicate new and potential applications of these herbal remedies as cancer chemopreventive agents since they are already in clinical use in the human population.

  18. SAG/ROC2/Rbx2 is a novel activator protein-1 target that promotes c-Jun degradation and inhibits 12-O-tetradecanoylphorbol-13-acetate-induced neoplastic transformation.

    PubMed

    Gu, Qingyang; Tan, Mingjia; Sun, Yi

    2007-04-15

    SAG (sensitive to apoptosis gene) was first identified as a stress-responsive protein that, when overexpressed, inhibited apoptosis both in vitro and in vivo. SAG was later found to be the second family member of ROC1 or Rbx1, a RING component of SCF and DCX E3 ubiquitin ligases. We report here that SAG/ROC2/Rbx2 is a novel transcriptional target of activator protein-1 (AP-1). AP-1 bound both in vitro and in vivo to two consensus binding sites in a 1.3-kb region of the mouse SAG promoter. The SAG promoter activity, as measured by luciferase reporter assay, was dependent on these sites. Consistently, endogenous SAG is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) with an induction time course following the c-Jun induction in both mouse epidermal JB6-Cl.41 and human 293 cells. TPA-mediated SAG induction was significantly reduced in JB6-Cl.41 cells overexpressing a dominant-negative c-Jun, indicating a requirement of c-Jun/AP-1. On the other hand, SAG seemed to modulate the c-Jun levels. When overexpressed, SAG remarkably reduced both basal and TPA-induced c-Jun levels, whereas SAG small interfering RNA (siRNA) silencing increased substantially the levels of both basal and TPA-induced c-Jun. Consistently, SAG siRNA silencing reduced c-Jun polyubiquitination and blocked c-Jun degradation induced by Fbw7, an F-box protein of SCF E3 ubiquitin ligase. Finally, SAG overexpression inhibited, whereas SAG siRNA silencing enhanced, respectively, the TPA-induced neoplastic transformation in JB6-Cl.41 preneoplastic model. Thus, AP-1/SAG establishes an autofeedback loop, in which on induction by AP-1, SAG promotes c-Jun ubiquitination and degradation, thus inhibiting tumor-promoting activity of AP-1.

  19. Anti-inflammatory effect of aqueous extracts of spent Pleurotus ostreatus substrates in mouse ears treated with 12-O-tetradecanoylphorbol-13-acetate

    PubMed Central

    Rivero-Pérez, Nallely; Ayala-Martínez, Maricela; Zepeda-Bastida, Armando; Meneses-Mayo, Marcos; Ojeda-Ramírez, Deyanira

    2016-01-01

    Aims: To evaluate the application of spent Pleurotus ostreatus substrates, enriched or not with medicinal herbs, as a source of anti-inflammatory compounds. Subjects and Methods: P. ostreatus was cultivated on five different substrates: Barley straw (BS) and BS combined 80:20 with medicinal herbs (Chenopodium ambrosioides L. [BS/CA], Rosmarinus officinalis L. [BS/RO], Litsea glaucescens Kunth [BS/LG], and Tagetes lucida Cav. [BS/TL]). The anti-inflammatory activity of aqueous extracts of spent mushroom substrates (SMSs) (4 mg/ear) was studied using an acute inflammation model in the mouse ear induced with 2.5 μg/ear 12-O-tetradecanoylphorbol13-acetate (TPA). Results: Groups treated with BS/CA, BS/RO, and BS/LG aqueous extracts exhibited the best anti-inflammatory activity (94.0% ± 5.5%, 92.9% ± 0.6%, and 90.4% ± 5.0% inhibition of auricular edema [IAO], respectively), and these effects were significantly different (P < 0.05) from that of the positive control indomethacin (0.5 mg/ear). BS/TL and BS were also able to reduce TPA-induced inflammation but to a lesser extent (70.0% ± 6.7% and 43.5% ± 6.6% IAO, respectively). Conclusions: Spent P. ostreatus substrate of BS possesses a slight anti-inflammatory effect. The addition of CA L. to mushroom substrate showed a slightly synergistic effect while RO L. had an additive effect. In addition, LG Kunth and TL Cav. enhanced the anti-inflammatory effect of SMS. However, to determine whether there is a synergistic or additive effect, it is necessary to determine the anti-inflammatory effect of each medicinal herb. PMID:27127316

  20. Pro-Oxidant Role of Silibinin in DMBA/TPA Induced Skin Cancer: 1H NMR Metabolomic and Biochemical Study

    PubMed Central

    Sati, Jasmine; Mohanty, Biraja Prasad; Garg, Mohan Lal; Koul, Ashwani

    2016-01-01

    Silibinin, a major bioactive flavonolignan in Silybum marianum, has received considerable attention in view of its anticarcinogenic activity. The present study examines its anticancer potential against 7, 12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin cancer. Male LACA mice were randomly segregated into 4 groups: Control, DMBA/TPA, Silibinin and Silibinin+DMBA/TPA. Tumors in DMBA/TPA and Silibinin+DMBA/TPA groups were histologically graded as squamous cell carcinoma. In the Silibinin+DMBA/TPA group, significant reduction in tumor incidence (23%), tumor volume (64.4%), and tumor burden (84.8%) was observed when compared to the DMBA/TPA group. The underlying protective mechanism of Silibinin action was studied at pre-initiation (2 weeks), post-initiation (10 weeks) and promotion (22 weeks) stages of the skin carcinogenesis. The antioxidant nature of Silibinin was evident at the end of 2 weeks of its treatment. However, towards the end of 10 and 22 weeks, elevated lipid peroxidation (LPO) levels indicate the pro-oxidative nature of Silibinin in the cancerous tissue. TUNEL assay revealed enhanced apoptosis in the Silibinin+DMBA/TPA group with respect to the DMBA/TPA group. Therefore, it may be suggested that raised LPO could be responsible for triggering apoptosis in the Silibinin+DMBA/TPA group. 1H Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine the metabolic profile of the skin /skin tumors. Dimethylamine (DMA), glycerophosphocholine (GPC), glucose, lactic acid, taurine and guanine were identified as the major contributors for separation between the groups from the Principal Component Analysis (PCA) of the metabolite data. Enhanced DMA levels with no alteration in GPC, glucose and lactate levels reflect altered choline metabolism with no marked Warburg effect in skin tumors. However, elevated guanine levels with potent suppression of taurine and glucose levels in the Silibinin+DMBA/TPA group are

  1. Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA.

    PubMed

    Wang, Xiao; Ramirez, Angel; Budunova, Irina

    2010-07-01

    Connexin 26 is important in keratinocyte proliferation, differentiation and skin pathologies. Cx26 is barely expressed in normal adult epidermis, but its expression is induced during wound healing, psoriasis, and skin hyperplasia stimulated by tumor promoters. In hyperplastic proliferating epidermis, Cx26 is co-expressed with Cx43 typical for basal and suprabasal keratinocytes. As Cx26 and Cx43 can not form permeable gap junctions, their co-expression may alter the gap junctional communication between keratinocytes and induce proliferation. To test the effect of persistent co-expression of Cx26 and Cx43 in epidermis, we generated transgenic mice using keratin5 promoter to target Cx26 to basal Cx43-positive keratinocytes. We evaluated the effect of ectopic Cx26 on keratinocyte proliferation and differentiation in normal and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated skin. The ectopic Cx26 expression in epidermis did not significantly affect skin development, keratinocyte differentiation and proliferation in newborn and adult skin. Unexpectedly, the proliferative effect of tumor promoter TPA was strongly decreased in epidermis of K5.Cx26 transgenics. This correlated with significant down-regulation of TPA-induced activity of protein kinase C (PKC) in K5.Cx26 mice.

  2. Loss of responsiveness of an AP1-related factor, PEBP1, to 12-O-tetradecanoylphorbol-13-acetate after transformation of NIH 3T3 cells by the Ha-ras oncogene

    SciTech Connect

    Sataka, Masanobu; Ibaraki, Tamotsu; Yamaguchi, Yuko; Ito, Yoshiaki )

    1989-09-01

    The function of the A element (nucleotides 5107 to 5130) of the polyomavirus enhancer is augmented in NIH 3T3 cells by a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). One of its targets is an AP1 consensus sequence motif recognized by a nuclear factor, PEBP1. In Ha-ras-transformed NIH 3T3 cells, however, A element function was not enhanced by TPA treatment, and at the same time PEBP1 was not detected in the nuclear extract by a mobility shift assay. PEBP1 was not detected in either the extract from NIH 3T3 cells treated in vivo with a protein kinase inhibitor, staurosporine, or the extract from NIH 3T3 cells after treatment in vitro with phosphatase. These results suggest that PEBP1 is required to be properly phosphorylated for DNA binding and that it is underphosphorylated, possibly due to the downregulation of protein kinase C in Ha-ras-transformed cells. In addition, it was observed that PEBP2, which bound to the A element adjacent to PEBP1, was converted to apparently related PEBP3 when conditions favored underphosphorylation.

  3. The 5' flanking region of the pS2 gene contains a complex enhancer region responsive to oestrogens, epidermal growth factor, a tumour promoter (TPA), the c-Ha-ras oncoprotein and the c-jun protein.

    PubMed Central

    Nunez, A M; Berry, M; Imler, J L; Chambon, P

    1989-01-01

    Expression of the pS2 gene which is transcriptionally controlled by oestrogens in the breast cancer cell line MCF-7 is oestrogen independent in stomach mucosa. We show here that the level of MCF-7 cell pS2 mRNA can also be increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). We further demonstrate, using transient transfection assays, that the -428 to -332 5' flanking sequence of the pS2 gene contains DNA enhancer elements responsive to oestrogens, TPA, EGF, the c-Ha-ras oncoprotein and the c-jun protein. Images PMID:2498085

  4. 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

    SciTech Connect

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

    1984-02-01

    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 (/sup 3/H)uridine into RNA did not alter appreciably from those in the control tissues; while the rates of (/sup 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 (/sup 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 (/sup 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.

  5. Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism.

    PubMed

    Park, Kwang Kyun; Park, Jae Hee; Jung, Youn Joo; Chung, Won Yoon

    2003-12-09

    Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his(+) reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH). DNA strand breaks by ROS derived from H(2)O(2)/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H(2)O(2)/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H(2)O(2) formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.

  6. Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells

    SciTech Connect

    Wen, Longthung; Tanaka, Akiko; Nonoyama, Meihan )

    1989-08-01

    Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors the authors called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.

  7. Inhibitory effect of the rhizomes of Alpinia officinarum on TPA-induced inflammation and tumor promotion in two-stage carcinogenesis in mouse skin.

    PubMed

    Yasukawa, Ken; Sun, Yi; Kitanaka, Susumu; Tomizawa, Naoyuki; Miura, Motofumi; Motohashi, Shigeyasu

    2008-07-01

    The methanol extract of galangal (the rhizomes of Alpinia officinarum L.) exhibited remarkable antitumor-promoting activity on an in vivo two-stage carcinogenesis test of mice using 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Seven diarylheptanoids (1-7) were isolated and identified from the active fraction of the methanol extracts of the galangal. These compounds, 1-7, were evaluated for their inhibitory effects on TPA-induced inflammation (1 microg/ear) in mice. These compounds (1-7) tested showed marked anti-inflammatory effects, with a 50% inhibitory dose of 0.8-2.7 micromol/ear.

  8. mXBP/CRE-BP2 and c-Jun form a complex which binds to the cyclic AMP, but not to the 12-O-tetradecanoylphorbol-13-acetate, response element.

    PubMed Central

    Ivashkiv, L B; Liou, H C; Kara, C J; Lamph, W W; Verma, I M; Glimcher, L H

    1990-01-01

    Proto-oncogene products c-Fos and c-Jun form a complex which binds with high affinity to the 12-O-tetradecanoylphorbol-13-acetate (TPA) response DNA element and which stimulates transcription of phorbol ester- inducible genes. We have previously identified, by screening a lambda gt11 expression library, murine protein mXBP, which binds to a sequence which overlaps the 3' end of the murine class II major histocompatibility complex A alpha gene X box, a conserved transcription element found upstream of all class II genes. Here, we demonstrate that the target sequence for mXBP is a consensus cyclic AMP response element (CRE). mXBP is a member of the leucine zipper family of DNA-binding proteins and has significant homology to oncoproteins c-Fos and c-Jun. The inferred amino acid sequence of mXBP shows near identity to human CRE-BP1, except it does not contain an internal proline-rich domain. Immunoprecipitation and glutaraldehyde cross-linking studies show that mXBP/CRE-BP2 can form a complex with c-Jun. Complex formation is dependent on intact leucine zipper domains in both proteins. mXBP-c-Jun complexes can coexist with c-Fos-c-Jun complexes and can bind with high affinity to CRE, but not to TPA response DNA element, sequences. These results suggest that changes in the expression of mXBP/CRE-BP2, c-Fos, and c-Jun, which alter the ratio of mXBP-c-Jun to c-Fos-c-Jun complexes, would affect the relative expression of cyclic AMP and phorbol ester-responsive genes. This provides support for a combinatorial model of gene regulation, whereby protein-protein interactions which alter the DNA binding specificity of protein complexes can expand the flexibility of cellular transcriptional responses. Images PMID:2138707

  9. The Consequences of edTPA

    ERIC Educational Resources Information Center

    Greenblatt, Deborah

    2016-01-01

    States and teacher preparation programs across the country are increasingly using a teacher candidate assessment called edTPA. The purpose? To make sure that teacher candidates are ready and able to teach before they begin their careers. The teacher performance assessment requires candidates to compile a portfolio that consists of lesson plans,…

  10. Fisetin regulates TPA-induced breast cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways.

    PubMed

    Noh, Eun-Mi; Park, Yeon-Ju; Kim, Jeong-Mi; Kim, Mi-Seong; Kim, Ha-Rim; Song, Hyun-Kyung; Hong, On-Yu; So, Hong-Seob; Yang, Sei-Hoon; Kim, Jong-Suk; Park, Samg Hyun; Youn, Hyun-Jo; You, Yong-Ouk; Choi, Ki-Bang; Kwon, Kang-Beom; Lee, Young-Rae

    2015-10-05

    Invasion and metastasis are among the main causes of death in patients with malignant tumors. Fisetin (3,3',4',7-tetrahydroxyflavone), a natural flavonoid found in the smoke tree (Cotinus coggygria), is known to have antimetastatic effects on prostate and lung cancers; however, the effect of fisetin on breast cancer metastasis is unknown. The aim of this study was to determine the anti-invasive activity of fisetin in human breast cancer cells. Matrix metalloproteinase (MMP)-9 is a major component facilitating the invasion of many cancer tumor cell types, and thus the inhibitory effect of fisetin on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human breast cancer cells was investigated in this study. Fisetin significantly attenuated TPA-induced cell invasion in MCF-7 human breast cancer cells, and was found to inhibit the activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways. This effect was furthermore associated with reduced NF-κB activation, suggesting that the anti-invasive effect of fisetin on MCF-7 cells may result from inhibited TPA activation of NF-κB and reduced TPA activation of PKCα/ROS/ERK1/2 and p38 MAPK signals, ultimately leading to the downregulation of MMP-9 expression. Our findings indicate the role of fisetin in MCF-7 cell invasion, and clarify the underlying molecular mechanisms of this role, suggesting fisetin as a potential chemopreventive agent for breast cancer metastasis.

  11. Cancer-promoting effect of capsaicin on DMBA/TPA-induced skin tumorigenesis by modulating inflammation, Erk and p38 in mice.

    PubMed

    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.

  12. Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

    SciTech Connect

    Hara, T.; Jung, L.K.L.; FU, S.M.

    1986-03-01

    With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% of the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.

  13. Comparison of altered expression of histocompatibility antigens with altered immune function in murine spleen cells treated with ultraviolet radiation and/or TPA

    SciTech Connect

    Pretell, J.O.; Cone, R.E.

    1985-02-01

    Previous studies in our laboratory demonstrated that several treatments that inhibited the ability of cells to stimulate the mixed lymphocyte reaction (MLR) also blocked the shedding of histocompatibility antigens and Ia antigens from murine spleen cells. In the present studies, one of these treatments, ultraviolet radiation (UV), was shown to cause an initial loss in the density of H-2K, IA, and IE antigens prior to the block in shedding observed after culture of these cells. Further analysis revealed that the UV-induced loss of antigens could be prevented by the presence of colchicine during irradiation. Biosynthetic analyses revealed the IA antigen synthesis was also inhibited in the UV-irradiated cells. Examination of the effects of a second agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) on the turnover of histocompatibility antigens revealed that the biosynthesis and shedding of these antigens were accelerated by this agent. However, addition of TPA to UV-irradiated cells did not result in a reversal of the UV-induced block in biosynthesis of IA antigens. Results of immune function assays correlated with the biochemical studies: UV-irradiation inhibited the generation of the MLR, but TPA enhanced this reaction, and addition of TPA to mixed lymphocyte cultures with UV-irradiated stimulators did not reverse the UV-induced inhibition. These results suggest that, although the turnover of histocompatibility antigens may be affected by TPA and UV in an antagonistic fashion, additional factors other than the expression of histocompatibility antigens are operating in the inhibition of stimulation of an MLR by UV radiation or its enhancement by TPA.

  14. Protective effects of Mangifera indica L. extract, mangiferin and selected antioxidants against TPA-induced biomolecules oxidation and peritoneal macrophage activation in mice.

    PubMed

    Sánchez, G M; Re, L; Giuliani, A; Núñez-Sellés, A J; Davison, G P; León-Fernández, O S

    2000-12-01

    We compared the protective abilities of Mangifera indica L. stem bark extract (Vimang) 50-250 mgkg(-1), mangiferin 50 mgkg(-1), vitamin C 100 mgkg(-1), vitamin E 100 mgkg(-1)and beta -carotene 50 mgkg(-1)against the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative damage in serum, liver, brain as well as in the hyper-production of reactive oxygen species (ROS) by peritoneal macrophages. The treatment of mice with Vimang, vitamin E and mangiferin reduced the TPA-induced production of ROS by the peritoneal macrophages by 70, 17 and 44%, respectively. Similarly, the H(2)O(2)levels were reduced by 55-73, 37 and 40%, respectively, when compared to the control group. The TPA-induced sulfhydryl group loss in liver homogenates was attenuated by all the tested antioxidants. Vimang, mangiferin, vitamin C plus E and beta -carotene decreased TPA-induced DNA fragmentation by 46-52, 35, 42 and 17%, respectively, in hepatic tissues, and by 29-34, 22, 41 and 17%, in brain tissues. Similar results were observed in respect to lipid peroxidation in serum, in hepatic mitochondria and microsomes, and in brain homogenate supernatants. Vimang exhibited a dose-dependent inhibition of TPA-induced biomolecule oxidation and of H(2)O(2)production by peritoneal macrophages. Even if Vimang, as well as other antioxidants, provided significant protection against TPA-induced oxidative damage, the former lead to better protection when compared with the other antioxidants at the used doses. Furthermore, the results indicated that Vimang is bioavailable for some vital target organs, including liver and brain tissues, peritoneal exudate cells and serum. Therefore, we conclude that Vimang could be useful to prevent the production of ROS and the oxidative tissue damages in vivo.

  15. Regulation of ectodermal differentiation in Xenopus laevis animal caps treated with TPA and ammonium chloride.

    PubMed

    Sotgia, C; Fascio, U; Pennati, R; De Bernardi, F

    1998-02-01

    Animal caps isolated from Xenopus laevis embryos at the blastula stage were treated sequentially with NH4Cl, a known cement gland inducer, and with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a known neural inducer. The two artificial inducers were also used in reverse order to see if they can mimic the natural inducers acting during the progressive determination of the ectodermal organ. Immunofluorescence and whole-mount in situ hybridization were used to study the expression of tubulin, taken to indicate an early step on the pathway of cell elongation, and neural cell adhesion molecule (N-CAM) taken to indicate an early step in the determination of the nervous system. The expression of XCG-1, a marker of early specification of the cement gland, was also studied. The results showed that the two artificial inducers can mimic the effects of the natural inducers in animal cap explants. The TPA behaves like a neural inducer, reducing the number and the extension of the cement gland when added to the medium in addition to NH4Cl, before or after NH4Cl treatment. In the process of cement gland/neural induction, it is possible to redirect the ectoderm already specified as cement gland to neural tissue, but it does not seem possible to respecify the neural tissue as cement gland. Moreover, the animal caps were also cut into dorsal and ventral parts and the two halves were treated separately. The results were similar to those obtained with treatment of the entire animal cap, suggesting that a dorsal-ventral pattern is not yet established before the gastrula stage, and that in normal embryos there are boundaries between the effects of different inducers.

  16. Protective effect of fermented soybean dried extracts against TPA-induced oxidative stress in hairless mice skin.

    PubMed

    Georgetti, Sandra R; Casagrande, Rúbia; Vicentini, Fabiana T M C; Baracat, Marcela M; Verri, Waldiceu A; Fonseca, Maria J V

    2013-01-01

    This study evaluated the chemical properties (polyphenol and genistein contents) of soybean extracts obtained by biotransformation and dried by spray dryer at different conditions and their in vivo ability to inhibit 12-O-tetradecanoylphorbol-13-acetate- (TPA-) induced biochemical alterations in the skin of hairless mice. By comparing the obtained data with that of the well-known active soybean extract Isoflavin beta, we evaluated the influence of the fermentation and drying process in the extracts efficacy. The results demonstrated that inlet gas temperature and adjuvant concentration for the extract drying process have significantly affected the total polyphenol contents and, to a minor degree, the genistein contents. However, the effect of topical stimulus with TPA, an oxidative stress inducer, which caused significant depletion of reduced glutathione (GSH) and catalase, with increased levels of H2O2 and lipid peroxidation (MDA) in the skin of hairless mice, was significantly prevented by the soybean extracts treatment. These results indicate that the spray drying processing resulted in a product capable of limiting the oxidative stress with possible therapeutic applicability as an antioxidant in pharmaceutical forms.

  17. Protective Effect of Fermented Soybean Dried Extracts against TPA-Induced Oxidative Stress in Hairless Mice Skin

    PubMed Central

    Georgetti, Sandra R.; Casagrande, Rúbia; Vicentini, Fabiana T. M. C.; Baracat, Marcela M.; Verri, Waldiceu A.; Fonseca, Maria J. V.

    2013-01-01

    This study evaluated the chemical properties (polyphenol and genistein contents) of soybean extracts obtained by biotransformation and dried by spray dryer at different conditions and their in vivo ability to inhibit 12-O-tetradecanoylphorbol-13-acetate- (TPA-) induced biochemical alterations in the skin of hairless mice. By comparing the obtained data with that of the well-known active soybean extract Isoflavin beta, we evaluated the influence of the fermentation and drying process in the extracts efficacy. The results demonstrated that inlet gas temperature and adjuvant concentration for the extract drying process have significantly affected the total polyphenol contents and, to a minor degree, the genistein contents. However, the effect of topical stimulus with TPA, an oxidative stress inducer, which caused significant depletion of reduced glutathione (GSH) and catalase, with increased levels of H2O2 and lipid peroxidation (MDA) in the skin of hairless mice, was significantly prevented by the soybean extracts treatment. These results indicate that the spray drying processing resulted in a product capable of limiting the oxidative stress with possible therapeutic applicability as an antioxidant in pharmaceutical forms. PMID:24073399

  18. Phytosphingosine stimulates the differentiation of human keratinocytes and inhibits TPA-induced inflammatory epidermal hyperplasia in hairless mouse skin.

    PubMed

    Kim, Sujong; Hong, Il; Hwang, Jung Sun; Choi, Jin Kyu; Rho, Ho Sik; Kim, Duck Hee; Chang, Ihseop; Lee, Seung Hun; Lee, Mi-Ock; Hwang, Jae Sung

    2006-01-01

    The binding of sphingoid bases to peroxisome proliferator-activated receptor (PPAR) has been detected in a solid-phase binding assay. However, sphingoid base-induced changes in PPAR transactivation activity have not been examined. In this report, we show by reporter gene analyses that phytosphingosine (PS), a natural sphingoid base, activates the transcriptional activity of PPARs in the immortalized human keratinocyte, HaCaT. Real-time PCR analyses showed that the mRNA level of PPARgamma was increased after PS treatment in HaCaT cells in a dose- and time-dependent manner. Because PPARs play important roles in skin barrier homeostasis by regulating epidermal cell growth, terminal differentiation, and inflammatory response, we examined the effect of PS on normal human epidermal keratinocytes (NHEKs) and mouse skin. PS increased the production of cornified envelope in NHEKs by approximately 1.8-fold compared with controls. Epidermal differentiation marker proteins such as involucrin, loricrin, and keratin1 were also increased in PS-treated NHEKs, by ELISA or Western blotting analysis. A [(3)H]thymidine incorporation assay showed that PS inhibited DNA synthesis in NHEKs to 20% compared with controls. The antiproliferative and anti-inflammatory effects of PS were examined in a mouse model of irritant contact dermatitis produced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). PS blocked epidermal thickening and edema and the infiltration of inflammatory cells into the dermis in the skin of TPA-treated hairless mice. The anti-inflammatory effects of PS were confirmed by the observation that PS blocked the TPA-induced generation of prostaglandin E(2) in peripheral mononuclear leukocytes. Taken together, our results provide an insight into the multiple regulatory roles of PS in epidermal homeostasis, and furthermore point to the potential use of PS as a therapeutic agent in the treatment of inflammatory and proliferative cutaneous diseases.

  19. Phytosphingosine Stimulates the Differentiation of Human Keratinocytes and Inhibits TPA-Induced Inflammatory Epidermal Hyperplasia in Hairless Mouse Skin

    PubMed Central

    Kim, Sujong; Hong, Il; Hwang, Jung Sun; Choi, Jin Kyu; Rho, Ho Sik; Kim, Duck Hee; Chang, Ihseop; Lee, Seung Hun; Lee, Mi-Ock; Hwang, Jae Sung

    2006-01-01

    The binding of sphingoid bases to peroxisome proliferator-activated receptor (PPAR) has been detected in a solid-phase binding assay. However, sphingoid base–induced changes in PPAR transactivation activity have not been examined. In this report, we show by reporter gene analyses that phytosphingosine (PS), a natural sphingoid base, activates the transcriptional activity of PPARs in the immortalized human keratinocyte, HaCaT. Real-time PCR analyses showed that the mRNA level of PPARγ was increased after PS treatment in HaCaT cells in a dose- and time-dependent manner. Because PPARs play important roles in skin barrier homeostasis by regulating epidermal cell growth, terminal differentiation, and inflammatory response, we examined the effect of PS on normal human epidermal keratinocytes (NHEKs) and mouse skin. PS increased the production of cornified envelope in NHEKs by approximately 1.8-fold compared with controls. Epidermal differentiation marker proteins such as involucrin, loricrin, and keratin1 were also increased in PS-treated NHEKs, by ELISA or Western blotting analysis. A [3H]thymidine incorporation assay showed that PS inhibited DNA synthesis in NHEKs to 20% compared with controls. The antiproliferative and anti-inflammatory effects of PS were examined in a mouse model of irritant contact dermatitis produced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). PS blocked epidermal thickening and edema and the infiltration of inflammatory cells into the dermis in the skin of TPA-treated hairless mice. The anti-inflammatory effects of PS were confirmed by the observation that PS blocked the TPA-induced generation of prostaglandin E2 in peripheral mononuclear leukocytes. Taken together, our results provide an insight into the multiple regulatory roles of PS in epidermal homeostasis, and furthermore point to the potential use of PS as a therapeutic agent in the treatment of inflammatory and proliferative cutaneous diseases. PMID:16838068

  20. PKM2 inhibitor shikonin suppresses TPA-induced mitochondrial malfunction and proliferation of skin epidermal JB6 cells.

    PubMed

    Li, Wenjuan; Liu, Joan; Zhao, Yunfeng

    2014-05-01

    Chemoprevention has been a pivotal and effective strategy during the skin cancer treatment. Using human skin normal and tumor samples, we demonstrated that both the expression and activity levels of pyruvate kinase M2 (PKM2) were higher in skin tumor tissues than normal tissues, suggesting that PKM2, one of important metabolic enzyme, might serve as a target for skin cancer prevention and/or therapy. Shikonin, a small-molecule active chemical, has been studied as an anti-cancer drug candidate in human cancer models. However, the mechanism of action and the chemopreventive potential of shikonin are unclear. Herein, we used the skin epidermal JB6 P+ cells and demonstrated that shikonin suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induced neoplastic cell transformation and PKM2 activation in the early stage of carcinogenesis. Mitochondrial functions were inhibited by TPA treatment, as indicated by reduced mitochondrial membrane potential and mitochondrial respiration, which were restored by shikonin. We also examined the levels of lactate as a glycolysis marker, and shikonin suppressed its increase caused by tumor promoter treatment. Modulation of cell metabolism by shikonin was associated with G2-M phase accumulation, and Fra-1 (a major subunit of activator protein 1 in skin tumorigenesis) downregulation. In addition, we demonstrated that AMP-activated protein kinase (AMPK), an energy sensor, which is inactivated by TPA, shikonin could reverse AMPK activity. These results suggest that shikonin bears chemopreventive potential for human skin cancers in which PKM2 is upregulated, which might be mediated by inhibiting oncogenic activation, PKM2 activation, and mitochondrial dysfunction.

  1. Fractionation of a tumor-initiating UV dose introduces DNA damage-retaining cells in hairless mouse skin and renders subsequent TPA-promoted tumors non-regressing

    PubMed Central

    van de Glind, Gerline; Rebel, Heggert; van Kempen, Marika; Tensen, Kees; de Gruijl, Frank

    2016-01-01

    Sunburns and especially sub-sunburn chronic UV exposure are associated with increased risk of squamous cell carcinomas (SCCs). Here we focus on a possible difference in tumor initiation from a single severe-sunburn dose (on day 1, 21 hairless mice) and from an equal dose fractionated into very low sub-sunburn doses not causing any (growth-promoting) epidermal hyperplasia (40 days daily exposure, n=20). From day 47 all mice received 12-O-Tetradecanoylphorbol-13-acetate (TPA) applications (2x/wk) for 20 weeks to promote tumor development within the lifetime of the animals. After the sub-sunburn regimen sparse DNA damage-retaining basal cells (quiescent stem cells, QSCs) remained in the non-hyperplastic epidermis. These cells were forced to divide by TPA. After discontinuation of TPA tumors regressed and disappeared in the ‘sunburn group’ but persisted and grew in the ‘sub-sunburn group’ (0.06 vs 2.50 SCCs and precursors ≥4mm/mouse after 280 days, p=0.03). As the tumors carried no mutations in p53, H/K/N-Ras and Notch1/2, these ‘usual suspects' were not involved in the UV-driven tumor initiation. Although we could not selectively eliminate QSCs (unknown phenotype) to establish causality, our data suggest that forcing specifically DNA damage-retaining QSCs to divide – with high mutagenic risk - gives rise to persisting (mainly ‘in situ’) skin carcinomas. PMID:26797757

  2. Modulation of survival and proliferation of BSC-1 cells through changes in spreading behavior caused by the tumor-promoting phorbol ester TPA.

    PubMed

    Shiba, Y; Kanno, Y

    1989-12-01

    The effect of a tumor-promoting phorbol ester on spreading behavior was investigated to clarify the involvement of the interactions between cells and substratum in the maintenance of cell viability and the control of cell proliferation. BSC-1 cells did not spread and lost cell viability after a 24-h incubation in the absence of calf serum. Addition of calf serum initially induced radial spreading and then polarized spreading, with the formation on stress fibers and focal contact-like structure, and enhanced survival. Vitronectin also induced both radial spreading and polarized spreading, and enhanced cell survival. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced radial spreading with actin ribbons in the absence of serum. It improved the survival of cells attached to the substratum, but not in suspension. TPA suppressed polarized spreading, formation of stress fibers and of focal contact-like structure, and cell proliferation, in the presence of serum. Phorbol did not have any effect. These results suggest that enhancement of radial spreading and inhibition of polarized spreading of BSC-1 cells by TPA are closely related to the enhancement of cell survival and inhibition of cell growth.

  3. Tissue Plasminogen Activator (tPA) Mediates Neurotoxin-Induced Cell Death and Microglial Activation

    DTIC Science & Technology

    2001-07-01

    Alzheimer’s disease and stroke. Tissue plasminogen activator (tPA), a protease converting plasminogen to plasmin, is necessary for neurodegeneration. In mice lacking tPA (tPA-/1), neurons are resistant to neurotoxic death. Delivery of tPA into tpA-/- mice restores susceptibility to neuronal death, indicating that tPA is neurotoxic in the context of excitotoxic injury. Although tPA is synthesized by neurons, the increase in tPA upon injury derives primarily from activated microglia, the immune cells of the brain. Microglia in tPA-/- mice demonstrate reduced activation.

  4. Beginning Teachers' Perceptions of the California Teaching Performance Assessment (TPA)

    ERIC Educational Resources Information Center

    Campbell, Conni; Ayala, Carlos Cuauhtémoc; Railsback, Gary; Freking, Frederick W.; McKenna, Corey; Lausch, David

    2016-01-01

    The teaching performance assessment (TPA) seeks to measure the knowledge, skills, and competencies of teachers during the credential phase of their training. The TPA was introduced in California in 2004 with programs piloting it and then became mandatory for candidates enrolling in preliminary programs in 2008. Although California has multiple…

  5. Enhancing Mitochondrial Respiration Suppresses Tumor Promoter TPA-Induced PKM2 Expression and Cell Transformation in Skin Epidermal JB6 Cells

    PubMed Central

    Wittwer, Jennifer A.; Robbins, Delira; Wang, Fei; Codarin, Sarah; Shen, Xinggui; Kevil, Christopher G.; Huang, Ting-Ting; Van Remmen, Holly; Richardson, Arlan; Zhao, Yunfeng

    2016-01-01

    Differentiated cells primarily metabolize glucose for energy via the tricarboxylic acid cycle and oxidative phosphorylation, but cancer cells thrive on a different mechanism to produce energy, characterized as the Warburg effect, which describes the increased dependence on aerobic glycolysis. The M2 isoform of pyruvate kinase (PKM2), which is responsible for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells and may contribute to the Warburg effect. However, whether PKM2 plays a contributing role during early cancer development is unclear. In our studies, we have made an attempt to elucidate the effects of varying mitochondrial respiration substrates on skin cell transformation and expression of PKM2. Tumorigenicity in murine skin epidermal JB6 P+ (promotable) cells was measured in a soft agar assay using 12-O-tetradecanoylphorbol-13-acetate (TPA) as a tumor promoter. We observed a significant reduction in cell transformation upon pretreatment with the mitochondrial respiration substrate succinate or malate/pyruvate. We observed that increased expression and activity of PKM2 in TPA-treated JB6 P+ cells and pretreatment with succinate or malate/pyruvate suppressed the effects. In addition, TPA treatment also induced PKM2 whereas PKM1 expression was suppressed in mouse skin epidermal tissues in vivo. In comparison with JB6 P+ cells, the nonpromotable JB6 P− cells showed no increase in PKM2 expression or activity upon TPA treatment. Knockdown of PKM2 using a siRNA approach significantly reduced skin cell transformation. Thus, our results suggest that PKM2 activation could be an early event and play a contributing role in skin tumorigenesis. PMID:21673231

  6. Reversal of the TPA-induced inhibition of gap junctional intercellular communication by Chaga mushroom (Inonotus obliquus) extracts: effects on MAP kinases.

    PubMed

    Park, Jung-Ran; Park, Joon-Suk; Jo, Eun-Hye; Hwang, Jae-Woong; Kim, Sun-Jung; Ra, Jeong-Chan; Aruoma, Okezie I; Lee, Yong-Soon; Kang, Kyung-Sun

    2006-01-01

    Chaga mushroom (Inonotus obliquus) has continued to receive attention as a folk medicine with indications for the treatment of cancers and digestive diseases. The anticarcinogenic effect of Chaga mushroom extract was investigated using a model system of gap junctional intercellular communication (GJIC) in WB-F344 normal rat liver epithelial cells. The cells were pre-incubated with Chaga mushroom extracts (5, 10, 20 microg/ml) for 24 h and this was followed by co-treatment with Chaga mushroom extracts and TPA (12-O-tetradecanoylphorbol-13-acetate, 10 ng/ml) for 1 h. The inhibition of GJIC by TPA (12-O-tetradecanoylphorbol-13-acetate), promoter of cancer, was prevented with treatment of Chaga mushroom extracts. Similarly, the increased phosphorylated ERK1/2 and p38 protein kinases were markedly reduced in Chaga mushroom extracts-treated cells. There was no change in the JNK kinase protein level, suggesting that Chaga mushroom extracts could only block the activation of ERK1/2 and p38 MAP kinase. The Chaga mushroom extracts further prevented the inhibition of GJIC through the blocking of Cx43 phosphorylation. Indeed cell-to-cell communication through gap junctional channels is a critical factor in the life and death balance of cells because GJIC has an important function in maintaining tissue homeostasis through the regulation of cell growth, differentiation, apoptosis and adaptive functions of differentiated cells. Thus Chaga mushroom may act as a natural anticancer product by preventing the inhibition of GJIC through the inactivation of ERK1/2 and p38 MAP kinase.

  7. Embryonic mutation as a possible cause of in utero carcinogenesis in mice revealed by postnatal treatment with 12-O-tetradecanoylphorbol-13-acetate

    SciTech Connect

    Nomura, T.; Nakajima, H.; Hatanaka, T.; Kinuta, M.; Hongyo, T. )

    1990-04-01

    Although in utero irradiation at early stages induced a high incidence of somatic mutations at coat color genes in the embryos of a specified tester strain (PT x HT F1) of mice, it was not carcinogenic by itself. However, in utero-irradiated animals did develop skin tumors and hepatomas (but not leukemias) by the postnatal administration of 12-O-tetradecanoylphorbol-13-acetate. The incidence of both tumors and embryonic mutations increased with in utero doses of X-rays. Furthermore, a large reduction of tumor incidence, about 80%, was observed by low-dose-rate irradiation, similar to the 75% reduction in spot size found for embryonic mutations. The tumor nodule size was also dramatically reduced by low-dose-rate irradiation. Consequently, the induced incidence and size of tumors produced by 12-O-tetradecanoylphorbol-13-acetate treatment parallel those which are observed for coat color mutations as expected, because somatic mutations observed in the pigment cells must similarly occur in embryonic cells of other organs. The larger the clone of mutant cells, the greater their chance of becoming tumorigenic by 12-O-tetradecanoylphorbol-13-acetate posttreatment. These results strongly support the recent epidemiological survey showing that adult types of cancers, but not leukemias, are increasing in the atomic bomb survivors exposed in utero, since humans are continuously exposed to a variety of cancer-promoting agents in contrast to experimental animals reared without such exposures.

  8. Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA

    SciTech Connect

    Friedmann, P.S.; Wren, F.E.; Matthews, J.N. )

    1990-02-01

    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.

  9. Effects of C-Phycocyanin on the representative genes of tumor development in mouse skin exposed to 12-O-tetradecanoyl-phorbol-13-acetate.

    PubMed

    Gupta, Naresh Kumar; Gupta, Krishna P

    2012-11-01

    C-Phycocyanin (C-PC), a biliprotein from the sea weed, has been shown to have the beneficial effects like antioxidant, anti-inflammatory, neuroprotective, and hepatoprotective properties and is used as food supplement. We are showing the effect of C-Phycocyanin on the early events altered by tumor promoter. TPA induced the expression of critical events of tumorigenesis like ornithine decarboxylase, cyclooxygenase-2, interleukin-6 and pSTAT3 in mouse skin after 5h of application, whereas expression of transglutaminase2 was decreased at this time point. This TPA-caused altered expression of genes was prevented in presence of C-Phycocyanin. This prevention by C-Phycocyanin appeared to be dependent on the dose of C-Phycocyanin used. The results are useful for the detailed study on the preventive effect of C-Phycocyanin on TPA induced tumor promotion.

  10. Selaginella tamariscina extract suppresses TPA-induced invasion and metastasis through inhibition of MMP-9 in human nasopharyngeal carcinoma HONE-1 cells

    PubMed Central

    2013-01-01

    Background Nasopharyngeal carcinoma (NPC) is known for its high incidence of neck lymph node metastasis, which represents poor prognosis. The present study aimed to examine the anti-metastatic properties of Selaginella tamariscina extract (STE) in human nasopharyngeal carcinoma HONE-1 cells in vitro. Methods Cell viability was examined by MTT assay, whereas cell motility was measured by invasive, migration and would healing assays. Real-time PCR, and promoter assays confirmed the inhibitory effects of STE on matrix metalloproteinase-9 (MMP-9) mRNA level in HONE-1 cells. Results The STE inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HONE-1 cell migration and invasion in a concentration-dependent manner. By zymographic and Western blot analyses, STE was shown to inhibit the activities and expression of MMP-9. Treatment of STE on TPA-induced HONE-1 cells inhibited MMP-9 expression and ERK1/2 phosphorylation without affecting JNK and p38 phosphorylation. Conclusions STE inhibits MMP-9 expression and HONE-1 cell metastasis. Its inhibitory effects may involve the Src/FAK/ERK 1/2 pathway. STE may have the potential of being an anti-metastatic agent against NPC. PMID:24053256

  11. DMBA/TPA treatment is necessary for BCC formation from patched deficient epidermal cells in Ptch(flox/flox)CD4Cre(+/-) mice.

    PubMed

    Uhmann, Anja; Heß, Ina; Frommhold, Anke; König, Simone; Zabel, Sebastian; Nitzki, Frauke; Dittmann, Kai; Lühder, Fred; Christiansen, Hans; Reifenberger, Julia; Schulz-Schaeffer, Walter; Hahn, Heidi

    2014-10-01

    The development of basal cell carcinoma (BCC), the most frequently diagnosed tumor among persons with European ancestry, is closely linked to mutations in the Hedgehog (Hh) receptor and tumor suppressor Patched1 (Ptch). Using Ptch(flox/flox)CD4Cre(+/-) mice, in which Ptch was ablated in CD4Cre-expressing cells, we demonstrate that the targeted cells can give rise to BCC after treatment with DMBA (7,12-dimethylbenz(a)anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate), but not after wounding of the skin. In addition, in this model, BCC are not caused by malfunctioning of Ptch-deficient T cells, as BCC did not develop when bone marrow (BM) of Ptch(flox/flox)CD4Cre(+/-) mice was transplanted into Ptch wild-type mice. Instead, lineage-tracing experiments and flow cytometric analyses suggest that the tumors are initiated from rare Ptch-deficient stem cell-like cells of the epidermis that express CD4. As DMBA/TPA is a prerequisite for BCC development in this model, the initiated cells need a second stimulus for expansion and tumor formation. However, in contrast to papilloma, this stimulus seems to be unrelated to alterations in the Ras signaling cascade. Together, these data suggest that biallelic loss of Ptch in CD4(+) cells does not suffice for BCC formation and that BCC formation requires a second so far unknown event, at least in the Ptch(flox/flox)CD4Cre(+/-) BCC mouse model.

  12. DHA blocks TPA-induced cell invasion by inhibiting MMP-9 expression via suppression of the PPAR-γ/NF-κB pathway in MCF-7 cells

    PubMed Central

    Hwang, Jin-Ki; Yu, Hong-Nu; Noh, Eun-Mi; Kim, Jeong-Mi; Hong, On-Yu; Youn, Hyun Jo; Jung, Sung Hoo; Kwon, Kang-Beom; Kim, Jong-Suk; Lee, Young-Rae

    2017-01-01

    Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is considered to have applications in cancer prevention and treatment. The beneficial effects of DHA against cancer metastasis are well established; however, the mechanisms underlying these effects in breast cancer are not clear. Cell invasion is critical for neoplastic metastasis, and involves the degradation of the extracellular matrix by matrix metalloproteinase (MMP)-9. The present study investigated the inhibitory effect of DHA on MMP-9 expression and cell invasion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the MCF-7 breast cancer cell line. DHA inhibited the TPA-induced activation of mitogen-activated protein kinase (MAPK) and the transcription of nuclear factor (NF)-κB, but did not inhibit the transcription of activator protein-1. DHA increased the activity of peroxisome proliferator-activated receptor (PPAR)-γ, an effect that was reversed by the application of the PPAR-γ antagonist GW9662. In addition, combined treatment with GW9662 and DHA increased NF-κB-related protein expression. These results indicate that DHA regulates MMP-9 expression and cell invasion via modulation of the MAPK signaling pathway and PPAR-γ/NF-κB activity. This suggests that DHA could be a potential therapeutic agent for the prevention of breast cancer metastasis. PMID:28123548

  13. TPA enhances growth hormone (GH) secretion effect of GH-releasing hormone (GHRH) by human gsp-positive pituitary somatotrophinomas.

    PubMed

    Lei, T; Bai, X; Hu, W; Xue, D; Jiang, X

    1999-01-01

    In recent years, one of the most exciting advances in the researches of pituitary adenomas is the discovery that 30%-40% of human pituitary somatotrophinomas carry somatic mutations of the gene for the alpha-subunit of the stimulatory GTP-binding protein, Gs (Gs alpha). These mutations, termed gsp oncogenes, may play an important role in the tumorigenesis of pituitary adenomas. Of 10 somatotrophinomas examined, 3 (30%) were proved to be gsp positive, as determined by sequence analysis of DNA generated by the polymerase chain reaction (PCR). GHRH exerted a significant stimulatory effect on GH secretion in 2 of 3 gsp-positive and 4 of 7 gsp-negative tumors. Moreover, phorbol ester, 1, 2-tetradecanoylphorbol-13-acetate (TPA), enhanced stimulation of lated the GH secretion effect exerted by GHRH in gsp-positive somatotrophinomas, whereas this effect was not observed in gsp-negative tumors. This result suggests that the protein kinase C signal system as well as adenylyl cyclase-cAMP-protein kinase A intracellular signal transduction system plays a pivotal role in GH secretory control of GHRH, which may work together via a cross-talk mechanism.

  14. TPA induction leads to a Th17-like response in transgenic K14/VEGF mice: a novel in vivo screening model of psoriasis.

    PubMed

    Hvid, Henning; Teige, Ingrid; Kvist, Peter Helding; Svensson, Lars; Kemp, Kåre

    2008-08-01

    Psoriasis is a common chronic inflammatory skin disease, characterized by epidermal hyperplasia, immune cell infiltration, increased dermal angiogenesis and local up-regulation of a variety of inflammatory mediators. Psoriasis is thought to be driven primarily by CD4(+) T cells with a T(h)1 and/or T(h)17 phenotype. Transgenic keratin 14 (K14)/vascular endothelial growth factor (VEGF) mice have previously been reported to develop a psoriasis-like phenotype. The aim of this study was to further characterize the model for validation as an in vivo screening model of psoriasis. Inflammation was induced in the ear skin with five topical applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and a significantly increased inflammation was found in TPA-induced K14/VEGF transgenic animals compared with wild-type mice. The amount of VEGF in the ear tissue was significantly elevated resulting in increased dermal angiogenesis. Furthermore, intense epidermal hyperplasia, CD3(+) infiltration and significantly increased amounts of (TNF) tumor necrosis factor alpha, IL-1 beta, IL-6, IL-12/23p40, IL-12p70, IL-22 and IL-17 were detected in the inflamed ear skin. This cytokine profile strongly suggests a T(h)17-mediated inflammation. All findings were a result of induced over-expression of VEGF. Topical treatment with betamethasone-17-valerate (BMS) significantly reduced ear skin inflammation and epidermal hyperplasia and also decreased the CD3(+) infiltration. In conclusion, the TPA-induced phenotype in K14/VEGF animals displayed several features of psoriasis, including a T(h)17 cytokine profile and a chronic-like progression, and can be used as an in vivo screening model of psoriasis.

  15. Potential O-acyl-substituted (-)-Epicatechin gallate prodrugs as inhibitors of DMBA/TPA-induced squamous cell carcinoma of skin in Swiss albino mice.

    PubMed

    Vyas, Sandeep; Manon, Benu; Vir Singh, Tej; Dev Sharma, Pritam; Sharma, Manu

    2011-04-01

    (-)-Epicatechin-3-gallate (1) is one of the principal catechins of green tea and exhibits cancer-preventive activities in various animal models. However, this compound is unstable in neutral or alkaline medium and, therefore, has a poor bioavailability. To improve its stability, O-acyl derivatives of 1 were prepared by isolating the partially purified tea catechin fraction from green tea extract and treating it with a variety of acylating agents. The resulting derivatives, compounds 2-6, were screened for their antitumor potential against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced squamous cell carcinogenesis of skin in mice. The results showed that the antitumor activity decreased with the increase in size of the chain length of the acyl groups, i.e., from compound 2, derivative with an Ac group, to compound 6, possessing a valeryl group. Moreover, the C(4) derivative with a branched acyl chain, 5, had a lower activity than the linear C(4) derivative 4. This reduction in the inhibitory activity may be due to the steric hindrance by the two Me groups. Moreover, significant increases in the protein levels analyzed by ELISA of c-Jun, p65, and p53 were observed in the skin of DMBA/TPA treated mice, whereas mice treated with 2 and DMBA/TPA had a similar expression of these transcription factors than the control mice. The prodrug potential of the O-acyl derivatives 2-6 showed that they were adequately stable to be absorbed intact from the intestine, more stable at gastric pH, and suitable for oral administration.

  16. Suppression of TPA-induced cancer cell invasion by Peucedanum japonicum Thunb. extract through the inhibition of PKCα/NF-κB-dependent MMP-9 expression in MCF-7 cells

    PubMed Central

    KIM, JEONG-MI; NOH, EUN-MI; KIM, HA-RIM; KIM, MI-SEONG; SONG, HYUN-KYUNG; LEE, MINOK; YANG, SEI-HOON; LEE, GUEM-SAN; MOON, HYOUNG-CHUL; KWON, KANG-BEOM; LEE, YOUNG-RAE

    2016-01-01

    Metastatic cancers spread from their site of origin (the primary site) to other parts of the body. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is important in metastatic cancers as it plays a major role in cancer cell invasion. The present study examined the inhibitory effect of an ethanol extract of Peucedanum japonicum Thunb. (PJT) on MMP-9 expression and the invasion of MCF-7 breast cancer cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis, gelatin zymography, and reverse transcription-quantitative PCR revealed that PJT significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, PJT attenuated TPA-induced nuclear translocation and the transcriptional activation of nuclear factor (NF)-κB. The results indicated that the PJT-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involved the suppression of the PKCα/NF-κB pathway in MCF-7 cells. Thus, the inhibition of MMP-9 expression by PJT may have potential value as a therapy for restricting the invasiveness of breast cancer. PMID:26717978

  17. Suppression of TPA-induced cancer cell invasion by Peucedanum japonicum Thunb. extract through the inhibition of PKCα/NF-κB-dependent MMP-9 expression in MCF-7 cells.

    PubMed

    Kim, Jeong-Mi; Noh, Eun-Mi; Kim, Ha-Rim; Kim, Mi-Seong; Song, Hyun-Kyung; Lee, Minok; Yang, Sei-Hoon; Lee, Guem-San; Moon, Hyoung-Chul; Kwon, Kang-Beom; Lee, Young-Rae

    2016-01-01

    Metastatic cancers spread from their site of origin (the primary site) to other parts of the body. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is important in metastatic cancers as it plays a major role in cancer cell invasion. The present study examined the inhibitory effect of an ethanol extract of Peucedanum japonicum Thunb. (PJT) on MMP-9 expression and the invasion of MCF-7 breast cancer cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis, gelatin zymography, and reverse transcription-quantitative PCR revealed that PJT significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, PJT attenuated TPA-induced nuclear translocation and the transcriptional activation of nuclear factor (NF)-κB. The results indicated that the PJT-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involved the suppression of the PKCα/NF-κB pathway in MCF-7 cells. Thus, the inhibition of MMP-9 expression by PJT may have potential value as a therapy for restricting the invasiveness of breast cancer.

  18. Perceived Influence of Cooperating Teachers on edTPA Performance

    ERIC Educational Resources Information Center

    Behney, Jennifer

    2016-01-01

    The Education Teaching Performance Assessment (edTPA) is a performance assessment of teacher effectiveness that is increasingly used to make decisions about licensure for teacher candidates, including candidates seeking certification in world languages. Because of the high-stakes nature of this assessment, it is important to isolate and better…

  19. Candidate Success and edTPA: Looking at the Data

    ERIC Educational Resources Information Center

    Evans, Lesley A.; Kelly, Mary K.; Baldwin, Joni L.; Arnold, Jackie M.

    2016-01-01

    This descriptive study looks at the correlations between Teacher Performance Assessment (edTPA) data and numerous program data points, including GPA, major GPA, and benchmark assignment scores, gathered in an Early Childhood Education (ECE) program. Previous studies have looked to correlate grade point average (GPA) with pre-service teacher…

  20. Teaching Elementary School Social Studies Methods under edTPA

    ERIC Educational Resources Information Center

    An, Sohyun

    2016-01-01

    This article reports a self-study that analyzes my experience as a teacher educator navigating a turbulent educational landscape with the advent of edTPA. The data consist of my journal entries, the syllabi, handouts, work submitted by my students, and course evaluations. Data were analyzed by using an inductive process to describe how the edTPA…

  1. Three Ways edTPA Prepared Me for the Classroom

    ERIC Educational Resources Information Center

    Butler, Matthew

    2015-01-01

    edTPA, a capstone assessment designed to assess whether new teachers are ready for the job by evaluating their teaching and their analysis of their teaching, helped prepare the author for the classroom in three ways. First, he became accountable to his students. Second, he learned to analyze his teaching. Third, he discovered how to relate…

  2. Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in the skin of SKH-1 mice.

    PubMed

    Wang, Z Y; Huang, M T; Ferraro, T; Wong, C Q; Lou, Y R; Reuhl, K; Iatropoulos, M; Yang, C S; Conney, A H

    1992-03-01

    Green tea was prepared by extracting 12.5 g of green tea leaves twice with 500 ml of boiling water, and the extracts were combined. This 1.25% green tea extract (1.25 g of tea leaves/100 ml of water) contained 4.69 mg of green tea extract solids per ml and was similar in composition to some green tea beverages consumed by humans. A 2.5% green tea extract (2.5 g of tea leaves/100 ml of water) was prepared similarly. Treatment of female SKH-1 mice with 180 mJ/cm2 of ultraviolet B light (UVB) once daily for 7 days resulted in red sunburn lesions of the skin. The intensity of red color and area of these lesions were inhibited in a dose-dependent fashion by the administration of 1.25 or 2.5% green tea extract as the sole source of drinking water before and during UVB treatment. Treatment of female SKH-1 mice with 180 mJ/cm2 of UVB once daily for 10 days followed 1 wk later by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate for 25 wk resulted in the development of skin tumors. The formation of skin tumors was inhibited by administration of 1.25% green tea extract as the sole source of drinking water prior to and during the 10 days of UVB treatment and for 1 wk after UVB treatment. In additional experiments, female SKH-1 mice were treated with 200 nmol of 7,12-dimethylbenz(a)anthracene followed 3 wk later by irradiation with 180, 60, or 30 mJ/cm2 of UVB twice weekly for 30 wk. UVB-induced formation of skin tumors and increased spleen size were inhibited by administration of 1.25% green tea extract as the sole source of drinking water prior to and during the 30 wk of UVB treatment. In these experiments, treatment of the animals with the green tea extract not only decreased the number of skin tumors but also decreased substantially the size of the tumors. In additional studies, SKH-1 mice were initiated by topical application of 200 nmol of 7,12-dimethylbenz(a)anthracene followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate for 25 wk

  3. A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair.

    PubMed

    Shivange, Gururaj; Kodipelli, Naveena; Monisha, Mohan; Anindya, Roy

    2014-12-26

    Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.

  4. TPA - A COMPUTER PROGRAM TO BALANCE MAPPED TURBOPUMP ASSEMBLIES

    NASA Technical Reports Server (NTRS)

    Walton, J. T.

    1994-01-01

    Accurate simulation of nuclear thermal propulsion systems using computational methods will permit reductions in testing and, thus, the time and cost of achieving a flight ready status for systems utilizing this advanced technology. An accurate simulation must maintain a "balance-of-plant" where the required pump work equals the supplied turbine work. This turbopump assembly balancing must be integrated into the overall system analysis models. TPA was developed to balance turbine and pump work using performance maps. It requires the inlet properties, performance maps, and shaft speed. TPA then computes the exit conditions and work terms. The work terms can then be balanced by varying the input shaft speed. The objective of the pump analysis is to determine the propellant state properties at the pump exit and the pump work. The pump analysis algorithm for liquid flow assumes that the shaft speed, the propellant state properties at the pump entrance, the propellant flow rate, the pump entrance and exit areas, as well as performance curves, are all known. The analysis of both the pump pressure rise and pump efficiency curves is required. The objective of the turbine analysis is to determine the propellant state properties at the turbine exit and the turbine work. The turbine analysis algorithm assumes that the shaft speed, the propellant state properties at the turbine entrance, the propellant flow rate, the turbine root mean square blade diameter, the turbine entrance and exit areas, as well as performance curves, are all known. The analysis also requires the turbine flow parameter curve and the turbine total efficiency curve. TPA is written in FORTRAN 77 to be machine independent. The TPA package includes the NBS+_PH2 code, which is also available separately (LEW-15505). TPA has been successfully implemented on a DEC VAX series computer running VMS, a Sun4 series computer running SunOS, and an IBM PC compatible computer running MS-DOS. Lahey F77L3 EM/32 v. 5.01 or

  5. 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.

    PubMed

    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.

  6. Topical (+)-catechin emulsified gel prevents DMBA/TPA-induced squamous cell carcinoma of the skin by modulating antioxidants and inflammatory biomarkers in BALB/c mice.

    PubMed

    Monga, Jitender; Aggarwal, Vaibhav; Suthar, Sharad Kumar; Monika; Nongalleima, Khumukcham; Sharma, Manu

    2014-12-01

    An emulsified gel of (+)-catechin was developed and evaluated topically against 7,12-dimethylbenz(a)anthracene-induced and 12-O-tetradecanoylphorbol-13-acetate-promoted (DMBA-induced and TPA-promoted) squamous cell carcinoma of the skin in BALB/c mice. The biological evaluation outcome indicated that the (+)-catechin emulsified gel increased the activity of oxidative stress biomarkers glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPx), whereas it decreased the level of malondialdehyde (MDA). The mechanistic study showed that genes implicated in the inflammation and cancer, such as cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB), and inducible nitric-oxide synthase (iNOS), were down-regulated by (+)-catechin emulsified gel while inhibiting an inflammatory mediator prostaglandin E2 (PGE2). The (+)-catechin emulsified gel further suppressed the activity of pro-inflammatory cytokines, viz. tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). The in vitro permeation study revealed that release of (+)-catechin from an emulsified gel base reached a steady state after 6 h, while pH of the entire emulsified gel was found to be between 6.2 and 6.5 that falls well within the normal pH range of the skin.

  7. Bioassay-guided chemical study of the anti-inflammatory effect of Senna villosa (Miller) H.S. Irwin & Barneby (Leguminosae) in TPA-induced ear edema.

    PubMed

    Susunaga-Notario, Ana del Carmen; Pérez-Gutiérrez, Salud; Zavala-Sánchez, Miguel Angel; Almanza-Pérez, Julio Cesar; Gutiérrez-Carrillo, Atilano; Arrieta-Báez, Daniel; López-López, Ana Laura; Román-Ramos, Rubén; Flores-Sáenz, José Luis Eduardo; Alarcón-Aguilar, Francisco Javier

    2014-07-15

    Senna villosa (Miller) is a plant that grows in México. In traditional Mexican medicine, it is used topically to treat skin infections, pustules and eruptions and to heal wounds by scar formation. However, studies of its potential anti-inflammatory effects have not been performed. The aim of the present study was to determine the anti-inflammatory effect of extracts from the leaves of Senna villosa and to perform a bioassay-guided chemical study of the extract with major activity in a model of ear edema induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). The results reveal that the chloroform extract from Senna villosa leaves has anti-inflammatory and anti-proliferative properties. Nine fractions were obtained from the bioassay-guided chemical study, including a white precipitate from fractions 2 and 3. Although none of the nine fractions presented anti-inflammatory activity, the white precipitate exhibited pharmacological activity. It was chemically characterized using mass spectrometry and infrared and nuclear magnetic resonance spectroscopy, resulting in a mixture of three aliphatic esters, which were identified as the principal constituents: hexyl tetradecanoate (C20H40O2), heptyl tetradecanoate (C21H42O2) and octyl tetradecanoate (C22H44O2). This research provides, for the first time, evidence of the anti-inflammatory and anti-proliferative properties of compounds isolated from Senna villosa.

  8. Intravenous tPA Therapy Does Not Worsen Acute Intracerebral Hemorrhage in Mice

    PubMed Central

    Foerch, Christian; Rosidi, Nathanael L.; Schlunk, Frieder; Lauer, Arne; Cianchetti, Flor A.; Mandeville, Emiri; Arai, Ken; Yigitkanli, Kazim; Fan, Xiang; Wang, Xiaoying; van Leyen, Klaus; Steinmetz, Helmuth; Schaffer, Chris B.; Lo, Eng H.

    2013-01-01

    Tissue plasminogen activator (tPA) is the only FDA-approved treatment for reperfusing ischemic strokes. But widespread use of tPA is still limited by fears of inadvertently administering tPA in patients with intracerebral hemorrhage (ICH). Surprisingly, however, the assumption that tPA will worsen ICH has never been biologically tested. Here, we assessed the effects of tPA in two models of ICH. In a mouse model of collagenase-induced ICH, hemorrhage volumes and neurological deficits after 24 hrs were similar in saline controls and tPA-treated mice, whereas heparin-treated mice had 3-fold larger hematomas. In a model of laser-induced vessel rupture, tPA also did not worsen hemorrhage volumes, while heparin did. tPA is known to worsen neurovascular injury by amplifying matrix metalloproteinases during cerebral ischemia. In contrast, tPA did not upregulate matrix metalloproteinases in our mouse ICH models. In summary, our experimental data do not support the assumption that intravenous tPA has a deleterious effect in acute ICH. However, due to potential species differences and the inability of models to fully capture the dynamics of human ICH, caution is warranted when considering the implications of these findings for human therapy. PMID:23408937

  9. Intravenous tPA therapy does not worsen acute intracerebral hemorrhage in mice.

    PubMed

    Foerch, Christian; Rosidi, Nathanael L; Schlunk, Frieder; Lauer, Arne; Cianchetti, Flor A; Mandeville, Emiri; Arai, Ken; Yigitkanli, Kazim; Fan, Xiang; Wang, Xiaoying; van Leyen, Klaus; Steinmetz, Helmuth; Schaffer, Chris B; Lo, Eng H

    2013-01-01

    Tissue plasminogen activator (tPA) is the only FDA-approved treatment for reperfusing ischemic strokes. But widespread use of tPA is still limited by fears of inadvertently administering tPA in patients with intracerebral hemorrhage (ICH). Surprisingly, however, the assumption that tPA will worsen ICH has never been biologically tested. Here, we assessed the effects of tPA in two models of ICH. In a mouse model of collagenase-induced ICH, hemorrhage volumes and neurological deficits after 24 hrs were similar in saline controls and tPA-treated mice, whereas heparin-treated mice had 3-fold larger hematomas. In a model of laser-induced vessel rupture, tPA also did not worsen hemorrhage volumes, while heparin did. tPA is known to worsen neurovascular injury by amplifying matrix metalloproteinases during cerebral ischemia. In contrast, tPA did not upregulate matrix metalloproteinases in our mouse ICH models. In summary, our experimental data do not support the assumption that intravenous tPA has a deleterious effect in acute ICH. However, due to potential species differences and the inability of models to fully capture the dynamics of human ICH, caution is warranted when considering the implications of these findings for human therapy.

  10. Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum.

    PubMed Central

    Barber, J R; Verma, I M

    1987-01-01

    We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed. Images PMID:3110603

  11. Using edTPA to Compare Online and Face to Face Teacher Preparation Programs

    ERIC Educational Resources Information Center

    Heafner, Tina; Petty, Teresa

    2016-01-01

    Central to determining the effectiveness of technology to support learning and the value of technology-mediated instruction is the quality of programs. edTPA is a widely accepted, national measure of teacher readiness and preparation. Using edTPA score reports for teacher candidates completing a teacher education program, this study provides data…

  12. Unstandardized Responses to a "Standardized" Test: The edTPA as Gatekeeper and Curriculum Change Agent

    ERIC Educational Resources Information Center

    Ledwell, Katherine; Oyler, Celia

    2016-01-01

    We examine edTPA (a teacher performance assessment) implementation at one private university during the first year that our state required this exam for initial teaching certification. Using data from semi-structured interviews with 19 teacher educators from 12 programs as well as public information on edTPA pass rates, we explore whether the…

  13. "What about Bilingualism?" A Critical Reflection on the edTPA with Teachers of Emergent Bilinguals

    ERIC Educational Resources Information Center

    Kleyn, Tatyana; López, Dina; Makar, Carmina

    2015-01-01

    Amidst the debates surrounding teacher quality and preparation programs, the edTPA (education Teaching Performance Assessment) has emerged to assess future teachers through a portfolio-based certification process. This study offers the perspective of three faculty members who participated in an experimental configuration of edTPA implementation…

  14. Impacts of tissue-type plasminogen activator (tPA) on neuronal survival

    PubMed Central

    Chevilley, Arnaud; Lesept, Flavie; Lenoir, Sophie; Ali, Carine; Parcq, Jérôme; Vivien, Denis

    2015-01-01

    Tissue-type plasminogen activator (tPA) a serine protease is constituted of five functional domains through which it interacts with different substrates, binding proteins, and receptors. In the last years, great interest has been given to the clinical relevance of targeting tPA in different diseases of the central nervous system, in particular stroke. Among its reported functions in the central nervous system, tPA displays both neurotrophic and neurotoxic effects. How can the protease mediate such opposite functions remain unclear but several hypotheses have been proposed. These include an influence of the degree of maturity and/or the type of neurons, of the level of tPA, of its origin (endogenous or exogenous) or of its form (single chain tPA versus two chain tPA). In this review, we will provide a synthetic snapshot of our current knowledge regarding the natural history of tPA and discuss how it sustains its pleiotropic functions with focus on excitotoxic/ischemic neuronal death and neuronal survival. PMID:26528141

  15. Polypyrrole layered SPEES/TPA proton exchange membrane for direct methanol fuel cells

    NASA Astrophysics Data System (ADS)

    Neelakandan, S.; Kanagaraj, P.; Sabarathinam, R. M.; Nagendran, A.

    2015-12-01

    Hybrid membranes based on sulfonated poly(1,4-phenylene ether ether sulfone) (SPEES)/tungstophosphoric acid (TPA) were prepared. SPEES/TPA membrane surfaces were modified with polypyrrole (Ppy) by in situ polymerization method to reduce the TPA leaching. The morphology and electrochemical property of the surface coated membranes were studied by SEM, AFM, water uptake, ion exchange capacity, proton conductivity, methanol permeability and tensile strength. The water uptake and the swelling ratio of the surface coated membranes decreased with increasing the Ppy layer. The surface roughness of the hybrid membrane was decreased with an increase in Ppy layer on the membrane surface. The methanol permeability of SPEES/TPA-Ppy4 hybrid membrane was significantly suppressed and found to be 2.1 × 10-7 cm2 s-1, which is 1.9 times lower than pristine SPEES membrane. The SPEES/TPA-Ppy4 membrane exhibits highest relative selectivity (2.86 × 104 S cm-3 s) than the other membrane with low TPA leaching. The tensile strength of hybrid membranes was improved with the introduction of Ppy layer. Combining their lower swelling ratio, high thermal stability and selectivity, SPEES/TPA-Ppy4 membranes could be a promising material as PEM for DMFC applications.

  16. Mesenchymal Stromal Cells Promote Axonal Outgrowth Alone and Synergistically with Astrocytes via tPA

    PubMed Central

    Qian, Jian-Yong; Chopp, Michael

    2016-01-01

    We reported that mesenchymal stromal cells (MSCs) enhance neurological recovery from experimental stroke and increase tissue plasminogen activator (tPA) expression in astrocytes. Here, we investigate mechanisms by which tPA mediates MSC enhanced axonal outgrowth. Primary murine neurons and astrocytes were isolated from wild-type (WT) and tPA-knockout (KO) cortices of embryos. Mouse MSCs (WT) were purchased from Cognate Inc. Neurons (WT or KO) were seeded in soma side of Xona microfluidic chambers, and astrocytes (WT or KO) and/or MSCs in axon side. The chambers were cultured as usual (normoxia) or subjected to oxygen deprivation. Primary neurons (seeded in plates) were co-cultured with astrocytes and/or MSCs (in inserts) for Western blot. In chambers, WT axons grew significantly longer than KO axons and exogenous tPA enhanced axonal outgrowth. MSCs increased WT axonal outgrowth alone and synergistically with WT astrocytes at both normoxia and oxygen deprivation conditions. The synergistic effect was inhibited by U0126, an ERK inhibitor, and receptor associated protein (RAP), a low density lipoprotein receptor related protein 1 (LRP1) ligand antagonist. However, MSCs exerted neither individual nor synergistic effects on KO axonal outgrowth. Western blot showed that MSCs promoted astrocytic tPA expression and increased neuronal tPA alone and synergistically with astrocytes. Also, MSCs activated neuronal ERK alone and synergistically with astrocytes, which was inhibited by RAP. We conclude: (1) MSCs promote axonal outgrowth via neuronal tPA and synergistically with astrocytic tPA; (2) neuronal tPA is critical to observe the synergistic effect of MSC and astrocytes on axonal outgrowth; and (3) tPA mediates MSC treatment-induced axonal outgrowth through the LRP1 receptor and ERK. PMID:27959956

  17. tPA Prescription and Administration Errors within a Regional Stroke System

    PubMed Central

    Chung, Lee S; Tkach, Aleksander; Lingenfelter, Erin M; Dehoney, Sarah; Rollo, Jeannie; de Havenon, Adam; DeWitt, Lucy Dana; Grantz, Matthew Ryan; Wang, Haimei; Wold, Jana J; Hannon, Peter M; Weathered, Natalie R; Majersik, Jennifer J

    2015-01-01

    Background IV tPA utilization in acute ischemic stroke (AIS) requires weight-based dosing and a standardized infusion rate. In our regional network, we have tried to minimize tPA dosing errors. We describe the frequency and types of tPA administration errors made in our comprehensive stroke center (CSC) and at community hospitals (CHs) prior to transfer. Methods Using our stroke quality database, we extracted clinical and pharmacy information on all patients who received IV tPA from 2010–11 at the CSC or CH prior to transfer. All records were analyzed for the presence of inclusion/exclusion criteria deviations or tPA errors in prescription, reconstitution, dispensing, or administration, and analyzed for association with outcomes. Results We identified 131 AIS cases treated with IV tPA: 51% female; mean age 68; 32% treated at CSC, 68% at CH (including 26% by telestroke) from 22 CHs. tPA prescription and administration errors were present in 64% of all patients (41% CSC, 75% CH, p<0.001), the most common being incorrect dosage for body weight (19% CSC, 55% CH, p<0.001). Of the 27 overdoses, there were 3 deaths due to systemic hemorrhage or ICH. Nonetheless, outcomes (parenchymal hematoma, mortality, mRS) did not differ between CSC and CH patients nor between those with and without errors. Conclusion Despite focus on minimization of tPA administration errors in AIS patients, such errors were very common in our regional stroke system. Although an association between tPA errors and stroke outcomes was not demonstrated, quality assurance mechanisms are still necessary to reduce potentially dangerous, avoidable errors. PMID:26698642

  18. Structure of Molybdenum Under Dynamic Compression to 1 TPa

    NASA Astrophysics Data System (ADS)

    Duffy, Thomas; Wang, Jue; Coppari, Federica; Smith, Raymond; Eggert, Jon; Lazicki, Amy; Fratanduono, Dayne; Rygg, Ryan; Boehly, Thomas; Collins, Gilbert

    2015-06-01

    Molybdenum (Mo) is a refractory 4d transition metal that is widely used as a standard in static and dynamic high-pressure experiments. However, there are significant unanswered questions and unresolved discrepancies about the melting curve and high-pressure phase stability of this fundamental material. Similar questions surround the melting curve and phase stabilities of other transition metals including Ta and Fe, and so a better understanding of Mo has broad implications for high-pressure science and geophysics. Here we use x-ray diffraction to determine the crystal structure of molybdenum under both shock and ramp compression to pressures as high as 1 TPa. Under shock loading, we find that Mo remains in body centered cubic (BCC) structure until melting begins at near 390 GPa. Our results are in good agreement with recent theoretical calculations and recent re-measurement of sound speeds along the Hugoniot. We also carried out x-ray diffraction measurements of ramp-loaded molybdenum up to 1050 GPa. Our x-ray diffraction patterns are consistent with the persistence of the BCC phase up to the highest pressure achieved. The measured densities under ramp loading are intermediate between those achieved under shock compression and those expected from extrapolation of room-temperature data. We do not observe evidence for the theoretically predicted transition to face centered cubic or double hexagonal close packed phases above 600 GPa.

  19. Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice

    SciTech Connect

    Kim, Young Nam; Kim, Dae Won; Jo, Hyo Sang; Shin, Min Jea; Ahn, Eun Hee; Ryu, Eun Ji; Yong, Ji In; Cha, Hyun Ju; Kim, Sang Jin; Yeo, Hyeon Ji; Youn, Jong Kyu; Hwang, Jae Hyeok; Jeong, Ji-Heon; Kim, Duk-Soo; Cho, Sung-Woo; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2015-07-15

    Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases. - Highlights: • Transduced Tat-CBR1 reduces LPS-induced inflammatory mediators and cytokines. • Tat-CBR1 inhibits MAPK and NF-κB activation. • Tat-CBR1 ameliorates inflammation response in vitro and in vivo. • Tat-CBR1 may be useful as potential therapeutic agent for inflammation.

  20. X-ray diffraction of solid tin to 1.2 TPa

    DOE PAGES

    Lazicki, A.; Rygg, J. R.; Coppari, F.; ...

    2015-08-12

    In this study, we report direct in situ measurements of the crystal structure of tin between 0.12 and 1.2 TPa, the highest stress at which a crystal structure has ever been observed. Using angle-dispersive powder x-ray diffraction, we find that dynamically compressed Sn transforms to the body-centered-cubic (bcc) structure previously identified by ambient-temperature quasistatic-compression studies and by zero-kelvin density-functional theory predictions between 0.06 and 0.16 TPa. However, we observe no evidence for the hexagonal close-packed (hcp) phase found by those studies to be stable above 0.16 TPa. Instead, our results are consistent with bcc up to 1.2 TPa. We conjecturemore » that at high temperature bcc is stabilized relative to hcp due to differences in vibrational free energy.« less

  1. X-ray diffraction of solid tin to 1.2 TPa

    SciTech Connect

    Lazicki, A.; Rygg, J. R.; Coppari, F.; Smith, R.; Fratanduono, D.; Kraus, R. G.; Collins, G. W.; Briggs, R.; Braun, D. G.; Swift, D. C.; Eggert, J. H.

    2015-08-12

    In this study, we report direct in situ measurements of the crystal structure of tin between 0.12 and 1.2 TPa, the highest stress at which a crystal structure has ever been observed. Using angle-dispersive powder x-ray diffraction, we find that dynamically compressed Sn transforms to the body-centered-cubic (bcc) structure previously identified by ambient-temperature quasistatic-compression studies and by zero-kelvin density-functional theory predictions between 0.06 and 0.16 TPa. However, we observe no evidence for the hexagonal close-packed (hcp) phase found by those studies to be stable above 0.16 TPa. Instead, our results are consistent with bcc up to 1.2 TPa. We conjecture that at high temperature bcc is stabilized relative to hcp due to differences in vibrational free energy.

  2. X-Ray Diffraction of Solid Tin to 1.2 TPa.

    PubMed

    Lazicki, A; Rygg, J R; Coppari, F; Smith, R; Fratanduono, D; Kraus, R G; Collins, G W; Briggs, R; Braun, D G; Swift, D C; Eggert, J H

    2015-08-14

    We report direct in situ measurements of the crystal structure of tin between 0.12 and 1.2 TPa, the highest stress at which a crystal structure has ever been observed. Using angle-dispersive powder x-ray diffraction, we find that dynamically compressed Sn transforms to the body-centered-cubic (bcc) structure previously identified by ambient-temperature quasistatic-compression studies and by zero-kelvin density-functional theory predictions between 0.06 and 0.16 TPa. However, we observe no evidence for the hexagonal close-packed (hcp) phase found by those studies to be stable above 0.16 TPa. Instead, our results are consistent with bcc up to 1.2 TPa. We conjecture that at high temperature bcc is stabilized relative to hcp due to differences in vibrational free energy.

  3. Toxicological review and oral risk assessment of terephthalic acid (TPA) and its esters: A category approach.

    PubMed

    Ball, Gwendolyn L; McLellan, Clifton J; Bhat, Virunya S

    2012-01-01

    Polyethylene terephthalate, a copolymer of terephthalic acid (TPA) or dimethyl terephthalate (DMT) with ethylene glycol, has food, beverage, and drinking water contact applications. Di-2-ethylhexyl terephthalate (DEHT) is a plasticizer in food and drinking water contact materials. Oral reference doses (RfDs) and total allowable concentrations (TACs) in drinking water were derived for TPA, DMT, and DEHT. Category RfD and TAC levels were also established for nine C(1)-C(8) terephthalate esters. The mode of action of TPA, and of DMT, which is metabolized to TPA, involves urinary acidosis, altered electrolyte elimination and hypercalciuria, urinary supersaturation with calcium terephthalate or calcium hydrogen terephthalate, and crystallization into bladder calculi. Weanling rats were more sensitive to calculus formation than dams. Calculi-induced irritation led to bladder hyperplasia and tumors in rats fed 1000 mg/kg-day TPA. The lack of effects at 142 mg/kg-day supports a threshold for urine saturation with calcium terephthalate, a key event for calculus formation. Chronic dietary DMT exposure in rodents caused kidney inflammation, but not calculi. Chronic dietary DEHT exposure caused general toxicity unrelated to calculi, although urine pH was reduced suggesting the TPA metabolite was biologically-active, but of insufficient concentration to induce calculi. Respective oral reference doses of 0.5, 0.5, and 0.2 mg/kg-day and total allowable drinking water concentrations of 3, 3, and 1 mg/L were derived for TPA, DMT, and DEHT. An oral RfD of 0.2 mg/kg-day for the terephthalate category chemicals corresponded to a drinking water TAC of 1 mg/L.

  4. Suppression of endothelial t-PA expression by prolonged high laminar shear stress

    SciTech Connect

    Ulfhammer, Erik; Carlstroem, Maria; Bergh, Niklas; Larsson, Pia; Karlsson, Lena; Jern, Sverker

    2009-02-06

    Primary hypertension is associated with an impaired capacity for acute release of endothelial tissue-type plasminogen activator (t-PA), which is an important local protective response to prevent thrombus extension. As hypertensive vascular remodeling potentially results in increased vascular wall shear stress, we investigated the impact of shear on regulation of t-PA. Cultured human endothelial cells were exposed to low ({<=}1.5 dyn/cm{sup 2}) or high (25 dyn/cm{sup 2}) laminar shear stress for up to 48 h in two different experimental models. Using real-time RT-PCR and ELISA, shear stress was observed to time and magnitude-dependently suppress t-PA transcript and protein secretion to approximately 30% of basal levels. Mechanistic experiments revealed reduced nuclear protein binding to the t-PA specific CRE element (EMSA) and an almost completely abrogated shear response with pharmacologic JNK inhibition. We conclude that prolonged high laminar shear stress suppresses endothelial t-PA expression and may therefore contribute to the enhanced risk of arterial thrombosis in hypertensive disease.

  5. Adenylate cyclase regulation in the spermatogenic cell plasma membrane: Modulating effects of TPA and TCDD

    SciTech Connect

    Beebe, L.E.

    1989-01-01

    This research was designed to compare the effects of TPA, a phorbol ester, and TCDD in a spermatogenic cell population, a target of TCDD toxicity. Membrane-bound adenylate cyclase activity was used an index of membrane function, and was quantified by the amount of {sup 32}P-cAMP formed from {sup 32}P-ATP following chromatographic separation. Exposure to male germ cells in-vitro to TPA and TCDD followed by direct measurement of enzyme activity was used to investigate the potential of each agent to perturb membrane function. TPA and TCDD consistently inhibited adenylate cyclase activity at the levels of G{sub s}-catalytic unit coupling and hormone-receptor activation, as measured by the stimulation of enzyme activity by concomitant addition of forskolin and GTP and FSH and GTP, respectively. The effect on coupling required at least 60 minutes of exposure to TPA or TCDD. Concentration-response curves demonstrated a progressive desensitization with increasing TPA concentration, while TCDD exhibited consistent inhibition over the same concentration range.

  6. TPA-inducible proteins may account for sensitivity to promotion of transformation

    SciTech Connect

    Hirano, K.; Smith, B.; Colburn, N.H.

    1986-05-01

    The preneoplastic JB6 mouse epidermal cell system includes cell lines sensitive (P/sup +/) or resistant (P/sup -/) to tumor promoter induced neoplastic transformation. The authors investigated whether a difference in TPA-inducible proteins may explain this differential sensitivity. The synthesis of a 39 Kd cytoplasmic protein (Major Excreted Protein) was TPA-inducible, but to a similar extent in both P/sup +/ and P/sup -/ cells. TPA stimulated phosphorylation but not synthesis of the previously described stress protein pp80 in both P/sup +/ and P/sup -/ cells from 1 to 5 hr after treatment. Pulse labelling of P/sup +/ and P/sup -/ cells with /sup 35/S-methionine revealed a TPA dependent P/sup +/ specific transient increase in the synthesis of 58Kd protein. Induction was observed at 1 hr, and returned to basal levels by 4 hr and 20 hr, in nuclear and cytoplasmic fractions, respectively. This protein is not phosphorylated in response to TPA treatment. P/sup +/ cells differ from P/sup -/ cells in one or more genes that specify sensitivity to promotion of transformation, designated pro genes. Antibodies to three peptides representing the pro-1 open reading frame were used in immunoprecipitation and Western blotting to isolate the pro-1 gene product. A 43 Kd protein was immunologically responsive to the pro-1 peptide antibodies, and showed an increased signal 40 min after TPA treatment. Since the predicted molecular weight of a pro-1 gene product is only 7 Kd, the possibility of a modification of the protein by poly(ADP-ribosylation) or glycosylation is being investigated.

  7. CpG and TpA frequencies in the plant system.

    PubMed Central

    Boudraa, M; Perrin, P

    1987-01-01

    Higher plant nuclear sequences reveal avoidance of CpG and TpA doublets. Chloroplast sequences avoid the TpA doublet in all codon positions. The chloroplast genome is not methylated but codon positions II-III and untranslated regions avoid CpG. The mitochondrial genome, also unmethylated, avoids CpG in all codon positions. We therefore deduce that methylation is not sufficient to explain CpG avoidance in the higher plant systems. Other factors must be taken into account such as amino acid composition, codon choices and perhaps stability of the DNA helix. PMID:3497385

  8. Where Practicum Meets Test Preparation: Supporting Teacher Candidates through EdTPA

    ERIC Educational Resources Information Center

    Clark-Gareca, Beth

    2015-01-01

    A strong focus on teacher performance is resurfacing in teacher-preparation programs across the US. EdTPA, a teacher-performance assessment designed to determine K-12 teacher candidates' readiness for the classroom, has become central in teacher-preparation programs in several states and promises to be implemented in more states in the coming…

  9. Breakers, Benders, and Obeyers: Inquiring into Teacher Educators' Mediation of edTPA

    ERIC Educational Resources Information Center

    Ratner, Andrew R.; Kolman, Joni S.

    2016-01-01

    This article reflects a qualitative exploratory inquiry into the lived experiences of faculty members working within a system of urban schools of education as they supported diverse teacher candidates in completing the Educative Teacher Performance Assessment (edTPA) during its first semesters of high-stakes implementation. Drawing upon…

  10. Next Generation Science Standards and edTPA: Evidence of Science and Engineering Practices

    ERIC Educational Resources Information Center

    Brownstein, Erica M.; Horvath, Larry

    2016-01-01

    Science teacher educators in the United States are currently preparing future science teachers to effectively implement the "Next Generation Science Standards" (NGSS) and, in thirteen states, to successfully pass a content-specific high stakes teacher performance assessment, the edTPA. Science education and teacher performance assessment…

  11. Developing a Culture of Learning around the edTPA: One University's Journey

    ERIC Educational Resources Information Center

    Miller, Matthew; Carroll, David; Jancic, Mitchell; Markworth, Kimberly

    2015-01-01

    In this article, we discuss how an interdisciplinary faculty team at a midsized public university created supports for the Teacher Performance Assessment (edTPA), a high-stakes performance assessment for preservice candidates being adopted by many states. We provide a general description of our work in contending with the challenge of developing a…

  12. Serum tissue polypeptide antigen (TPA) and prostatic acid phosphatase (PAP) in patients with prostatic cancer.

    PubMed

    Marczyńska, A; Kulpa, J; Leńko, J; Augustyn, M

    1988-01-01

    Concurrent measurements of serum TPA and PAP concentrations by double antibody radioimmunoassays were done in 49 patients with prostatic cancer in different clinical stages. The reference group comprised patients suffering from BPH. Positive TPA was found in 32.7% of cancer patients, the lowest percentage in stage A (11.1%) and the highest in stage D (55.6%). The additional value as a diagnostic aid of the TPA test was revealed on the basis of examination of the selected group of patients with not increased PAP. Positive TPA was found in 16.7% of patients: none in stage A, 22.2% in stage B, and 33.3% in stage D. Prostatic cancer remains the most common malignancy of the genitourinary tract. The improvement in the results of treatment involves not only a modernization of treatment modalities but also the introduction of laboratory tests which give the most ample information on the stage of tumour development and improve possibilities to control tumour therapy. Besides the refinement of the determination procedures of specific prostatic markers, prostatic acid phosphatase (PAP), through radio- and enzyme-immunological methods, there is a search for additional markers which might be helpful in diagnosis and follow-up of treatment.

  13. Buyer Beware: Lessons Learned from EdTPA Implementation in New York State

    ERIC Educational Resources Information Center

    Greenblatt, Deborah; O'Hara, Kate E.

    2015-01-01

    As states across the country continue their implementation of the Teacher Performance Assessment Portfolio (edTPA), a complex and high-stakes certification requirement for teacher certification, there are important lessons for educators and education advocates to learn from New York State's implementation. As Linda Darling-Hammond, developer and…

  14. Racist Ordering, Settler Colonialism, and EdTPA: A Participatory Policy Analysis

    ERIC Educational Resources Information Center

    Tuck, Eve; Gorlewski, Julie

    2016-01-01

    This article tells the story of an intervention by a collective of teacher educators on New York State's adoption of edTPA. Too often in education policy analysis, issues of race are discussed briefly, if at all. This article argues that attending to constructions of race specific to settler colonialism is an important approach to education policy…

  15. Draft Genome Sequence of an Anicemycin Producer, Streptomyces sp. TP-A0648

    PubMed Central

    Hosoyama, Akira; Kimura, Akane; Ichikawa, Natsuko; Igarashi, Yasuhiro

    2017-01-01

    ABSTRACT We report the draft genome sequence of Streptomyces sp. TP-A0648 isolated from a leaf of Aucuba japonica. This strain produces a new tumor cell growth inhibitor designated anicemycin. The genome harbors at least 12 biosynthetic gene clusters for polyketides and nonribosomal peptides, suggesting the potential to produce diverse secondary metabolites. PMID:28082502

  16. Labeling of human clots in vitro with an active-site mutant of t-PA

    SciTech Connect

    Fry, E.T.; Mack, D.L.; Monge, J.C.; Billadello, J.J.; Sobel, B.E. )

    1990-02-01

    Prompt detection of acute thrombosis and its response to treatment with thrombolytic agents generally require angiography. Scintigraphic approaches with labeled antibodies to or components of the coagulation and fibrinolytic systems have been disappointing because of prolonged circulating half-lives of tracers and relatively slow or limited binding to thrombi. Accordingly, we developed and characterized a thrombolytically inactive, active-site mutant (Ser-478----Thr) of tissue-type plasminogen activator (t-PA) designed to detect thrombi in vivo. Binding of iodine-125-({sup 125}I) labeled Ser----Thr t-PA to thrombi in vitro was time- and concentration-dependent, and specific judging from inhibition by pre-incubation with anti-t-PA IgG. Clearance of 125I-labeled mutant t-PA in rabbits was rapid and biexponential (alpha t1/2 = 1.9 +/- 0.4 min, beta t1/2 = 39.8 +/- 11.2 min). Thus, the amidolytically inactive mutant of t-PA designed binds rapidly and specifically to human thrombi in vitro and is cleared rapidly from the circulation in vivo--properties rendering it attractive as a potentially useful clot imaging agent.

  17. Experiences of t-PA use in moderate-to-severe hepatic veno-occlusive disease after hematopoietic SCT: is it still reasonable to use t-PA?

    PubMed

    Yoon, J-H; Min, W-S; Kim, H-Je; Kim, J-H; Shin, S-H; Yahng, S-A; Lee, S-E; Cho, B-S; Eom, K-S; Kim, Y-J; Lee, S; Min, C-K; Cho, S-G; Kim, D-W; Lee, J-W; Park, C-W

    2013-11-01

    Hepatic veno-occlusive disease (VOD) remains one of the most severe complications of hematopoietic SCT (HSCT). Anticoagulation and thrombolytic therapies using tissue-plasminogen activator (t-PA) have been used, but are reported to be ineffective and are associated with significant bleeding complications. We analyzed 56 moderate-to-severe post HSCT hepatic VOD cases treated with t-PA. We analyzed clinical outcomes according to the maximal daily dose of t-PA (t-PAmax) and the severity of VOD. Patients were stratified by t-PAmax10 mg (n=37) vs t-PAmax>10 mg (n=19). A higher t-PAmax was associated with increased mortality. Bleeding complications were more likely at higher t-PAmax in both moderate and severe VOD (P=0.036, 0.063), especially if patients had concomitant use of anticoagulants (36.4% vs 13.3%). In moderate VOD, the response rate was 86.4% for t-PAmax10 mg/day and 80% for t-PAmax>10 mg compared with 33.3% and 7.1%, respectively, for severe VOD (P=0.106). The 5-year OS in moderate and severe VOD was 49% and 7%, respectively, and it was 32% for t-PAmax10 mg and 18% for t-PAmax>10 mg. Our data demonstrate that lower bleeding complications and bleeding-related deaths may result from strict limitations on the t-PAmax without concomitant use of anticoagulation therapy. However, the overall response and survival outcomes should be re-evaluated by a well-validated study in the future.

  18. What Do We Really Know about the EdTPA? Research, PACT, and Packaging a Local Teacher Performance Assessment for National Use

    ERIC Educational Resources Information Center

    Hébert, Cristyne

    2017-01-01

    This article calls attention to the overreliance on research about the Performance Assessment for California Teachers (PACT)--often labeled edTPA's predecessor--as justification for the edTPA. The article argues that the distinctions between the assessments are too vast to rely on PACT data to support the edTPA, given the localized nature of PACT…

  19. Preservice Teachers' Adaptations to Tensions Associated with the edTPA during Its Early Implementation in New York and Washington States

    ERIC Educational Resources Information Center

    Meuwissen, Kevin W.; Choppin, Jeffrey M.

    2015-01-01

    The edTPA is a teaching performance assessment (TPA) that the states of New York and Washington implemented as a licensure requirement in 2013. While TPAs are not new modes of assessment, New York and Washington are the first states to use the edTPA specifically as a compulsory, high-stakes policy lever in an effort to strengthen the quality and…

  20. Phorbol ester and spontaneous activity in SHR aorta

    SciTech Connect

    Moisey, D.M.; Cox, R.H.

    1986-03-01

    Thoracic aortas (TA) were excised from 6-week old SHR and WKY. 2mm rings were mounted isometrically at optimum preload. Spontaneous rhythmical activity developed in TA from SHR and had a frequency of 3-4/min with varying periods of quiescence between bursts of activity. The spontaneous activity often produced an increase in tension development which was associated with increased frequency of oscillations. Verapamil (10/sup -7/ M) or Ca/sup + +/-free solution added during the contractile phase resulted in an immediate loss of tension and spontaneous activity. Addition of ouabain (10/sup -4/ M) during the contractile phase of spontaneous activity, increased the frequency of oscillations which appeared to fuse into a tetanus. Spontaneous rhythmical activity was infrequently observed in TA from WKY. However, addition of phorbol 12-myristate-13 acetate (TPA), frequently induced spontaneous rhythmic oscillations associated with tension development in TA from WKY. TPA contracted the SHR TA and increased the frequency of oscillations. SHR TA were more sensitive to TPA than WKY. This study demonstrates (1) spontaneous rhythmical activity, independent of agonist stimulation in TA from 6-week old SHR and (2) TPA induced spontaneous oscillatory activity. The mechanism underlying the spontaneous oscillatory activity may involve membrane coupling events and Na-pump difference between SHR and WKY.

  1. A unique enhancer element for the trans activator (p40 sup tax ) of human T-cell leukemia virus type I that is distinct from cyclic AMP- and 12-O-tetradecanoylphobol-13-acetate-responsive elements

    SciTech Connect

    Fujisawa, Junichi; Toita, Masami; Yoshida, Mitsuaki )

    1989-08-01

    The trans activator (p40{sup tax}) of human T-cell leukemia virus type I (HTLV-I) is a transcriptional factor that activates the long terminal repeat (LTR) of HTLV-I and interleukin-2 receptor {alpha}. The authors examined the HTLV-I enhancer responsible for tax-mediated trans activation and identified (A/T)(G/C)(G/C)CNNTGACG(T/A) as a plausible tax-responsive element (TRE). The putative TRE in the LTR was found to be different from the elements required for activation by cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate, although these elements overlapped each other. The TRE was also different from a binding site of N-{kappa}B-like factor that was identified was identified in the interleukin-2 receptor {alpha} promoter and human immunodeficiency virus LTR as a TRE. The latter result was further demonstrated by the failure of the NF-{kappa}B sequence to compete with the TRE of the LTR in a protein-binding assay. These findings indicate that tax function and its cascade can modulate activities of various enhancer sequences, which are probably regulated by distinct DNA-binding factors.

  2. Polymorphism in the spin-crossover ferric complexes [(TPA)Fe(III)(TCC)]PF6.

    PubMed

    Collet, Eric; Boillot, Marie Laure; Hebert, Johan; Moisan, Nicolas; Servol, Marina; Lorenc, Maciej; Toupet, Loïc; Buron-Le Cointe, Marylise; Tissot, Antoine; Sainton, Joelle

    2009-08-01

    We have identified two polymorphs of the molecular complex [(TPA)Fe((III))(TCC)]PF(6) [TPA = tris(2-pyridylmethyl)amine and TCC = 3,4,5,6-tetrachlorocatecholate dianion]: one is monoclinic and the other is orthorhombic. By lowering the temperature both undergo a thermal spin-crossover between a high-spin (S = 5/2) and a low-spin (S = 1/2) state, which we detected by magnetic, optical and X-ray diffraction measurements. The thermal crossover is only slightly shifted between the polymorphs. Their crystalline structures consist of similar cation layers alternating with PF(6) anion layers, packed differently in the two polymorphs. The magnetic and optical properties of the polymorphs are presented.

  3. Shock wave equation of state experiments at multi-TPa pressures on NIF

    NASA Astrophysics Data System (ADS)

    Celliers, P. M.; Fratanduono, D. E.; Peterson, J. L.; Meezan, N. B.; MacKinnon, A. J.; Braun, D. G.; Millot, M.; Fry, J.; Boehm, K. J.; Collins, G. W.; Nikroo, A.; Fitzsimmons, P.

    2015-06-01

    The National Ignition Facility provides an unprecedented capability to generate steady, planar, ultra-high pressure shock waves (around 10 TPa) in solid samples. Building on successful laser shock equation of state experiments performed on a variety of other laser facilities, we have designed and fielded experiments to perform impedance match experiments on samples of C, Be, quartz and CH, in the range of 3 to 7 TPa. The experiments use a line-imaging VISAR as the primary diagnostic to measure the shock velocity in an Al reference standard and in an array of the four samples. Initial tests with the line-imaging VISAR show that the NIF is capable of driving shocks that are steady for several ns, with smooth planar breakout patterns over a 2 mm diameter spot. Initial results will be discussed. Prepared by LLNL under Contract DE-AC52-07NA27344.

  4. Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.

    PubMed

    Ghosh, S; Strum, J C; Sciorra, V A; Daniel, L; Bell, R M

    1996-04-05

    Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

  5. Modulation of ADPase and t-PA release by radiographic contrast media in bovine aortic endothelium.

    PubMed

    Caprino, L; Togna, A R; Zappacosta, B; Giardina, B; Togna, G

    1997-05-01

    Vascular endothelial injuries induced by intravascular administration of radiographic contrast agents may be clinically relevant to the development of thrombosis and platelet activation. In this connection, we investigated the in vitro effects induced by iodamide, iopamidol, and ioxaglate on vascular endothelial ADPase activity and tissue plasminogen activator (t-PA) release in bovine aortic endothelium, in order to extend knowledge required to evaluate endothelial compatibility of radiographic contrast media. Undiluted and Tris-diluted contrast agent formulations were employed, and mannitol and sucrose hyperosmolar solutions were used as comparison. Results demonstrated that the high-osmolar ionic contrast agent iodamide, and to a lesser extent, the low-osmolar nonionic agent iopamidol, stimulated endothelial ADPase activity of the aortic endothelium; the low-osmolar ionic agent ioxaglate left endothelial ADPase activity unchanged. Furthermore, the diluted formulations of iodamide and iopamidol, as well as high-osmolar mannitol and sucrose solutions, were devoid of activity in ADPase. This suggests that the endothelial ADPase stimulation induced by both radiographic contrast media was a hyperosmolar-independent pharmacodynamic activity. Iopamidol and ioxaglate reduced endogenous t-PA release from bovine aortic endothelium only in undiluted formulation, while iodamide showed this inhibiting action in both diluted and undiluted formulations. No effect was observed when using mannitol solutions at different osmolarity values. Our in vitro findings agree with published data on the different thrombotic tendency attributed to the contrast agents used, suggesting endothelial enzymatic activities (ADPase and t-PA release) as suitable tools for evaluating endothelial vessel wall compatibility with radiographic contrast media.

  6. Inhibitory Effects of Lysine Analogues on t-PA Induced Whole Blood Clot Lysis

    DTIC Science & Technology

    1994-01-01

    Tween - 80 , pH 7.4 at 4 0C for 2 min in a 12x75 mm glass tube precoated with 10 pg iodogen. The specific activity of t-PA was 3.6x10’ cpm/mg and...plasminogen (0.6-15 nM) in phosphate buffered saline with 0.01% Tween - 80 , pH 7.4 was incubated with the fibrin-coated wells for 1 hr at 37 0C. The

  7. Catheter-directed Thrombolysis with Argatroban and tPA for Massive Iliac and Femoropopliteal Vein Thrombosis

    SciTech Connect

    Sharifi, Mohsen; Bay, Curt; Nowroozi, Sasan; Bentz, Suzanne; Valeros, Gayle; Memari, Sara

    2013-12-15

    Purpose: Catheter-directed thrombolysis (CDT) is a highly effective approach in the treatment of deep venous thrombosis (DVT). There are no data on the primary use of CDT with argatroban and tissue plasminogen activator (tPA) in patients without heparin-induced thrombocytopenia (HIT). The aim of this study was to evaluate the efficacy and safety of the combined administration of argatroban and tPA during CDT for massive DVT in patients without HIT. Methods: Thirty-three patients with massive symptomatic iliac and femoropopliteal DVT underwent CDT with tPA and argatroban within 28 {+-} 6 h of presentation. The dose of tPA was 0.75-1 mg/h through the infusion port and that of argatroban at 0.3-1 {mu}g/kg/min through the side port of the sheath. The patients were evaluated for the efficacy and safety of CDT and recurrent symptomatic venous thromboembolism (VTE) at a mean follow-up of 22 months. Results: There was no bleeding or iatrogenic pulmonary embolism with the CDT regimen we used. Grade III lysis (complete resolution of thrombus on venography) was achieved in 30 patients (91 %). In 3 patients with additional inferior vena cava filter thrombosis, further thrombectomy of the filter was required. No patient developed recurrent VTE. Conclusion: Concomitant administration of argatroban and tPA is a highly safe and effective regimen for CDT for massive DVT.

  8. Shock wave equation of state experiments at multi-TPa pressures on NIF

    NASA Astrophysics Data System (ADS)

    Celliers, P. M.; Fratanduono, D. E.; Peterson, J. L.; Meezan, N. B.; MacKinnon, A. J.; Braun, D. G.; Millot, M.; Fry, J.; Boehm, K. J.; Sterne, P. A.; Collins, G. W.; Nikroo, A.; Fitzsimmons, P.

    2015-11-01

    The National Ignition Facility provides an unprecedented capability to generate steady, planar, ultra-high pressure shock waves (up to 10 TPa or more) in solid samples. Building on successful laser shock equation of state experiments performed on a variety of other laser facilities, we have designed and fielded experiments to perform impedance match experiments on samples of C, Be, SiO2 and CH, in the range of 3 to 7 TPa. The experiments use a line-imaging VISAR as the primary diagnostic to measure the shock velocity in an Al reference standard and in an array of the four samples. Initial tests with the line-imaging VISAR show that the NIF is capable of driving shocks that are steady to better than 2% in velocity for several ns, with smooth planar breakout patterns over a 2 mm diameter spot. Hugoniot data points will be compared to current equation-of-state models for the various materials under study. This work was performed under the auspices of the U.S. Department of Energy by LLNL under contract DE-AC52-07NA27344.

  9. Enhanced histamine production through the induction of histidine decarboxylase expression by phorbol ester in Jurkat cells.

    PubMed

    Nagashima, Yusuke; Kako, Koichiro; Kim, Jun-Dal; Fukamizu, Akiyoshi

    2012-11-01

    Histamine (HA), a mediator of inflammation, type I allergic responses and neurotransmission, is synthesized from L-histidine, the reaction of which is catalyzed by histidine decarboxylase (HDC). HDC has been reported to be induced by various stimuli, not only in mast cells and basophils, but also in T lymphocytes and macrophages. Although its mRNA has been shown to be increased in Jurkat cells when treated with phorbol 12-myristate 13-acetate (TPA), little is known concerning the induced production of HA by HDC. The present study quantified the trace amounts of intracellular HA using ultra-high liquid chromatography in combination with the 6-aminoquinoline carbamate-derivatization technique. To test whether the cellular level of HA is elevated by the induction of HDC in Jurkat cells treated with TPA, the peak corresponding to authentic HA in the cell lysate was fractioned and its molecular weight determined by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry. The results of this study show that the HA level is increased by the induction of HDC expression by TPA in Jurkat cells. Therefore, this method is useful in elucidating the physiological significance of HA production.

  10. Stability of protein-encapsulating DRV liposomes after freeze-drying: A study with BSA and t-PA.

    PubMed

    Ntimenou, Vassiliki; Mourtas, Spyridon; Christodoulakis, Emmanouil V; Tsilimbaris, Miltiadis; Antimisiaris, Sophia G

    2006-01-01

    Stability of protein-encapsulating DRV (dried-rehydrated vesicle) liposomes is evaluated after freeze-drying vesicles in presence (or not) of trehalose. Two proteins, bovine serum albumin (BSA) and tissue-type plasminogen activator (t-PA), are used, and protein-encapsulating liposomes with different lipid compositions are prepared by DRV technique. Encapsulation efficiencies are calculated, after measuring BSA with a fluorescence technique and t-PA's amidolytic activity toward a chromogenic substrate. Experimental results show that encapsulation of BSA in vesicles ranges between 35 and 53% of the protein and is only slightly affected by lipid composition. For t-PA, entrapment efficiencies are lower, ranging between 2 and 16%, while lipid composition has substantial effect on entrapment (cholesterol inclusion is very important). After freeze-drying, some lipid compositions remain stable, retaining most of initially entrapped proteins, while others do not, but they may be stabilized by trehalose. In the case of BSA, liposome behavior cannot be explained based on lipid membrane rigidity (more rigid = more stable). This may be connected with previously demonstrated interactions of BSA with membranes. Oppositely, t-PA behavior is more predictable, meaning that the lipid composition selected for the specific therapeutic application determines the need for cryoprotectant addition before freeze-drying t-PA containing DRV liposomes, perhaps due to the fact that under conditions applying minimum or no interactions between t-PA and lipid membranes occur.Thereby, interactions between proteins and membranes determine not only the encapsulation efficiency but also the need for cryopreservation of liposomal protein formulations.

  11. Stability of iron crystal structures at 0.3-1.5 TPa

    NASA Astrophysics Data System (ADS)

    Godwal, B. K.; González-Cataldo, F.; Verma, A. K.; Stixrude, Lars; Jeanloz, Raymond

    2015-01-01

    Ab initio molecular dynamics simulations carried out for tetragonal and orthorhombic distortions of iron closely follow the results of static-lattice electronic-structure calculations in revealing that the body-centered cubic (bcc) phase of Fe is mechanically unstable at pressures of 0.3-1.5 TPa and temperatures up to 7000 K. Crystal-structural instabilities originate in the static lattice for the bcc configuration, and are consistent with recent results from both static and dynamic high-pressure experiments. Both theory and experiment thus show that the close-packed (hexagonal, hcp and face-centered cubic, fcc) crystal structures of iron are those relevant to the cores of Earth-like planets.

  12. The quest for TPa Hugoniot data: using the DEMG in high velocity pulsed power experiments

    SciTech Connect

    Peterson, Jeff H; Rousculp, Christopher L; Holtkamp, David B; Oro, David M; Griego, Jeffrey R; Atchison, Walter L; Reinovsky, Robert E

    2010-12-20

    ALT-3 is an experiment being designed in collaboration between Russian VNIIEF scientists and LANL that aims to conduct high velocity material experiments to measure shock velocities at pressures near 1 TPa. The DEMG (Disk Explosive Magnetic Generator) is used to drive >60MA currents to accelerate an aluminum liner to speeds in excess of 20 km/s. The 1-D model of the DEMG has been refined from a given current profile to a time-varying inductance. Various techniques are used to model the FOS (Foil Opening Switch) on the DEMG and a refined DEMG model is then used to drive a liner into various targets to determine the optimum design for the experiment and analyze the possible conditions and complications.

  13. Improvement of photovoltaic performance by substituent effect of donor and acceptor structure of TPA-based dye-sensitized solar cells.

    PubMed

    Inostroza, Natalia; Mendizabal, Fernando; Arratia-Pérez, Ramiro; Orellana, Carlos; Linares-Flores, Cristian

    2016-01-01

    We report a computational study of a series of organic dyes built with triphenylamine (TPA) as an electron donor group. We designed a set of six dyes called (TPA-n, where n = 0-5). In order to enhance the electron-injection process, the electron-donor effect of some specific substituent was studied. Thus, we gave insights into the rational design of organic TPA-based chromophores for use in dye-sensitized solar cells (DSSCs). In addition, we report the HOMO, LUMO, the calculated excited state oxidized potential E(dye*)(eV) and the free energy change for electron-injection ΔGinject(eV), and the UV-visible absorption bands for TPA-n dyes by a time-dependent density functional theory (TDDFT) procedure at the B3LYP and CAM-B3LYP levels with solvent effect. The results demonstrate that the introduction of the electron-acceptor groups produces an intramolecular charge transfer showing a shift of the absorption wavelengths of TPA-n under studies. Graphical Abstract Several organic dyes TPA-n with different donors and acceptors are modeled. A strong conjugation acrros the donor and anchoring groips (TPA-n) bas been studied. Candidate TPA-3 shows a promising results.

  14. Increasing tPA Activity in Astrocytes Induced by Multipotent Mesenchymal Stromal Cells Facilitate Neurite Outgrowth after Stroke in the Mouse

    PubMed Central

    Xin, Hongqi; Li, Yi; Shen, Li Hong; Liu, Xianshuang; Wang, Xinli; Zhang, Jing; Pourabdollah-Nejad D, Siamak; Zhang, Chunling; Zhang, Li; Jiang, Hao; Zhang, Zheng Gang; Chopp, Michael

    2010-01-01

    We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke. PMID:20140248

  15. An Examination of the EeTPA Portfolio Assessment and Other Measures of Teacher Preparation and Readiness

    ERIC Educational Resources Information Center

    Russell, Victoria; Davidson Devall, Kelly F.

    2016-01-01

    The authors examined the outcomes on several measures of world language teacher preparedness, including university- and state-mandated summative evaluations and the edTPA portfolio assessment, for seven world language teacher candidates during their final semester of clinical practice. The candidates were enrolled in an initial certification…

  16. Prevention of neuronal apoptosis by phorbol ester-induced activation of protein kinase C: blockade of p38 mitogen-activated protein kinase.

    PubMed

    Behrens, M M; Strasser, U; Koh, J Y; Gwag, B J; Choi, D W

    1999-01-01

    Consistent with previous studies on cell lines and non-neuronal cells, specific inhibitors of protein kinase C induced mouse primary cultured neocortical neurons to undergo apoptosis. To examine the complementary hypothesis that activating protein kinase C would attenuate neuronal apoptosis, the cultures were exposed for 1 h to phorbol-12-myristate-13-acetate, which activated protein kinase C as evidenced by downstream enhancement of the mitogen-activated protein kinase pathway. Exposure to phorbol-12-myristate-13-acetate, or another active phorbol ester, phorbol-12,13-didecanoate, but not to the inactive ester, 4alpha-phorbol-12,13-didecanoate, markedly attenuated neuronal apoptosis induced by serum deprivation. Phorbol-12-myristate-13-acetate also attenuated neuronal apoptosis induced by exposure to beta-amyloid peptide 1-42, or oxygen-glucose deprivation in the presence of glutamate receptor antagonists. The neuroprotective effects of phorbol-12-myristate-13-acetate were blocked by brief (non-toxic) concurrent exposure to the specific protein kinase C inhibitors, but not by a specific mitogen-activated protein kinase 1 inhibitor. Phorbol-12-myristate-13-acetate blocked the induction of p38 mitogen-activated protein kinase activity and specific inhibition of this kinase by SB 203580 attenuated serum deprivation-induced apoptosis. c-Jun N-terminal kinase 1 activity was high at rest and not modified by phorbol-12-myristate-13-acetate treatment. These data strengthen the idea that protein kinase C is a key modulator of several forms of central neuronal apoptosis, in part acting through inhibition of p38 mitogen-activated protein kinase regulated pathways.

  17. Chemopreventive activity of sesquiterpene lactones (SLs) from yacon against TPA-induced Raji cells deformation.

    PubMed

    Siriwan, D; Miyawaki, C; Miyamoto, T; Naruse, T; Okazaki, K; Tamura, H

    2011-05-15

    Yacon is a medicinal plant used as a traditional medicine by the natives in South America. In Japan, it becomes popular as a health food. Sesquiterpene Lactones (SLs) from yacon leaves were investigated and the active SLs such as enhydrin, uvedalin and sonchifolin, bearing alpha-methylene-gamma-lactone and epoxides as the active functional groups, were identified by 1H-6000 MHz-NMR. Chemopreventive and cytotoxic activities were determined using different primary screening methods. In this study, all tested SLs strongly inhibited TPA-induced deformed of Raji cells. The IC50 values of yacon SLs from anti-deforming assay were 0.04-0.4 microM. Interestingly, yacon SLs showed more potential of chemo preventive activity than both curcumin and parthenolide. However, the cytotoxicity on Raji cells was observed at high concentration of yacon SLs. The degree of anti-deformation was ranked in order: enhydrin >uvedalin >sonchifolin >parthenolide >curcumin. As according to structure-activity relationship, the high activities of enhydrin, uvedalin and sonchifolin may be due to the 2-methyl-2-butenoate and its epoxide moiety.

  18. Russian Nesting Doll Complexes of Molecular Baskets and Zinc Containing TPA Ligands.

    PubMed

    Zhiquan, Lei; Polen, Shane; Hadad, Christopher M; RajanBabu, T V; Badjić, Jovica D

    2016-07-06

    In this study, we examined the structural and electronic complementarities of convex 1-Zn(II), comprising functionalized tris(2-pyridylmethyl)amine (TPA) ligand, and concave baskets 2 and 3, having glycine and (S)-alanine amino acids at the rim. With the assistance of (1)H NMR spectroscopy and mass spectrometry, we found that basket 2 would entrap 1-Zn(II) in water to give equimolar 1-Zn⊂2in complex (K = (2.0 ± 0.2) × 10(3) M(-1)) resembling Russian nesting dolls. Moreover, C3 symmetric and enantiopure basket 3, containing (S)-alanine groups at the rim, was found to transfer its static chirality to entrapped 1-Zn(II) and, via intermolecular ionic contacts, twist the ligand's pyridine rings into a left-handed (M) propeller (circular dichroism spectroscopy). With molecular baskets embodying the second coordination sphere about metal-containing TPAs, the here described findings should be useful for extending the catalytic function and chiral discrimination capability of TPAs.

  19. The impact of metal line reflections on through-wafer TPA SEE testing

    SciTech Connect

    Khachatrian, Ani; Roche, Nicolas J-H.; Dodds, Nathaniel A.; McMorrow, Dale; Warner, Jeffrey H.; Buchner, Stephen P.; Reed, Robert A.

    2015-12-17

    Charge-collection experiments and simulations designed to quantify the effects of reflections from metallization during through-wafer TPA testing are presented. The results reveal a strong dependence on metal line width and metal line position inside the SiO2 overlayer. The charge-collection enhancement is largest for the widest metal lines and the metal lines closest to the Si/SiO2 interface. The charge-collection enhancement is also dependent on incident laser pulse energy, an effect that is a consequence of higher-order optical nonlinearities induced by the ultrashort optical pulses. However, for the lines further away from the Si/SiO2 interface, variations in laser pulse energies affect the charge-collection enhancement to a lesser degree. Z-scan measurements reveal that the peak charge collection occurs when the axial position of the laser focal point is inside the Si substrate. There is a downward trend in peak collected-charge enhancement with the increase in laser pulse energies for the metal lines further away from the Si/SiO2 interface. Metallization enhances the collected charge by same amount regardless of the applied bias voltage. In conclusion, for thinner metal lines and laser pulse energies lower than 1 nJ, the collected charge enhancement due to metallization is negligible.

  20. The impact of metal line reflections on through-wafer TPA SEE testing

    DOE PAGES

    Khachatrian, Ani; Roche, Nicolas J-H.; Dodds, Nathaniel A.; ...

    2015-12-17

    Charge-collection experiments and simulations designed to quantify the effects of reflections from metallization during through-wafer TPA testing are presented. The results reveal a strong dependence on metal line width and metal line position inside the SiO2 overlayer. The charge-collection enhancement is largest for the widest metal lines and the metal lines closest to the Si/SiO2 interface. The charge-collection enhancement is also dependent on incident laser pulse energy, an effect that is a consequence of higher-order optical nonlinearities induced by the ultrashort optical pulses. However, for the lines further away from the Si/SiO2 interface, variations in laser pulse energies affect themore » charge-collection enhancement to a lesser degree. Z-scan measurements reveal that the peak charge collection occurs when the axial position of the laser focal point is inside the Si substrate. There is a downward trend in peak collected-charge enhancement with the increase in laser pulse energies for the metal lines further away from the Si/SiO2 interface. Metallization enhances the collected charge by same amount regardless of the applied bias voltage. In conclusion, for thinner metal lines and laser pulse energies lower than 1 nJ, the collected charge enhancement due to metallization is negligible.« less

  1. Cooperative enhancement of TPA in cruciform double-chain DSB derivation: a femtosecond transient absorption spectra study

    NASA Astrophysics Data System (ADS)

    He, X.; Wang, Y.; Yang, Z.; Ma, Y.; Yang, Y.

    2010-09-01

    Femtosecond time-resolved transient absorption (TA) spectra study was adopted to study the mechanism of the cooperative enhancement of two-photon absorption (TPA) cross section from the linear structure 1,4-di(4'-N,N-diphenylaminostyryl)benzene (DPA-DSB) to its cruciform double-chain dimer DPA-TSB. The results suggested that a non-emissive intramolecular charge-transfer (ICT) state, ICT’, was present upon excitation in the dimer, which was absent in the monomer. The existence of this non-emissive state, indicating the enhancement of the intramolecular charge-transfer of the dimer, should be the reason for the cooperative enhancement of the TPA cross section of the dimer compared to the monomer.

  2. Early recovery of microvascular perfusion induced by t-PA in combination with abciximab or eptifibatide during postischemic reperfusion

    PubMed Central

    Bertuglia, Silva; Giusti, Andrea

    2002-01-01

    Background GPIIb/IIIa inhibitors abciximab and eptifibatide have been shown to inhibit platelet aggregation in ischemic heart disease. Our aim was to test the efficacy of abiciximab (Reo Pro) or eptifibatide (Integrilin) alone or in combination with plasminogen activator (t-PA) in an experimental model of ischemia reperfusion (I/R) in hamster cheek pouch microcirculation visualized by fluorescence microscopy. Hamsters were treated with saline, or abiciximab or eptifibatide or these drugs combined with t-PA infused intravenously 10 minutes before ischemia and through reperfusion. We measured the microvessel diameter changes, the arteriolar red blood cell (RBC) velocity, the increase in permeability, the perfused capillary length (PCL), and the platelet and leukocyte adhesion on microvessels. Results I/R elicited large increases in the platelet and leukocyte adhesion and a decrease in microvascular perfusion. These responses were significantly attenuated by abiciximab or eptifibatide (PCL:70 and 65% at 5–10 mins of reperfusion and 85 and 87% at 30 mins of reperfusion, respectively, p < 0.001) while t-PA combined with abiciximab or eptifibatide, was more effective and microvascular perfusion recovered immediately after postischemic reperfusion. Conclusions Platelets are crucial in I/R injury, as shown by the treatment with abicixmab or eptifibatide, which decreased platelet aggregation in microvessels, and also decreased leukocyte adhesion in venules. Arterial vasoconstriction, decreased arterial RBC velocity and alterations in the endothelial barrier with increased permeability delayed the complete restoration of blood flow, while t-PA combined with inhibition of platelet aggregation speeded up the capillary perfusion after reperfusion. PMID:12086588

  3. Anti-inflammatory activity of flavonol glycosides from Erythrospermum monticolum depending on single or repeated local TPA administration.

    PubMed

    del Carmen Recio, M; Giner, R M; Máñez, S; Talens, A; Cubells, L; Gueho, J; Julien, H R; Hostettmann, K; Rios, J L

    1995-12-01

    Two anti-inflammatory principles were isolated from the methanol extract of the leaves of Erythrospermum monticolum (Flacourtiaceae). The isolation was based on a guided bioassay of the inhibitory activity on TPA-induced ear edema in mice. These compounds were identified as quercetin 3-O-xylosyl(1-->2) rhamnoside and quercetin 3-O-rhamnoside. In addition, their effects on a chronic topic inflammation model were evaluated.

  4. Intravitreal tPA Injection and Pneumatic Displacement for Submacular Hemorrhage in a 10-Year-Old Child

    PubMed Central

    Hirose, Hiroshi; Hattori, Tomohiro

    2016-01-01

    Background. Submacular hemorrhage can occur after blunt trauma to the eye. Intravitreal tissue plasminogen activator (tPA) and gas injection are often used for treatment and are effective for submacular hemorrhage caused by age-related macular degeneration. This report describes the clinical outcome in a child with submacular hemorrhage caused by traumatic choroidal rupture who underwent successful intravitreal tPA injection and pneumatic displacement. Case Presentation. A 10-year-old boy developed sudden decrease of vision and a central scotoma in his right eye after trauma. Submacular hemorrhage was found in the eye. Visual acuity was 20/70 OD. Tissue plasminogen activator (12.5 μg in 0.05 mL) and 0.3 mL of pure sulfur hexafluoride were injected into the vitreous cavity under general anesthesia. After surgery, the patient was instructed to maintain a prone position. Displacement of the submacular hemorrhage from the fovea revealed a choroidal rupture, presumed to be the cause of the hemorrhage. After 4 months of follow-up, visual acuity was restored and final visual acuity is 20/16. Conclusion. Intravitreal tPA and gas injection can be an effective treatment for children with submacular hemorrhage. PMID:27722001

  5. The TRAF-interacting protein (TRIP) is a regulator of keratinocyte proliferation.

    PubMed

    Almeida, Stéphanie; Ryser, Stephan; Obarzanek-Fojt, Magdalena; Hohl, Daniel; Huber, Marcel

    2011-02-01

    The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis factor-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in keratinocytes. TRIP mRNA expression was strongly downregulated in primary human keratinocytes undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-12-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative keratinocytes suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. Keratinocytes undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused growth arrest in the G1/S phase. Keratinocytes with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD keratinocytes, indicating that NF-κB activity in keratinocytes is not regulated by TRIP. TRIP expression was increased by ∼2-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to keratinocyte proliferation.

  6. PACAP Interacts with PAC1 Receptors to Induce Tissue Plasminogen Activator (tPA) Expression and Activity in Schwann Cell-Like Cultures

    PubMed Central

    Castorina, Alessandro; Waschek, James A.; Marzagalli, Rubina; Cardile, Venera; Drago, Filippo

    2015-01-01

    Regeneration of peripheral nerves depends on the abilities of rejuvenating axons to migrate at the injury site through cellular debris and altered extracellular matrix, and then grow along the residual distal nerve sheath conduit and reinnervate synaptic targets. Considerable evidence suggest that glial cells participate in this process, although the mechanisms remain to be clarified. In cell culture, regenerating neurites secrete PACAP, a peptide shown to induce the expression of the protease tissue plasminogen activator (tPA) in neural cell types. In the present studies, we tested the hypothesis that PACAP can stimulate peripheral glial cells to produce tPA. More specifically, we addressed whether or not PACAP promoted the expression and activity of tPA in the Schwann cell line RT4-D6P2T, which shares biochemical and physical properties with Schwann cells. We found that PACAP dose- and time-dependently stimulated tPA expression both at the mRNA and protein level. Such effect was mimicked by maxadilan, a potent PAC1 receptor agonist, but not by the PACAP-related homolog VIP, suggesting a PAC1-mediated function. These actions appeared to be mediated at least in part by the Akt/CREB signaling cascade because wortmannin, a PI3K inhibitor, prevented peptide-driven CREB phosphorylation and tPA increase. Interestingly, treatment with BDNF mimicked PACAP actions on tPA, but acted through both the Akt and MAPK signaling pathways, while causing a robust increase in PACAP and PAC1 expression. PACAP6-38 totally blocked PACAP-driven tPA expression and in part hampered BDNF-mediated effects. We conclude that PACAP, acting through PAC1 receptors, stimulates tPA expression and activity in a Akt/CREB-dependent manner to promote proteolytic activity in Schwann-cell like cultures. PMID:25658447

  7. Phorbol ester and epidermal growth factor enhance the expression of two inducible prostaglandin H synthase genes in rat tracheal epithelial cells.

    PubMed

    Hamasaki, Y; Kitzler, J; Hardman, R; Nettesheim, P; Eling, T E

    1993-07-01

    Previous studies from our laboratory suggested that phorbol 12-myristate 13-acetate (TPA) stimulates prostaglandin E2 (PGE2) production by inducing de novo synthesis of prostaglandin H synthase (PHS) in a rat tracheal cell line. We report here an extension of this work to further elucidate the mechanisms by which TPA (and epidermal growth factor) stimulates PGE2 production. We used the rat tracheal cell line EGV6, which has a lower basal level of PGE2 production and responds to TPA and EGF stimulation with a much greater increase in PGE2 synthesis than the previously used cell line, Incubation of EGV6 cultures with TPA or EGF resulted in a time- and dose-dependent increase in PGE2 synthesis up to 40-fold and 6-fold, respectively. Serum also stimulated PGE2 synthesis, while bombesin, retinoic acid, and bacterial lipopolysaccharide did not. PHS protein levels in microsomal preparations from the cells were estimated by Western analysis. Antibodies raised against murine PHS-2 cross reacted with the EGV-6 PHS while several antibody preparations that react with PHS-1 from ram or mouse reacted poorly with the cellular preparation. TPA treatment increased the de novo synthesis of PHS-2 while dexamethasone treatment reduced the response to TPA. Northern blot analysis of mRNA from EGV6 cultures using a ram PHS cDNA revealed a 2.8- and a 4.5- to 4.9-kb (designated 4.9 kb) transcript. Treatment with TPA or EGF increased the expression of both transcripts and this effect was further enhanced by cyclohexamide. To further define the PHS mRNA species of EGV6 cells, two well-characterized murine PHS cDNA probes were used. The constitutive murine PHS cDNA probe hybridized only with the 2.8-kb transcript, and the inducible murine PHS cDNA hybridized only with the 4.9-kb transcript. The rates of induction as well as degradation of the 4.9-kb PHS mRNA were much more rapid than those of the 2.8-kb mRNA species. Dexamethasone partially inhibited the induction of both PHS transcripts by

  8. Exogenous t-PA Administration Increases Hippocampal Mature BDNF Levels. Plasmin- or NMDA-Dependent Mechanism?

    PubMed Central

    Rodier, Marion; Prigent-Tessier, Anne; Béjot, Yannick; Jacquin, Agnès; Mossiat, Claude; Marie, Christine; Garnier, Philippe

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) through TrkB activation is central for brain functioning. Since the demonstration that plasmin is able to process pro-BDNF to mature BDNF and that these two forms have opposite effects on neuronal survival and plasticity, a particular attention has been paid to the link between tissue plasminogen activator (tPA)/plasmin system and BDNF metabolism. However, t-PA via its action on different N-methyl-D-aspartate (NMDA) receptor subunits is also considered as a neuromodulator of glutamatergic transmission. In this context, the aim of our study was to investigate the effect of recombinant (r)t-PA administration on brain BDNF metabolism in rats. In the hippocampus, we found that rt-PA (10 mg/kg) administration induced a progressive increase in mature BDNF levels associated with TrkB activation. In order to delineate the mechanistic involved, plasmin activity was assessed and its inhibition was attempted using tranexamic acid (30 or 300 mg/kg, i.v.) while NMDA receptors were antagonized with MK801 (0.3 or 3 mg/kg, i.p.) in combination with rt-PA treatment. Our results showed that despite a rise in rt-PA activity, rt-PA administration failed to increase hippocampal plasmin activity suggesting that the plasminogen/plasmin system is not involved whereas MK801 abrogated the augmentation in mature BDNF levels observed after rt-PA administration. All together, our results show that rt-PA administration induces increase in hippocampal mature BDNF expression and suggests that rt-PA contributes to the control of brain BDNF synthesis through a plasmin-independent potentiation of NMDA receptors signaling. PMID:24670989

  9. Exogenous t-PA administration increases hippocampal mature BDNF levels. plasmin- or NMDA-dependent mechanism?

    PubMed

    Rodier, Marion; Prigent-Tessier, Anne; Béjot, Yannick; Jacquin, Agnès; Mossiat, Claude; Marie, Christine; Garnier, Philippe

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) through TrkB activation is central for brain functioning. Since the demonstration that plasmin is able to process pro-BDNF to mature BDNF and that these two forms have opposite effects on neuronal survival and plasticity, a particular attention has been paid to the link between tissue plasminogen activator (tPA)/plasmin system and BDNF metabolism. However, t-PA via its action on different N-methyl-D-aspartate (NMDA) receptor subunits is also considered as a neuromodulator of glutamatergic transmission. In this context, the aim of our study was to investigate the effect of recombinant (r)t-PA administration on brain BDNF metabolism in rats. In the hippocampus, we found that rt-PA (10 mg/kg) administration induced a progressive increase in mature BDNF levels associated with TrkB activation. In order to delineate the mechanistic involved, plasmin activity was assessed and its inhibition was attempted using tranexamic acid (30 or 300 mg/kg, i.v.) while NMDA receptors were antagonized with MK801 (0.3 or 3 mg/kg, i.p.) in combination with rt-PA treatment. Our results showed that despite a rise in rt-PA activity, rt-PA administration failed to increase hippocampal plasmin activity suggesting that the plasminogen/plasmin system is not involved whereas MK801 abrogated the augmentation in mature BDNF levels observed after rt-PA administration. All together, our results show that rt-PA administration induces increase in hippocampal mature BDNF expression and suggests that rt-PA contributes to the control of brain BDNF synthesis through a plasmin-independent potentiation of NMDA receptors signaling.

  10. RBC-coupled tPA prevents cerebrovasodilatory impairment and tissue injury in pediatric cerebral hypoxia/ischemia through inhibition of ERK MAPK unregulation

    SciTech Connect

    Ganguly, Kumkum; Armstead, William M; Kiessling, J W; Chen, Xiao - Han; Smith, Douglas H; Higazi, Abd Ar; Cines, Douglas B; Bdeir, Khalil; Zaitsev, Sergei; Muzykantov, Vladimir R

    2008-01-01

    Babies experience hypoxia (H) and ischemia (I) from stroke. The only approved treatment for stroke is fibrinolytic therapy with tissue-type plasminogen activator (tPA). However, tPA potentiates H/I-induced impairment of responses to cerebrovasodilators such as hypercapnia and hypotension, and blockade of tPA-mediated vasoactivity prevents this deleterious effect. Coupling tPA to RBCs reduces its CNS toxicity through spatially confining the drug to the vasculature. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, is upregulated after H/I. In this study we determined if RBC-tPA given before or after cerebral H/I would preserve responses to cerebrovasodilators and prevent neuronal injury mediated through the ERK MAPK pathway. Animals given RBC-tPA maintained responses to cerebrovasodilators at levels equivalent to pre-H/I values. CSF and brain parenchymal ERK MAPK was elevated by H/I and this upregulation was potentiated by tPA, but blunted by RBC-tPA. U 0126, an ERK MAPK antagonist, also maintained cerebrovasodilation post H/I. Neuronal degeneration in CA1 hippocampus and parietal cortex after H/I was exacerbated by tPA, but ameliorated by RBC-tPA and U 0126. These data suggest that coupling tPA to RBCs may offer a novel approach towards increasing the benefit/risk ratio of thrombolytic therapy for CNS disorders associated with H/I.

  11. Genome-Wide Association Study for Circulating Tissue Plasminogen Activator (tPA) Levels and Functional Follow-up Implicates Endothelial STXBP5 and STX2

    PubMed Central

    Huang, Jie; Huffman, Jennifer E.; Yamkauchi, Munekazu; Trompet, Stella; Asselbergs, Folkert W.; Sabater-Lleal, Maria; Trégouët, David-Alexandre; Chen, Wei-Min; Smith, Nicholas L.; Kleber, Marcus E.; Shin, So-Youn; Becker, Diane M.; Tang, Weihong; Dehghan, Abbas; Johnson, Andrew D.; Truong, Vinh; Folkersen, Lasse; Yang, Qiong; Oudot-Mellakh, Tiphaine; Buckley, Brendan M.; Moore, Jason H.; Williams, Frances M.K.; Campbell, Harry; Silbernagel, Günther; Vitart, Veronique; Rudan, Igor; Tofler, Geoffrey H.; Navis, Gerjan J.; DeStefano, Anita; Wright, Alan F.; Chen, Ming-Huei; de Craen, Anton J.M.; Worrall, Bradford B.; Rudnicka, Alicja R.; Rumley, Ann; Bookman, Ebony B.; Psaty, Bruce M.; Chen, Fang; Keene, Keith L.; Franco, Oscar H.; Böhm, Bernhard O.; Uitterlinden, Andre G.; Carter, Angela M.; Jukema, J. Wouter; Sattar, Naveed; Bis, Joshua C.; Ikram, Mohammad A.; Sale, Michèle M.; McKnight, Barbara; Fornage, Myriam; Ford, Ian; Taylor, Kent; Slagboom, P. Eline; McArdle, Wendy L.; Hsu, Fang-Chi; Franco-Cereceda, Anders; Goodall, Alison H.; Yanek, Lisa R.; Furie, Karen L.; Cushman, Mary; Hofman, Albert; Witteman, Jacqueline CM.; Folsom, Aaron R.; Basu, Saonli; Matijevic, Nena; van Gilst, Wiek H.; Wilson, James F.; Westendorp, Rudi G.J.; Kathiresan, Sekar; Reilly, Muredach P.; Tracy, Russell P.; Polasek, Ozren; Winkelmann, Bernhard R.; Grant, Peter J.; Hillege, Hans L.; Cambien, Francois; Stott, David J.; Lowe, Gordon D.; Spector, Timothy D.; Meigs, James B.; Marz, Winfried; Eriksson, Per; Becker, Lewis C.; Morange, Pierre-Emmanuel; Soranzo, Nicole; Williams, Scott M.; Hayward, Caroline; van der Harst, Pim; Hamsten, Anders; Lowenstein, Charles J.; Strachan, David P.; O'Donnell, Christopher J.

    2014-01-01

    Objective Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some populations, elevated plasma levels of tPA have been associated with myocardial infarction and other cardiovascular diseases (CVD). We conducted a meta-analysis of genome-wide association studies (GWAS) to identify novel correlates of circulating levels of tPA. Approach and Results Fourteen cohort studies with tPA measures (N=26,929) contributed to the meta-analysis. Three loci were significantly associated with circulating tPA levels (P <5.0×10−8). The first locus is on 6q24.3, with the lead SNP (rs9399599, P=2.9×10−14) within STXBP5. The second locus is on 8p11.21. The lead SNP (rs3136739, P=1.3×10−9) is intronic to POLB and less than 200kb away from the tPA encoding gene PLAT. We identified a non-synonymous SNP (rs2020921) in modest LD with rs3136739 (r2 = 0.50) within exon 5 of PLAT (P=2.0×10−8). The third locus is on 12q24.33, with the lead SNP (rs7301826, P=1.0×10−9) within intron 7 of STX2. We further found evidence for association of lead SNPs in STXBP5 and STX2 with expression levels of the respective transcripts. In in vitro cell studies, silencing STXBP5 decreased release of tPA from vascular endothelial cells, while silencing of STX2 increased tPA release. Through an in-silico lookup, we found no associations of the three lead SNPs with coronary artery disease or stroke. Conclusions We identified three loci associated with circulating tPA levels, the PLAT region, STXBP5 and STX2. Our functional studies implicate a novel role for STXBP5 and STX2 in regulating tPA release. PMID:24578379

  12. Cortical-layer-specific effects of PACAP and tPA on interneuron migration during post-natal development of the cerebellum.

    PubMed

    Raoult, Emilie; Bénard, Magalie; Komuro, Hitoshi; Lebon, Alexis; Vivien, Denis; Fournier, Alain; Vaudry, Hubert; Vaudry, David; Galas, Ludovic

    2014-07-01

    During early post-natal development of the cerebellum, granule neurons (GN) execute a centripetal migration toward the internal granular layer, whereas basket and stellate cells (B/SC) migrate centrifugally to reach their final position in the molecular layer (ML). We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates in vitro the expression and release of the serine protease tissue-type plasminogen activator (tPA) from GN, but the coordinated role of PACAP and tPA during interneuron migration has not yet been investigated. Here, we show that endogenous PACAP is responsible for the transient arrest phase of GN at the level of the Purkinje cell layer (PCL) but has no effect on B/SC. tPA is devoid of direct effect on GN motility in vitro, although it is widely distributed along interneuron migratory routes in the ML, PCL, and internal granular layer. Interestingly, plasminogen activator inhibitor 1 reduces the migration speed of GN in the ML and PCL, and that of B/SC in the ML. Taken together, these results reveal for the first time that tPA facilitates the migration of both GN and fast B/SC at the level of their intersection in the ML through degradation of the extracellular matrix. Crucial role of tissue plasminogen activator (tPA) in interneuron migration. Interneuron migration is a critical step for normal establishment of neuronal network. This study indicates that, in the post-natal cerebellum, tPA facilitates the opposite migration of immature excitatory granule neurons (GN) and immature inhibitory basket/stellate cells (B/SC) along the same migratory route. These data show that tPA exerts a pivotal role in neurodevelopment.

  13. [t-PA and PAI in patients with Raynaud's syndrome in treatment with a stable prostacyclin analog].

    PubMed

    Pola, P; de Martini, D; Gerardino, L

    1992-01-01

    An study was made in order to determinate the relationship between the restoration of the local fibrinolytic activity and the clinical signs in patients with a Raynaud's phenomenon. It is known that local fibrinolytic activity is a system influenced by changes into its components produced by exogenous and endogenous factors. An important role is represented by the t-PA and PAI-1. On the contrary, u-PA doesn't change. Samples were all taken at the same time, approximately at the middle of the morning. In patients with Raynaud's phenomenon treated with a prostacyclin stable analogous, we have perceived a clinical improvement, corresponding with a fibrinolytic activity increase.

  14. Quantum molecular dynamics simulations of the thermophysical properties of shocked liquid ammonia for pressures up to 1.3 TPa.

    PubMed

    Li, Dafang; Zhang, Ping; Yan, Jun

    2013-10-07

    We investigate via quantum molecular-dynamics simulations the thermophysical properties of shocked liquid ammonia up to the pressure 1.3 TPa and temperature 120,000 K. The principal Hugoniot is predicted from the wide-range equation of state, which agrees well with the available experimental measurements up to 64 GPa. Our systematic study of the structural properties demonstrates that the liquid ammonia undergoes a gradual phase transition along the Hugoniot. At about 4800 K, the system transforms into a metallic, complex mixture state consisting of NH3, N2, H2, N, and H. Furthermore, we discuss the implications for the interiors of Uranus and Neptune.

  15. Tissue-type Plasminogen Activator (tPA) Modulates the Postsynaptic Response of Cerebral Cortical Neurons to the Presynaptic Release of Glutamate

    PubMed Central

    Jeanneret, Valerie; Wu, Fang; Merino, Paola; Torre, Enrique; Diaz, Ariel; Cheng, Lihong; Yepes, Manuel

    2016-01-01

    Tissue-type plasminogen activator (tPA) is a serine proteinase released by the presynaptic terminal of cerebral cortical neurons following membrane depolarization (Echeverry et al., 2010). Recent studies indicate that the release of tPA triggers the synaptic vesicle cycle and promotes the exocytosis (Wu et al., 2015) and endocytic retrieval (Yepes et al., 2016) of glutamate-containing synaptic vesicles. Here we used electron microscopy, proteomics, quantitative phosphoproteomics, biochemical analyses with extracts of the postsynaptic density (PSD), and an animal model of cerebral ischemia with mice overexpressing neuronal tPA to study whether the presynaptic release of tPA also has an effect on the postsynaptic terminal. We found that tPA has a bidirectional effect on the composition of the PSD of cerebral cortical neurons that is independent of the generation of plasmin and the presynaptic release of glutamate, but depends on the baseline level of neuronal activity and the extracellular concentrations of calcium (Ca2+). Accordingly, in neurons that are either inactive or incubated with low Ca2+ concentrations tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-mediated phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in neurons with previously increased baseline levels of pCaMKIIα in the PSD due to neuronal depolarization in vivo or incubation with high concentrations of either Ca2+ or glutamate in vitro, tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. We found that these effects of tPA are mediated by synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. Our data indicate that by regulating the pCaMKIIα/PP1 balance in the PSD tPA acts

  16. Evaluation of adult dTPaP vaccination coverage in France: experience in Lyon city, 2010–2011

    PubMed Central

    2012-01-01

    Background Compliance with official recommendations can be assessed by evaluating vaccination coverage (VC) in populations. The main objective of our study was to assess VC of adults against diphtheria, tetanus, poliomyelitis and pertussis (dTPaP) according to age. The second objective was to explore if vaccination status could be confirmed by documentation. Methods A cross-sectional study was conducted in 680 adults consulting for biological examination in private laboratories in Lyon (France) to evaluate VC for diphtheria, tetanus, poliomyelitis and pertussis (dTPaP) and enabled reported vaccinations to be compared with documented, confirmed vaccinations. Results Verification of documented, confirmed vaccinations disclosed VC of 78.7% for tetanus, 63.6% for poliomyelitis, 57.8% for diphtheria and 10.7% for pertussis. Comparison of confirmed and self-reported vaccinations revealed that a large percentage of people who thought that they were vaccinated were not. VC significantly decreased with age for diphtheria and poliomyelitis and did not vary by gender. The VC rate for pertussis has increased since the 2008 recommendations were made. Conclusions The main thrust of this study was to compare reported and confirmed data. A significant percentage of people wrongly believed that they were up to date with their vaccination. PMID:23114050

  17. Melatonin inhibits TPA-induced oral cancer cell migration by suppressing matrix metalloproteinase-9 activation through the histone acetylation

    PubMed Central

    Yeh, Chia-Ming; Lin, Chiao-Wen; Yang, Jia-Sin; Yang, Wei-En; Su, Shih-Chi; Yang, Shun-Fa

    2016-01-01

    Melatonin exerts antimetastatic effects on liver and breast cancer and also inhibits matrix metalloproteinase (MMP) activity. However, the detailed impacts and underlying mechanisms of melatonin on oral cancer cell metastasis are still unclear. This study showed that melatonin attenuated the 12-O-tetradecanoylphorbol-13-acetate-induced migration of oral cancer cell lines, HSC-3 and OECM-1. Zymography, quantitative real-time PCR, and Western blotting analyses revealed that melatonin lessened MMP-9 enzyme activity as well as the expression of MMP-9 mRNA and protein. Furthermore, melatonin suppressed the phosphorylation of the ERK1/2 signalling pathway, which dampened MMP-9 gene transcription by affecting the expression of transcriptional coactivators, such as CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300), and decreasing histone acetylation in HSC-3 and OECM-1 cells. Examinations on clinical samples exhibited that MMP-9, CREBBP, and EP300 were significantly increased in oral cancer tissues. Moreover, the relative level of CREBBP was positively correlated with the expression of MMP-9 and EP300. In conclusion, we demonstrated that melatonin inhibits the motility of HSC-3 and OECM-1 cells in vitro through a molecular mechanism that involves attenuation of MMP-9 expression and activity mediated by decreased histone acetylation. PMID:26980735

  18. Prostate Cell-Specific Regulation of Androgen Receptor Phosphorylation In Vivo

    DTIC Science & Technology

    2007-11-01

    phosphorylation of S650 is enhanced by treatment with forskolin (FSK), Epidermal Growth Factor (EGF) and phorbol-12-myristate-13-acetate (PMA)[Gioeli, D., J. Biol...phosphorylation. - 7 - Figure 2: A PMA but not R1881 or Forskolin induces S650 phosporylation. LNCaP cells were steroid starved and

  19. [The expression of tPA directed by the bovine BLG regulatory elements in the mammary gland of transgenic mice].

    PubMed

    Chen, H X; Chen, X; Yang, X; Deng, J X; Su, G F; Huang, P T

    2001-03-01

    In order to get the regulatory elements which are essential for generating mammary gland bioreactors, the whole 8.4 kb bovine BLG gene was obtained by PCR amplification. The 1.6 kb chicken lysozyme matrix attachment region (MAR) was used to overcome position effects. The bovine BLG-tPA expression vector was constructed and the BLG-tPA fusion gene was introduced into fertilized eggs of mice by microinjection to generate transgenic mouse. 170 offsprings were obtained, of which 9 were proved to be transgenic mice based on PCR and Southern-blot analysis. The tPA expression level amounted to 12 micrograms/mL in the milk of mice. The bovine BLG-tPA fusion gene integrated in the founders was inheritable.

  20. Photovoltaic Small Molecules of TPA(FxBT-T-Cz)3: Tuning Open-Circuit Voltage over 1.0 V for Their Organic Solar Cells by Increasing Fluorine Substitution.

    PubMed

    Wang, Qiong; Duan, Linrui; Tao, Qiang; Peng, Wenhong; Chen, Jianhua; Tan, Hua; Yang, Renqiang; Zhu, Weiguo

    2016-11-09

    To simultaneously improve both open-circuit voltage (Voc) and short-circuit current density (Jsc) for organic solar cells, a novel D(A-π-Ar)3 type of photovoltaic small molecules of TPA(FxBT-T-3Cz)3 was designed and synthesized, which contain central triphenylamine (TPA), terminal carbazole (Cz), armed fluorine-substituted benzothiadiazole (FxBT, where x = 1 or 2), and bridged thiophene (T) units. A narrowed ultraviolet-visible absorption and a decreasing highest occupied molecular orbital energy level were observed from TPA(F1BT-T-3Cz)3 to TPA(F2BT-T-3Cz)3 with increasing fluorine substitution. However, the TPA(F2BT-T-3Cz)3/PC71BM-based solar devices showed a rising Voc of 1.01 V and an enhanced Jsc of 10.84 mA cm(-2) as well as a comparable power conversion efficiency of 4.81% in comparison to the TPA(F1BT-T-3Cz)3/PC71BM-based devices. Furthermore, in comparison to the parent TPA(BT-T-3Cz)3 molecule without fluorine substitution, the fluorine-substituted TPA(FxBT-T-3Cz)3 molecules exhibited significantly incremental Voc and Jsc values in their bulk heterojunction organic solar cells, owing to fluorine incorporation in the electron-deficient benzothiadiazole unit.

  1. Pretreatment Blood Brain Barrier Damage and Post Treatment Intracranial Hemorrhage in Patients Receiving IV tPA

    PubMed Central

    Leigh, Richard; Jen, Shyian S.; Hillis, Argye E.; Krakauer, John W.; Barker, Peter B.

    2014-01-01

    Background and Purpose Early blood brain barrier (BBB) damage after acute ischemic stroke (AIS) has previously been qualitatively linked to subsequent intracranial hemorrhage (ICH). In this quantitative study, it was investigated whether the amount of BBB damage evident on pre-tPA MRI scans was related to the degree of post-tPA ICH in patients with AIS. Methods Analysis was performed on a database of patients with AIS provided by the STIR and VISTA Imaging Investigators. Patients with perfusion-weighted imaging (PWI) lesions >10mL and negative gradient-recalled echo (GRE) imaging prior to IV tPA were included. Post processing of the PWI source images was performed to estimate changes in BBB permeability within the perfusion deficit relative to the unaffected hemisphere. Follow-up GRE images were reviewed for evidence of ICH and divided into three groups according to ECASS criteria: no hemorrhage (NH), hemorrhagic infarction (HI), and parenchymal hematoma (PH). Results 75 patients from the database met the inclusion criteria, 28 of whom experienced ICH, of which 19 were classified as HI, and nine were classified as PH. The mean permeability (±standard deviations), expressed as an index of contrast leakage, was 17.0%±8.8 in the NH group, 19.4%±4.0 in the HI group, and 24.6%±4.5 in the PH group. Permeability was significantly correlated with ICH grade in univariate (p=0.007) and multivariate (p=0.008) linear regression modeling. Conclusions A PWI-derived index of BBB damage measured prior to IV tPA is associated with the severity of ICH after treatment in patients with AIS. PMID:24876245

  2. Outcomes of Patients Requiring Blood Pressure Control Before Thrombolysis with tPA for Acute Ischemic Stroke

    PubMed Central

    Darger, Bryan; Gonzales, Nicole; Banuelos, Rosa C.; Peng, Hui; Radecki, Ryan P.; Doshi, Pratik B.

    2015-01-01

    Introduction The purpose of this study was to assess safety and efficacy of thrombolysis in the setting of aggressive blood pressure (BP) control as it compares to standard BP control or no BP control prior to thrombolysis. Methods We performed a retrospective review of patients treated with tissue plasminogen activator (tPA) for acute ischemic stroke (AIS) between 2004–2011. We compared the outcomes of patients treated with tPA for AIS who required aggressive BP control prior to thrombolysis to those requiring standard or no BP control prior to thrombolysis. The primary outcome of interest was safety, defined by all grades of hemorrhagic transformation and neurologic deterioration. The secondary outcome was efficacy, determined by functional status at discharge, and in-hospital deaths. Results Of 427 patients included in the analysis, 89 received aggressive BP control prior to thrombolysis, 65 received standard BP control, and 273 required no BP control prior to thrombolysis. Patients requiring BP control had more severe strokes, with median arrival National Institutes of Health Stroke Scale of 10 (IQR [6–17]) in patients not requiring BP control versus 11 (IQR [5–16]) and 13 (IQR [7–20]) in patients requiring standard and aggressive BP lowering therapies, respectively (p=0.048). In a multiple logistic regression model adjusting for baseline differences, there were no statistically significant differences in adverse events between the three groups (P>0.10). Conclusion We observed no association between BP control and adverse outcomes in ischemic stroke patients undergoing thrombolysis. However, additional study is necessary to confirm or refute the safety of aggressive BP control prior to thrombolysis. PMID:26759644

  3. Novel non-invasive distribution measurement of texture profile analysis (TPA) in salmon fillet by using visible and near infrared hyperspectral imaging.

    PubMed

    Wu, Di; Sun, Da-Wen; He, Yong

    2014-02-15

    This study developed a pushbroom visible and near-infrared hyperspectral imaging system in the wavelength range of 400-1758 nm to determine the spatial distribution of texture profile analysis (TPA) parameters of salmon fillets. Six TPA parameters (hardness, adhesiveness, chewiness, springiness, cohesiveness, and gumminess) were analysed. Five spectral features (mean, standard deviation, skew, energy, and entropy) and 22 image texture features obtained from graylevel co-occurrence matrix (GLCM) were extracted from hyperspectral images. Quantitative models were established with the extracted spectral and image texture signatures of samples based on partial least squares regression (PLSR). The results indicated that spectral features had better ability to predict TPA parameters of salmon samples than image texture features, and Spectral Set I (400-1000 nm) performed better than Spectral II (967-1634 nm). On the basis of the wavelengths selected by regression coefficients of PLSR models, instrumental optimal wavelengths (IOW) and predictive optimal wavelengths (POW) were further chosen to reduce the high dimensionality of the hyperspectral image data. Our results show that hyperspectral imaging holds promise as a reliable and rapid alternative to traditional universal testing machines for measuring the spatial distribution of TPA parameters.

  4. Politics of Policy: Assessing the Implementation, Impact, and Evolution of the Performance Assessment for California Teachers (PACT) and edTPA

    ERIC Educational Resources Information Center

    Reagan, Emilie Mitescu; Schram, Thomas; McCurdy, Kathryn; Chang, Te-Hsin; Evans, Carla M.

    2016-01-01

    Summative performance assessments in teacher education, such as the Performance Assessment for California Teachers (PACT) and the edTPA, have been heralded through polices intended to enhance the quality of the teaching profession and raise its stature among other professions. However, the development and implementation of the PACT, and…

  5. The AP-1 site is required for basal expression but is not necessary for TPA-response of the human stromelysin gene.

    PubMed Central

    Buttice, G; Quinones, S; Kurkinen, M

    1991-01-01

    We have studied the activity of the AP-1 site, a target for the Fos and Jun family of transcription factors, in the context of the human stromelysin promoter (-1303 to +4). In transiently transfected human HepG2, HeLa and fibroblast cell cultures, point-mutations in any position of the stromelysin AP-1 sequence TGAGTCA (-70 to -64) reduced both the basal level and TPA-induced expression from the stromelysin promoter. TPA-induction fold of the mutant promoters, however, was comparable to that of the wild-type promoter. Similarly, antisense c-Fos mRNA expression reduced basal activity but had no significant effect on the relative TPA-response of the stromelysin promoter. Further, in mouse F9 cells cotransfected with c-Fos and c-Jun expression plasmids, the transfected wild-type stromelysin promoter activity was increased 57-fold whereas no transactivation was detected for an AP-1 mutant stromelysin promoter. In gelshift assays, stromelysin promoter fragments (-101 to -11), containing the mutated AP-1 site, all failed to bind or compete for the in vitro synthesized Fos and Jun proteins. We interpret these data to suggest that the Fos and Jun proteins, or similar activity, and the AP-1 site are required for the basal level expression of the human stromelysin gene. Strikingly, these data also suggest that the stromelysin AP-1 site is not necessary for the TPA-response. Images PMID:1906606

  6. Critical Care Needs in Patients with Diffusion-Weighted Imaging Negative MRI after tPA - Does One Size Fit All?

    PubMed Central

    Faigle, Roland; Marsh, Elisabeth B.; Llinas, Rafael H.; Urrutia, Victor C.

    2015-01-01

    Background and Purpose Patients who receive intravenous (IV) tissue plasminogen activator (tPA) for ischemic stroke are currently monitored in an intensive care unit (ICU) or a comparable stroke unit for at least 24 hours due to the high frequency of neurological exams and vital sign checks. The present study evaluates ICU needs in patients with diffusion-weighted imaging (DWI) negative MRI after IV tPA. Methods A retrospective chart review was performed for 209 patients who received IV tPA for acute stroke. Data on stroke risk factors, physiologic parameters, stroke severity, MRI characteristics, and final diagnosis were collected. The timing and nature of ICU interventions, if needed, was recorded. Multivariable logistic regression was used to determine factors associated with subsequent ICU needs. Results Patients with cerebral infarct on MRI after tPA had over 9 times higher odds of requiring ICU care compared to patients with DWI negative MRI (OR 9.2, 95% CI 2.49–34.15). All DWI negative patients requiring ICU care did so by the end of tPA infusion (p = 0.006). Among patients with DWI negative MRI, need for ICU interventions was associated with higher NIH Stroke Scale (NIHSS) scores (p<0.001), uncontrolled hypertension (p<0.001), seizure at onset (p = 0.002), and reduced estimated glomerular filtration rate (eGFR) (p = 0.010). Conclusions Only a small number of DWI negative patients required ICU care. In patients without critical care needs by the end of thrombolysis, post-tPA MRI may be considered for triaging DWI negative patients to a less resource intense monitoring environment. PMID:26517543

  7. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA.

    PubMed Central

    Wagner, O F; de Vries, C; Hohmann, C; Veerman, H; Pannekoek, H

    1989-01-01

    Plasminogen activation is catalyzed both by tissue-type-(t-PA) and by urokinase-type plasminogen activator (u-PA). This reaction is controlled by plasminogen activator inhibitor type 1 (PAI-1) that is either present in plasma or bound to fibrin, present in a thrombus. We studied the mechanism of in vitro inhibition of both t-PA and u-PA activity by PAI-1 bound to fibrin. It is shown that activation of latent PAI-1 unmasks a specific fibrin-binding site that is distinct from its reactive site. This reactive site of activated PAI-1 bound to fibrin is fully exposed to form complexes with t-PA and u-PA, that are unable to activate plasminogen. Upon complex formation with either one of the plasminogen activators, PAI-1 apparently undergoes a conformational change and loses its affinity for fibrin. Consequently, complexes of u-PA and PAI-1 dissociate from the fibrin matrix and are encountered in the fluid phase. In contrast, t-PA/PAI-1 complexes remain bound to fibrin. By employing recombinant t-PA deletion-mutant proteins, that precisely lack domains involved in fibrin binding, we demonstrate that binding of t-PA/PAI-1 complexes is mediated by both the "finger" (F) and the "kringle-2" (K2) domain of t-PA. A model is proposed that explains inhibition of the fibrinolytic process, at the level of plasminogen activation by t-PA, directed by PAI-1 bound to fibrin. An implication of the proposed model is that t-PA/PAI-1 complexes and free t-PA compete for the same binding sites on fibrin. Images PMID:2503541

  8. Combination approaches to attenuate hemorrhagic transformation after tPA thrombolytic therapy in patients with poststroke hyperglycemia/diabetes.

    PubMed

    Fan, Xiang; Jiang, Yinghua; Yu, Zhanyang; Yuan, Jing; Sun, Xiaochuan; Xiang, Shuanglin; Lo, Eng H; Wang, Xiaoying

    2014-01-01

    To date, tissue type plasminogen activator (tPA)-based thrombolytic stroke therapy is the only FDA-approved treatment for achieving vascular reperfusion and clinical benefit, but this agent is given to only about 5% of stroke patients in the USA. This may be related, in part, to the elevated risk of symptomatic intracranial hemorrhage, and consequently limited therapeutic time window. Clinical investigations demonstrate that poststroke hyperglycemia is one of the most important risk factors that cause intracerebral hemorrhage and worsen neurological outcomes. There is a knowledge gap in understanding the underlying molecular mechanisms, and lack of effective therapeutics targeting the severe complication. This short review summarizes clinical observations and experimental investigations in preclinical stroke models of the field. The data strongly suggest that interactions of multiple pathogenic factors including hyperglycemia-mediated vascular oxidative stress and inflammation, ischemic insult, and tPA neurovascular toxicity in concert contribute to the BBB damage-intracerebral hemorrhagic transformation process. Development of combination approaches targeting the multiple pathological cascades may help to attenuate the hemorrhagic complication.

  9. NO-dependent attenuation of TPA-induced immunoinflammatory skin changes in Balb/c mice by pindolol, heptaminol or ATRA, but not by verapamil

    PubMed Central

    Chung, Jinhyuk F.; Yoon, Calvin J.; Cheon, Seon Ah; Seo, Eun Seok; Park, Sung Ho; Yang, Jae Seung; Kim, Bumju; Joo, Min Young; Park, Tae Jung; Kim, Ki Hean; Sood, Anil K.; Lee, Sang Joon

    2016-01-01

    Recently a mouse skin carcinogenesis study reported that a β-blocker carvedilol displayed antitumor-properties via antihyperplastic effects. However, the antihyperplastic mechanism is unclear as the β-blocker is characterized with multiple pleiotropic effects including stimulation of endothelial NO release and verapamil-like calcium channel blocking activity. To investigate the nature and the origin of the antihyperplastic effects, we tested topical pretreatment with pindolol, heptaminol, ATRA or verapamil against Balb/c mouse ear skin hyperplasia that was induced by TPA. We found that pindolol, heptaminol or ATRA, but not verapamil, inhibited the TPA-induced immunoinflammatory skin changes in an NO-dependent manner, which included epidermal hyperplasia, skin edema and fibrosis. Furthermore, we also observed NO-dependent alleviation of the TPA-induced NK cell depletion in the ear tissues by heptaminol pretreatment. Together our results suggest that stimulation of NO generation from constitutive synthases may be primarily responsible for the reported antihyperplastic and NK cell-preserving effects of the β-blockers, and that similar effects may be observed in other immunity normalizing compounds that also promote endothelial NO synthesis. PMID:27374093

  10. Role of a TPA-responsive element in hepcidin transcription induced by the bone morphogenetic protein pathway.

    PubMed

    Kanamori, Yohei; Murakami, Masaru; Matsui, Tohru; Funaba, Masayuki

    2015-10-16

    Systemic iron balance is governed by the liver-derived peptide hormone hepcidin. The transcription of hepcidin is primarily regulated by the bone morphogenetic protein (BMP) and inflammatory cytokine pathways through the BMP-response element (BMP-RE) and STAT-binding site, respectively. In addition to these elements, we previously identified a TPA-responsive element (TRE) in the hepcidin promoter and showed that it mediated the transcriptional activation of hepcidin through activator protein (AP)-1 induced by serum. In the present study, we examined the role of TRE in the BMP-induced transcription of hepcidin in HepG2 liver cells. The serum treatment increased the basal transcription of hepcidin; however, responsiveness to the expression of ALK3(QD), a constitutively active BMP type I receptor, was unaffected. Consistent with these results, mutations in TRE in the hepcidin promoter decreased basal transcription, whereas responsiveness to the expression of ALK3(QD) remained unchanged. HepG2 cells significantly expressed AP-1 components in the basal state, whereas BMP did not up-regulate the expression of these components. The expression of c-fos enhanced the basal transcription of hepcidin as well as ALK3(QD)-mediated hepcidin transcription, whereas that of dominant-negative c-fos decreased hepcidin transcription. The results of the present study suggested that the cis-elements of the hepcidin promoter, BMP-RE and TRE, individually transmitted BMP-mediated and AP-1-mediated signals, respectively, whereas transcription was synergistically increased by the stimulation of BMP-RE and TRE.

  11. Potent Anti-inflammatory and Analgesic Actions of the Chloroform Extract of Dendropanax morbifera Mediated by the Nrf2/HO-1 Pathway.

    PubMed

    Akram, Muhammad; Kim, Kyeong-A; Kim, Eun-Sun; Syed, Ahmed Shah; Kim, Chul Young; Lee, Jong Soo; Bae, Ok-Nam

    2016-01-01

    Dendropanax morbifera LEVEILLE (DP) has been used in traditional Korean medicines to treat a variety of inflammatory diseases. Although the in vitro anti-inflammatory potential of this plant is understood, its in vivo efficacy and underlying molecular mechanism of anti-inflammatory effects are largely unknown. We elucidated the anti-inflammatory and analgesic activities and the underlying molecular mechanisms of DP using in vitro and in vivo models. Lipopolysaccharide (LPS)-stimulated murine macrophages were used to analyze the in vitro anti-inflammatory potential of DP extract and to elucidate the underlying mechanisms. In vivo animal models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and acetic acid-induced writhing response tests were used to analyze the in vivo anti-inflammatory effects and anti-nociceptive effects of DP extract, respectively. Methanolic extract of DP (DPME) significantly inhibited the release of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-activated macrophages. Among the five sub-fractions, the chloroform fraction (DP-C) showed the most potent suppressive effects against pro-inflammatory mediators and cytokines in LPS-stimulated macrophages. These effects were attributed to inhibition of nuclear factor-κB (NF-κB) nuclear translocation and c-Jun N terminal kinase (JNK) 1/2 phosphorylation and to activation of NF-E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling. DP-C exhibited strong protective in vivo effects in TPA-induced ear edema mouse model and acetic acid-induced writhing response test. Our data suggest that DP-C has potent anti-inflammatory and analgesic activities and may be a promising treatment against a variety of inflammatory diseases.

  12. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    PubMed

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  13. Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.

    PubMed Central

    MacLeod, K T; Harding, S E

    1991-01-01

    1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac myocytes. Intracellular pH was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura-2. 2. Application of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 +/- 34% (n = 8) (p less than 0.02). The rate of change of cell length during contraction (i.e. +dL/dt) increased from 67.2 +/- 8.7 microns/s to 127.7 +/- 14.1 microns/s (n = 7). The rate of change of cell length during relaxation (-dL/dt) increased from 55.8 +/- 7.4 microns/s to 118.9 +/- 12.1 microns/s (n = 7). Time to peak shortening was unchanged. 3. Application of 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7.5 +/- 7.5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C. 4. In rat myocytes we have measured the changes of pHi and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p less than 0.05) enhanced by 15.1 +/- 6.9% (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p less than 0.05) by an average of 59.1 +/- 10.6% (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4-phorbol 12,13-didecanoate (which does not activate protein kinase C). 5. Since pHi recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM-amiloride and inhibited by removing extracellular Na

  14. Dynamic compression of dense oxide (Gd3Ga5O12) from 0.4 to 2.6 TPa: Universal Hugoniot of fluid metals

    DOE PAGES

    Ozaki, N.; Nellis, W. J.; Mashimo, T.; ...

    2016-05-19

    Materials at high pressures and temperatures are of great current interest for warm dense matter physics, planetary sciences, and inertial fusion energy research. Shock-compression equation-of-state data and optical reflectivities of the fluid dense oxide, Gd3Ga5O12 (GGG), were measured at extremely high pressures up to 2.6 TPa (26 Mbar) generated by high-power laser irradiation and magnetically-driven hypervelocity impacts. Above 0.75 TPa, the GGG Hugoniot data approach/reach a universal linear line of fluid metals, and the optical reflectivity most likely reaches a constant value indicating that GGG undergoes a crossover from fluid semiconductor to poor metal with minimum metallic conductivity (MMC). Thesemore » results suggest that most fluid compounds, e.g., strong planetary oxides, reach a common state on the universal Hugoniot of fluid metals (UHFM) with MMC at sufficiently extreme pressures and temperatures. Lastly, the systematic behaviors of warm dense fluid would be useful benchmarks for developing theoretical equation-of-state and transport models in the warm dense matter regime in determining computational predictions.« less

  15. The safety and reactogenicity of a reduced-antigen-content diphtheria-tetanus-acellular pertussis (dTpa) booster vaccine in healthy Vietnamese children.

    PubMed

    Anh, Dang Duc; Jayadeva, Girish; Kuriyakose, Sherine; Han, Htay Htay

    2016-08-17

    Despite effective infant immunization against pertussis, the disease continues to circulate due to waning immunity. Booster vaccinations against pertussis beyond infancy are widely recommended. In Vietnam, however, no recommendations for pertussis boosters beyond the second year of life exist. This open-label, single-centre study was designed to assess the safety of a single booster dose of reduced-antigen-content-diphtheria-tetanus-acellular-pertussis vaccine (dTpa) in 300 healthy Vietnamese children (mean age 7.9years), who had completed primary vaccination against diphtheria, tetanus and pertussis. Solicited symptoms were recorded for 4days and unsolicited and serious adverse events (SAEs) for 31days post-vaccination. Pain and fatigue were the most common solicited local and general symptoms in 35.0% and 14.0% of children, respectively. Grade 3 swelling occurred in 3 children; no large injection site reactions or SAEs were reported. The dTpa booster vaccine was well tolerated and this study supports its administration in school age Vietnamese children.

  16. The anticoagulant effect of PGI2S and tPA in transgenic umbilical vein endothelial cells is linked to up-regulation of PKA and PKC.

    PubMed

    Wang, Jian-Hua; Yuan, Lin-Jing; Zhong, Zhi-Min; Wen, Zhe-Sheng; Deng, Jian-Ming; Liang, Rong-Xin; Zheng, Min

    2014-02-19

    The selection of vascular grafts for coronary artery bypass surgery is crucial for a positive outcome. This study aimed to establish a novel line of vascular endothelial cells with a potent anticoagulant effect. A lentiviral vector was used to stably transfect human umbilical vein endothelial cells (HUVECs) with PGI2S alone (HUVEC-PGI2S) or both PGI2S and tPA (HUVEC-PGI2S-tPA). Both HUVEC-PGI2S and HUVEC-PGI2S-tPA cells over-expressing PGI2S and tPA were compared to mock-transfected cells. The enzyme-linked immuno sorbent assay (ELISAs) demonstrated that the anticoagulation components, ATIII and PLG, were up-regulated and coagulation factor FVIII was down-regulated in both cell lines. QRT-PCR and western blotting demonstrated the vasodilation and platelet disaggregation proteins PKA, PKC, and PTGIR were up-regulated in both cell lines, but MAPK expression was not altered in either cell line. However, cell viability and colony formation assays and cell cycle analysis demonstrated that both cell lines had a lower rate of cell growth and induced G1 phase arrest. HUVEC-PGI2S and HUVEC-PGI2S-tPA cells have a potent anticoagulant effect and their use in vascular heterografts may decrease the risk of thrombosis.

  17. Acute stroke therapy with tissue plasminogen activator (tPA) since it was approved by the U.S. Food and Drug Administration (FDA).

    PubMed

    Zivin, Justin A

    2009-07-01

    Tissue plasminogen activator (tPA) for acute ischemic stroke was approved by the U.S. Food and Drug Administration (FDA) in 1996. Since then it has been severely underutilized. At the time when most practitioners were first being exposed to the literature concerning tPA, there were many concerns about safety and the restrictions on use were quite onerous. Since then a good deal of further work has been done to loosen the restrictions and allay concerns about the risks. The true risk to benefit ratio is far better than is generally realized. Now it is mostly economic problems related to the costs of constantly supplying emergency care that is limiting access. Furthermore, in the current litigious environment, failure to treat is likely to be a more hazardous course of action than legal exposure due to poor outcomes. It must be emphasized that the drug is quite safe and highly effective, and current utilization rates are unacceptably low. Ann Neurol 2009;66:6-10.

  18. Dynamic compression of dense oxide (Gd3Ga5O12) from 0.4 to 2.6 TPa: Universal Hugoniot of fluid metals

    PubMed Central

    Ozaki, N.; Nellis, W. J.; Mashimo, T.; Ramzan, M.; Ahuja, R.; Kaewmaraya, T.; Kimura, T.; Knudson, M.; Miyanishi, K.; Sakawa, Y.; Sano, T.; Kodama, R.

    2016-01-01

    Materials at high pressures and temperatures are of great current interest for warm dense matter physics, planetary sciences, and inertial fusion energy research. Shock-compression equation-of-state data and optical reflectivities of the fluid dense oxide, Gd3Ga5O12 (GGG), were measured at extremely high pressures up to 2.6 TPa (26 Mbar) generated by high-power laser irradiation and magnetically-driven hypervelocity impacts. Above 0.75 TPa, the GGG Hugoniot data approach/reach a universal linear line of fluid metals, and the optical reflectivity most likely reaches a constant value indicating that GGG undergoes a crossover from fluid semiconductor to poor metal with minimum metallic conductivity (MMC). These results suggest that most fluid compounds, e.g., strong planetary oxides, reach a common state on the universal Hugoniot of fluid metals (UHFM) with MMC at sufficiently extreme pressures and temperatures. The systematic behaviors of warm dense fluid would be useful benchmarks for developing theoretical equation-of-state and transport models in the warm dense matter regime in determining computational predictions. PMID:27193942

  19. Biological responsiveness to the phorbol esters and specific binding of (/sup 3/H)phorbol 12,13-dibutyrate in the nematode Caenorhabditis elegans, a manipulable genetic system

    SciTech Connect

    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. (/sup 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.

  20. Effects of garlic on cellular doubling time and DNA strand breaks caused by UV light and BPL, enhanced with catechol and TPA

    SciTech Connect

    Baturay, N.Z.; Gayle, F.; Liu, S.; Kreidinger, C.

    1995-11-01

    3T3 cell cultures were exposed to UV light and Beta-Propiolactone. Neoplastic cell transformation (TF) was demonstrated after concurrent addition of catechol, or repeated addition of TPA. Addition of garlic to all fluences/concentrations of the carcinogen/cocarcinogen/promoter groups reduced the number of transformed foci/dish by at least 40%. Since the cell cycle is prolonged following exposure to carcinogens, it is likely the cell requires a longer time to repair this damage. The doubling time (DT) was extended from 12 to 36 hrs. when cells were exposed to BPL and from 12 o 28 hrs. when cells were exposed to 3.0J/M2/sec. If an anticarcinogenic compound is also added, it is reasonable to assume that the cell cycle may be further elongated. The cell cycle, denoted by DT was lengthened from 12 to 47 hrs and from 12 to 86 hrs for BPL and UVC, respectively. The extensions occurred in a dope dependent manner. The concentrations of the cocarcinogen and promoter remained constant throughout the experiment. When strand breaks were determined at the same dose sequences, by alkaline elution, more repair was seen with garlic where the lowest and middle doses of BPL were used and almost no decrease in % DNA eluted was seen with UVC exposed cells. With catechol, there was a two-fold decrease in % DNA eluted at the lowest and middle fluences. When TPA was added, all three fluences of UVC showed more than a threefold decrease in % DNA eluted. BPS with both TPA and catechol, again showed a reduction in strand breaks only low and middle doses. Both a direct-acting alkylating agent, BPL, and a physical carcinogen, UVC, were homogeneously affected, in terms of doubling time, but not when strand break repair was examined. A separate mechanism may be responsible for repair, and the mechanism associated with combinations of physical carcinogen enhancing agents combined with some non-carcinogens may be more profoundly affected by some natural products.

  1. Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors.

    PubMed

    Smeets, R L; Fouraux, M A; van Emst-de Vries, S E; De Pont, J J; Willems, P H

    1998-03-01

    was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

  2. Synthesis, fine structure of 19F NMR and fluorescence of novel amorphous TPA derivatives having perfluorinated cyclopentenyl and benzo[b]thiophene unit

    NASA Astrophysics Data System (ADS)

    Wu, Bian-Peng; Pang, Mei-Li; Tan, Ting-Feng; Meng, Ji-ben

    2013-04-01

    Three novel triphenylamine (TPA) derivatives having perfluorinated cyclopentenyl and benzo[b]thiophene unit are obtained from 4-bromo-N,N-diphenyl-2-methylbenzo[b]thiophen-5-amine. The new compounds are expected to find their use in thin film devices as charge transport materials and host organic light-emitting materials. It is found that the new compounds show relatively strong fluorescence either in solution or in solid state, and are amorphous due to a special conformation which is elucidated by the fine structure of 19F NMR. Molecular structure and properties of these compounds is characterized by 1H NMR, 13C NMR (broadband decoupled), ESI-HRMS, elemental analysis and thermal analysis (DSC). Fluorescent quantum yield in solution is measured using 9,10-diphenylanthrancene (DPA) as standard fluorescent substance.

  3. Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

    PubMed

    French, B T; Patrick, D E; Grever, M R; Trewyn, R W

    1991-01-15

    6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells.

  4. Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

    PubMed Central

    French, B T; Patrick, D E; Grever, M R; Trewyn, R W

    1991-01-01

    6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells. Images PMID:1988936

  5. The growth of cultured human foreskin keratinocytes is not stimulated by a tumor promoter.

    PubMed

    Fischer, S M; Viaje, A; Mills, G D; Wong, E W; Weeks, C E; Slaga, T J

    1984-01-01

    The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) does not stimulate the growth of human epidermal cells in foreskin explant cultures; a dose-dependent inhibition is seen at doses higher than 10(-5) micrograms/ml. TPA also inhibits epidermal growth factor-stimulated growth and does not induce ornithine decarboxylase activity or increase polyamine levels. This is not due to the rapid breakdown of TPA, since TPA is not metabolized to any appreciable extent.

  6. Control of c-fos and c-myc proto-oncogene induction in rat thyroid cells in culture

    SciTech Connect

    Isozaki, O.; Kohn, L.D. )

    1987-11-01

    Removal of TSH, insulin, and cortisol from the medium, and decreasing the serum content to 0.2%, abolishes both the proliferate and differentiated state of FRTL-5 rat thyroid cells in culture. In these basal conditions, the individual addition of TSH, insulin, insulin-like growth factor-I (IGF-I), phorbol 12-myristate 13-acetate (TPA), alpha 1-adrenergic agents, or A23187, increase c-myc and/or c-fos proto-oncogene expression. Under the same conditions, only the addition of TSH increased cAMP levels; 8-bromo-cAMP can increase c-myc or c-fos mRNA levels. Pretreatment of cells with phorbol 12,13-dibutyrate, an agent which down regulates the C-kinase, completely inhibits the effect of TPA on proto-oncogene expression but has no affect on the A23187 induced-increase. The sum of these results indicate that at least four separate signal systems independently increase c-myc or c-fos gene expression in FRTL-5 cells cAMP (TSH), C-kinase (TPA), Ca++/phosphoinositide (A23187), and that influenced by insulin/IGF-I. None of the ligands, when individually returned to cells in basal medium (no TSH, insulin, or cortisol and only 0.2% serum), increases cell number; norepinephrine, and A23187 do not increase (3H)thymidine incorporation into DNA under these conditions; and combinations of the ligands can be more than additive in effecting (3H)thymidine incorporation into DNA but are only additive in effecting proto-oncogene expression. Insulin/IGF-I plus TSH or insulin/IGF-I plus norepinephrine can increase both proto-oncogene expression and (3H)thymidine incorporation into DNA to the same extent; however, the former combination can increase cell number whereas the latter cannot. There is therefore no simple correlation between the ability of the above ligands to increase proto-oncogene expression and their ability to increase cell number or induce DNA synthesis.

  7. De novo protein synthesis is required for lytic cycle reactivation of Epstein-Barr virus, but not Kaposi's sarcoma-associated herpesvirus, in response to histone deacetylase inhibitors and protein kinase C agonists.

    PubMed

    Ye, Jianjiang; Gradoville, Lyndle; Daigle, Derek; Miller, George

    2007-09-01

    The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the lytic cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatment with cycloheximide (CHX), an inhibitor of protein synthesis. CHX blocked expression of mRNAs of EBV BZLF1 and BRLF1, the two EBV lytic cycle activator genes, when HH514-16 Burkitt lymphoma cells were treated with histone deacetylase (HDAC) inhibitors, sodium butyrate or trichostatin A, or a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine. CHX also inhibited EBV lytic cycle activation in B95-8 marmoset lymphoblastoid cells by phorbol ester phorbol-12-myristate-13-acetate (TPA). EBV lytic cycle induction became resistant to CHX between 4 and 6 h after application of the inducing stimulus. KSHV lytic cycle activation, as assessed by ORF50 mRNA expression, was rapidly induced by the HDAC inhibitors, sodium butyrate and trichostatin A, in HH-B2 primary effusion lymphoma cells. In HH-B2 cells, CHX did not inhibit, but enhanced, expression of the KSHV lytic cycle activator gene, ORF50. In BC-1, a primary effusion lymphoma cell line that is dually infected with EBV and KSHV, CHX blocked EBV BRLF1 lytic gene expression induced by TPA and sodium butyrate; KSHV ORF50 mRNA induced simultaneously in the same cells by the same inducing stimuli was resistant to CHX. The experiments show, for the cell lines and inducing agents studied, that the EBV BZLF1 and BRLF1 genes do not behave with "immediate-early" kinetics upon reactivation from latency. KSHV ORF50 is a true "immediate-early" gene. Our results indicate that the mechanism by which HDAC inhibitors and TPA induce lytic cycle

  8. Identification of chaperones in a MPP+-induced and ATRA/TPA-differentiated SH-SY5Y cell PD model

    PubMed Central

    Xie, Hongrong; Hu, Hui; Chang, Ming; Huang, Dongya; Gu, Xiaobo; Xiong, Xinli; Xiong, Ran; Hu, Linsen; Li, Gang

    2016-01-01

    Parkinson’s disease (PD) is characterized by the pathological accumulation of misfolded proteins. Molecular chaperones assist in the proper folding of proteins and removal of irreversibly misfolded proteins. This study aims to identify potential chaperones associated with protein misfolding and accumulation in PD. ATRA/TPA-differentiated SH-SY5Y cells were treated with 1 mM of MPP+ for 48 hours. Proteins were analyzed by 2D-DIGE followed by MALDI-ToF MS. The treatment of differentiated SH-SY5Y cells by MPP+ led to the unambiguous identification of 10 protein spots, which corresponds to six proteins. Among these six proteins, four were chaperone proteins including nucleophosmin (NPM1), chaperonin-containing TCP-1 subunit 2 (CCT2 or CCTβ), heat shock 90 kDa protein 1 beta (HSP90AB1 or HSP90-β), and tyrosin3/tryptopha5-monoxygenase activation protein, zeta polypeptide (14-3-3ζ, gene symbol: Ywhaz). To our knowledge, this is the first report that linked the upregulation of chaperones after MPP+ treatment with SH-SY5Y cells. However, the NPM1 protein was identified for the first time in the PD model. The upregulation of four chaperone proteins provided evidence that these chaperones have a complementary effect on protein misfolding in the pathogenesis of PD, and hold promise as a good therapeutic target for PD treatment. PMID:28078037

  9. Cloning of the staurosporine biosynthetic gene cluster from Streptomyces sp. TP-A0274 and its heterologous expression in Streptomyces lividans.

    PubMed

    Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu

    2002-12-01

    Staurosporine is a representative member of indolocarbazole antibiotics. The entire staurosporine biosynthetic and regulatory gene cluster spanning 20-kb was cloned from Streptomyces sp. TP-A0274 and sequenced. The gene cluster consists of 14 ORFs and the amino acid sequence homology search revealed that it contains three genes, staO, staD, and staP, coding for the enzymes involved in the indolocarbazole aglycone biosynthesis, two genes, staG and staN, for the bond formation between the aglycone and deoxysugar, eight genes, staA, staB, staE, staJ, staI, staK, staMA, and staMB, for the deoxysugar biosynthesis and one gene, staR is a transcriptional regulator. Heterologous gene expression of a 38-kb fragment containing a complete set of the biosynthetic genes for staurosporine cloned into pTOYAMAcos confirmed its role in staurosporine biosynthesis. Moreover, the distribution of the gene for chromopyrrolic acid synthase, the key enzyme for the biosynthesis of indolocarbazole aglycone, in actinomycetes was investigated, and rebD homologs were shown to exist only in the strains producing indolocarbazole antibiotics.

  10. Introducing [Mn(CO)3(tpa-κ(3)N)](+) as a novel photoactivatable CO-releasing molecule with well-defined iCORM intermediates - synthesis, spectroscopy, and antibacterial activity.

    PubMed

    Nagel, Christoph; McLean, Samantha; Poole, Robert K; Braunschweig, Holger; Kramer, Thomas; Schatzschneider, Ulrich

    2014-07-14

    [Mn(CO)3(tpa-κ(3)N)]Br was prepared as a novel photoactivatable CO-releasing molecule (PhotoCORM) from [MnBr(CO)5] and tris(2-pyridylmethyl)amine (tpa) for the delivery of carbon monoxide to biological systems, with the κ(3)N binding mode of the tetradentate tpa ligand demonstrated by X-ray crystallography. The title compound is a CORM prodrug stable in solution in the dark for up to 16 h. However, photoactivation at 365 nm leads to CO release from the metal coordination sphere and transfer to haem proteins, as demonstrated by the standard myoglobin assay. Different iCORM intermediates could be detected with solution IR spectroscopy and assigned using DFT vibrational calculations. The antibacterial activity of the complex was studied on Escherichia coli. No effects were observed when the cultures were either kept in the dark in the presence of PhotoCORM or illuminated in the absence of metal complex. However, photoactivation of [Mn(CO)3(tpa-κ(3)N)]Br at 365 nm led to the appearance of the spectral signatures of CO-coordinated haems in the terminal oxidases of the bacterial electron transport chain in whole-cell UV/Vis absorption spectra. Significant internalization of the PhotoCORM was demonstrated by ICP-MS measurement of the intracellular manganese concentration. In particular when using medium with succinate as the sole carbon source, a very pronounced and concentration-dependent decrease in the E. coli growth rate could be observed upon illumination in the presence of metal complex, which is attributed to the constrained energy metabolism under these conditions and a strong indicator of terminal oxidase inhibition by carbon monoxide delivered from the PhotoCORM.

  11. Oxygen binding and activation by the complexes of PY2- and TPA-appended diphenylglycoluril receptors with copper and other metals.

    PubMed

    Sprakel, Vera S I; Feiters, Martin C; Meyer-Klaucke, Wolfram; Klopstra, Marten; Brinksma, Jelle; Feringa, Ben L; Karlin, Kenneth D; Nolte, Roeland J M

    2005-11-07

    The copper(I) complexes of diphenylglycoluril basket receptors and , appended with bis(2-ethylpyridine)amine (PY2) and tris(2-methylpyridine)amine (TPA), respectively, and their dioxygen adducts were studied with low-temperature UV-vis and X-ray absorption spectroscopy (XAS). The copper(I) complex of, [.Cu(I)2] or, forms a micro-eta2:eta2 dioxygen complex, whereas the copper(I) complex of, [.Cu(I)2] or, does not form a well defined dioxygen complex, but is oxidized to Cu(II). Dioxygen is bound irreversibly to and the formed complex is stable over time. The coordination geometries of the above complexes were determined by XAS, which revealed that pyridyl groups and amine N-donors participate in the coordination to Cu(I) ions in the complexes of both receptors. The catalytic activities of various metal complexes of and , that were designed as mimics of dinuclear copper enzymes that can activate dioxygen, were investigated. Phenolic substrates that were expected to undergo aromatic hydroxylation, showed oxidative polymerization without insertion of oxygen. The mechanism of this polymerization turns out to be a radical coupling reaction as was established by experiments with the model substrate 2,4-di-tert-butylphenol. In addition to Cu(II), the Mn(III) complex of and the Fe(II) complex of were tested as oxidation catalysts. Oxidation of catechol was observed for the Cu(II) complex of receptor but the other metal complexes did not lead to oxidation.

  12. Prevention of EP Migratory Contamination in a Cluster Randomized Trial to Increase tPA Use in Stroke (The INSTINCT Trial)

    PubMed Central

    Weston, Victoria C.; Meurer, William J.; Frederiksen, Shirley M.; Fox, Allison K.; Scott, Phillip A.

    2016-01-01

    Objectives Cluster randomized trials (CRTs) are increasingly utilized to evaluate quality improvement interventions aimed at healthcare providers. In trials testing emergency department interventions, migration of emergency physicians (EPs) between hospitals is an important concern, as contamination may affect both internal and external validity. We hypothesized that geographically isolating emergency departments would prevent migratory contamination in a CRT designed to increase ED delivery of tPA in stroke (The INSTINCT Trial). Methods INSTINCT was a prospective, cluster randomized, controlled trial. 24 Michigan community hospitals were randomly selected in matched pairs for study. Contamination was defined at the cluster level, with substantial contamination defined a priori as >10% of EPs affected. Non-adherence, total crossover (contamination + non-adherence), migration distance and characteristics were determined. Results 307 emergency physicians were identified at all sites. Overall, 7 (2.3%) changed study sites. 1 moved between control sites, leaving 6 (2.0%) total crossovers. Of these, 2 (0.7%) moved from intervention to control (contamination) and 4 (1.3%) moved from control to intervention (non-adherence). Contamination was observed in 2 of 12 control sites, with 17% and 9% contamination of the total site EP workforce at follow-up, respectively. Average migration distance was 42 miles for all EPs moving in the study and 35 miles for EPs moving from intervention to control sites. Conclusion The mobile nature of emergency physicians should be considered in the design of quality improvement CRTs. Increased reporting of contamination in CRTs is encouraged to clarify thresholds and facilitate CRT design. PMID:25440230

  13. Effects of Ferumoxides – Protamine Sulfate Labeling on Immunomodulatory Characteristics of Macrophage-like THP-1 Cells

    PubMed Central

    Janic, Branislava; Iskander, A. S. M.; Rad, Ali M.; Soltanian-Zadeh, Hamid; Arbab, Ali S.

    2008-01-01

    Superparamagnetic Iron Oxide (SPIO) complexed with cationic transfection agent is used to label various mammalian cells. Labeled cells can then be utilized as an in vivo magnetic resonance imaging (MRI) probes. However, certain number of in vivo administered labeled cells may be cleared from tissues by the host's macrophages. For successful translation to routine clinical application of SPIO labeling method it is important that this mode of in vivo clearance of iron does not elicit any diverse immunological effects. The purpose of this study was to demonstrate that SPIO agent ferumoxides-protamine sulfate (FePro) incorporation into macrophages does not alter immunological properties of these cells with regard to differentiation, chemotaxis, and ability to respond to the activation stimuli and to modulate T cell response. We used THP-1 cell line as a model for studying macrophage cell type. THP-1 cells were magnetically labeled with FePro, differentiated with 100 nM of phorbol ester, 12-Myristate-13-acetate (TPA) and stimulated with 100 ng/ml of LPS. The results showed 1) FePro labeling had no effect on the changes in morphology and expression of cell surface proteins associated with TPA induced differentiation; 2) FePro labeled cells responded to LPS with slightly higher levels of NFκB pathway activation, as shown by immunobloting; TNF-α secretion and cell surface expression levels of CD54 and CD83 activation markers, under these conditions, were still comparable to the levels observed in non-labeled cells; 3) FePro labeling exhibited differential, chemokine dependent, effect on THP-1 chemotaxis with a decrease in cell directional migration to MCP-1; 4) FePro labeling did not affect the ability of THP-1 cells to down-regulate T cell expression of CD4 and CD8 and to induce T cell proliferation. Our study demonstrated that intracellular incorporation of FePro complexes does not alter overall immunological properties of THP-1 cells. The described experiments provide

  14. 12-O-Tetradecanoylphorbol-13-acetate in Treating Patients With Hematologic Cancer or Bone Marrow Disorder

    ClinicalTrials.gov

    2010-01-25

    Chronic Myeloproliferative Disorders; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Precancerous/Nonmalignant Condition

  15. Dynamic compression of dense oxide (Gd3Ga5O12) from 0.4 to 2.6 TPa: Universal Hugoniot of fluid metals

    SciTech Connect

    Ozaki, N.; Nellis, W. J.; Mashimo, T.; Ramzan, M.; Ahuja, R.; Kaewmaraya, T.; Kimura, T.; Knudson, M.; Miyanishi, K.; Sakawa, Y.; Sano, T.; Kodama, R.

    2016-05-19

    Materials at high pressures and temperatures are of great current interest for warm dense matter physics, planetary sciences, and inertial fusion energy research. Shock-compression equation-of-state data and optical reflectivities of the fluid dense oxide, Gd3Ga5O12 (GGG), were measured at extremely high pressures up to 2.6 TPa (26 Mbar) generated by high-power laser irradiation and magnetically-driven hypervelocity impacts. Above 0.75 TPa, the GGG Hugoniot data approach/reach a universal linear line of fluid metals, and the optical reflectivity most likely reaches a constant value indicating that GGG undergoes a crossover from fluid semiconductor to poor metal with minimum metallic conductivity (MMC). These results suggest that most fluid compounds, e.g., strong planetary oxides, reach a common state on the universal Hugoniot of fluid metals (UHFM) with MMC at sufficiently extreme pressures and temperatures. Lastly, the systematic behaviors of warm dense fluid would be useful benchmarks for developing theoretical equation-of-state and transport models in the warm dense matter regime in determining computational predictions.

  16. Morphofunctional study of 12-O-tetradecanoyl-13-phorbol acetate (TPA)-induced differentiation of U937 cells under exposure to a 6 mT static magnetic field.

    PubMed

    Dini, Luciana; Dwikat, Majdi; Panzarini, Elisa; Vergallo, Cristian; Tenuzzo, Bernadetta

    2009-07-01

    This study deals with the morphofunctional influence of 72 h exposure to a 6 mT static magnetic field (SMF) during differentiation induced by 50 ng/ml 12-O-tetradecanoyl-13-phorbol acetate (TPA) in human leukaemia U937 cells. The cell morphology of U937 cells was investigated by optic and electron microscopy. Specific antibodies and/or molecules were used to label CD11c, CD14, phosphatidylserine, F-actin and to investigate the distribution and activity of lysosomes, mitochondria and SER. [Ca(2+)](i) was evaluated with a spectrophotometer. The degree of differentiation in SMF-exposed cells was lower than that of non-exposed cells, the difference being exposure time-dependent. SMF-exposed cells showed cell shape and F-actin modification, inhibition of cell attachment, appearance of membrane roughness and large blebs and impaired expression of specific macrophagic markers on the cell surface. The intracellular localization of SER and lysosomes was only partially affected by exposure. A significant localization of mitochondria with an intact membrane potential at the cell periphery in non-exposed, TPA-stimulated cells was observed; conversely, in the presence of SMF, mitochondria were mainly localised near the nucleus. In no case did SMF exposure affect cell viability. The sharp intracellular increase of [Ca(2+)](i) could be one of the causes of the above-described changes.

  17. Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.

    PubMed Central

    Zaitsev, S V; Efendić, S; Arkhammar, P; Bertorello, A M; Berggren, P O

    1995-01-01

    Simultaneous measurements of cytosolic free Ca2+ concentration and insulin release, in mouse single pancreatic islets, revealed a direct correlation only initially after stimulation with glucose or K+. Later, there is an apparent dissociation between these two parameters, with translocation of alpha and epsilon isoenzymes of protein kinase C to membranes and simultaneous desensitization of insulin release in response to glucose. Recovery of insulin release, without any concomitant changes in cytosolic free Ca2+ concentration, after addition of phorbol 12-myristate 13-acetate, okadaic acid, and forskolin supports the notion that the desensitization process is accounted for by dephosphorylation of key regulatory sites of the insulin exocytotic machinery. Images Fig. 3 PMID:7568203

  18. Endomorphins 1 and 2 modulate chemotaxis, phagocytosis and superoxide anion production by microglia.

    PubMed

    Azuma, Y; Ohura, K; Wang, P L; Shinohara, M

    2001-09-03

    We evaluate the role of endomorphins 1 and 2 on microglial functions. Endomorphins 1 and 2 blocked phagocytosis of Escherichia coli. In addition, both markedly inhibited chemotaxis toward zymosan-activated serum. In contrast, when microglia was preincubated with these endomorphins, followed by incubation with LPS before stimulation with phorbol 12-myristate 13-acetate (PMA) at 200 nM, they potentiated superoxide anion production. Furthermore, when microglia was preincubated with these endomorphins together with PMA at 20 nM, followed by stimulation with PMA at 200 nM, superoxide anion production was potentiated. These results suggest that endomorphins 1 and 2 modulate phagocytosis, chemotaxis and superoxide anion production by microglia.

  19. Novel Quantitation of Autocrine/Paracrine Stimulation of Cell Motility in Vitro and Metastasis

    DTIC Science & Technology

    2008-09-01

    1) revealed that MDA-MB-435 cells metastatic ability is great than MDA-MB-231, which is then greater than MDA-MB-468. Therefore, to test if L1-CAM...cancer cells was tested using the same three cell lines in above tests . As presented in Fig.5, PMA (phorbol 12-myristate 13-acetate) did increased L1...Then virus was introduced firstly into 293T or QT6 cells to test correct L1 fragments expression. As shown in Fig. 6 different parts of L1 were

  20. Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis.

    PubMed

    Yasukawa, K; Takido, M; Matsumoto, T; Takeuchi, M; Nakagawa, S

    1991-01-01

    A single topical application of 1 microgram of 12-O-tetradecanoylphorbol- 13-acetate (TPA) to the ears of mice was shown to induce edema, and this TPA-induced inflammation was inhibited by 4-methylsterol and triterpene derivatives. The ED50 of these compounds against TPA-induced inflammation was 0.1-3 mumol. Phytosterols had only slight inhibitory effects. Furthermore, application of 5 micrograms TPA to mouse skin rapidly caused accumulation of ornithine decarboxylase (ODC). Similarly, sitosterol and lupane-type triterpene derivatives markedly inhibited this TPA-induced ODC accumulation. In addition, 5 mumol betulinic acid markedly inhibited the promoting effect of 2.5 micrograms TPA applied twice weekly on skin tumor formation in mice initiated with 50 micrograms of 7,12-dimethylbenz[a]anthracene, and 5 mumol of sitosterol caused slight suppression. Thus, the inhibitory effects of sterol and triterpene derivatives on TPA-induced inflammation roughly parallelled their inhibitory activities against tumor promotion.

  1. Interaction between constitutively expressed heat shock protein, Hsc 70, and cysteine string protein is important for cortical granule exocytosis in Xenopus oocytes.

    PubMed

    Smith, Geoffrey B; Umbach, Joy A; Hirano, Arlene; Gundersen, Cameron B

    2005-09-23

    In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by approximately 80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.

  2. Anti-inflammatory effects of zinc in PMA-treated human gingival fibroblast cells

    PubMed Central

    Kim, Sangwoo; Jeon, Sangmi; Hui, Zheng; Kim, Young; Im, Yeonggwan; Lim, Wonbong; Kim, Changsu; Choi, Hongran; Kim, Okjoon

    2015-01-01

    Objectives: Abnormal cellular immune response has been considered to be responsible for oral lesions in recurrent aphthous stomatitis. Zinc has been known to be an essential nutrient metal that is necessary for a broad range of biological activities including antioxidant, immune mediator, and anti-inflammatory drugs in oral mucosal disease. The objective of this study was to investigate the effects of zinc in a phorbol-12-myristate-13-acetate (PMA)-treated inflammatory model on human gingival fibroblast cells (hGFs). Study Design: Cells were pre-treated with zinc chloride, followed by PMA in hGFs. The effects were assessed on cell viability, cyclooxygenease-1,2(COX-1/2) protein expression, PGE2 release, ROS production and cytokine release, Results: The effects were assessed on cell viability, COX1/2 protein expression, PGE2 release, ROS production, cytokine release. The results showed that, in the presence of PMA, zinc treatment leads to reduce the production of ROS, which results in decrease of COX-2 expression and PGE2 release. Conclusions: Thus, we suggest that zinc treatment leads to the mitigation of oral inflammation and may prove to be an alternative treatment for recurrent aphthous stomatitis. Key words:Zinc, inflammatory response, cytokines, phorbol-12-myristate-13-acetate, gingival fibroblasts cells. PMID:25662537

  3. Interaction between Constitutively Expressed Heat Shock Protein, Hsc 70, and Cysteine String Protein Is Important for Cortical Granule Exocytosis in Xenopus Oocytes*

    PubMed Central

    Smith, Geoffrey B.; Umbach, Joy A.; Hirano, Arlene; Gundersen, Cameron B.

    2013-01-01

    In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by ~80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes. PMID:16055447

  4. Antimicrobial Activity of the Manganese Photoactivated Carbon Monoxide-Releasing Molecule [Mn(CO)3(tpa-κ3N)]+ Against a Pathogenic Escherichia coli that Causes Urinary Infections

    PubMed Central

    Tinajero-Trejo, Mariana; Rana, Namrata; Nagel, Christoph; Jesse, Helen E.; Smith, Thomas W.; Wareham, Lauren K.; Hippler, Michael; Schatzschneider, Ulrich

    2016-01-01

    Abstract Aims: We set out to investigate the antibacterial activity of a new Mn-based photoactivated carbon monoxide-releasing molecule (PhotoCORM, [Mn(CO)3(tpa-κ3N)]+) against an antibiotic-resistant uropathogenic strain (EC958) of Escherichia coli. Results: Activated PhotoCORM inhibits growth and decreases viability of E. coli EC958, but non-illuminated carbon monoxide-releasing molecule (CORM) is without effect. NADH-supported respiration rates are significantly decreased by activated PhotoCORM, mimicking the effect of dissolved CO gas. CO from the PhotoCORM binds to intracellular targets, namely respiratory oxidases in strain EC958 and a bacterial globin heterologously expressed in strain K-12. However, unlike previously characterized CORMs, the PhotoCORM is not significantly accumulated in cells, as deduced from the cellular manganese content. Activated PhotoCORM reacts avidly with hydrogen peroxide producing hydroxyl radicals; the observed peroxide-enhanced toxicity of the PhotoCORM is ameliorated by thiourea. The PhotoCORM also potentiates the effect of the antibiotic, doxycycline. Innovation: The present work investigates for the first time the antimicrobial activity of a light-activated PhotoCORM against an antibiotic-resistant pathogen. A comprehensive study of the effects of the PhotoCORM and its derivative molecules upon illumination is performed and mechanisms of toxicity of the activated PhotoCORM are investigated. Conclusion: The PhotoCORM allows a site-specific and time-controlled release of CO in bacterial cultures and has the potential to provide much needed information on the generality of CORM activities in biology. Understanding the mechanism(s) of activated PhotoCORM toxicity will be key in exploring the potential of this and similar compounds as antimicrobial agents, perhaps in combinatorial therapies with other agents. Antioxid. Redox Signal. 24, 765–780. PMID:26842766

  5. Protective effects of black rice bran against chemically-induced inflammation of mouse skin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Role of the Chemokine MCP-1 in Sensitization of PKC-Mediated Apoptosis in Prostate Cancer Cells

    DTIC Science & Technology

    2010-02-01

    expression of PKCs by Western blot. We have observed maxi - mum depletion at 48 h, lasting up to 96 h in some cases. Phorbol Ester–Induced Apoptosis...apoptosis mediated by 12-0-tetradeca- noylphorbol-13-acetate (TPA). Br J Cancer 2004;90: 2017 – 2024. 17. Aggarwal BB. Signalling pathways of the TNF

  7. Tumor promoter induces sister chromatid exchanges: relevance to mechanisms of carcinogenesis.

    PubMed Central

    Kinsella, A R; Radman, M

    1978-01-01

    12-O-Tetradecanoylphorbol 13-acetate (TPA), a powerful tumor promoter, is shown to induce sister chromatid exchanges (SCEs), whereas the nonpromoting derivative 4-O-methyl-TPA does not. Inhibitors of tumor promotion--antipain, leupeptin, and fluocinolone acetonide--inhibit formation of such TPA-induced SCEs. TPA is a unique agent in its induction of SCEs in the absence of DNA damage, chromosome aberrations, mutagenesis, or significant toxicity. Because TPA is known to induce several gene functions, we speculate that it might also induce enzymes involved in genetic recombination. Thus, the irreversible step in tumor promotion might be the result of an aberrant mitotic segregation event leading to the expression of carcinogen/mutagen-induced recessive genetic or epigenetic chromosomal changes. Images PMID:282631

  8. Biomimetic Oxidation Studies. 11. Alkane Functionalization in Aqueous Solution Utilizing in Situ Formed [Fe(2)O(eta(1)-H(2)O)(eta(1)-OAc)(TPA)(2)](3+), as an MMO Model Precatalyst, Embedded in Surface-Derivatized Silica and Contained in Micelles.

    PubMed

    Neimann, Karine; Neumann, Ronny; Rabion, Alain; Buchanan, Robert M.; Fish, Richard H.

    1999-07-26

    The biomimetic, methane monooxygenase enzyme (MMO) precatalyst, [Fe(2)O(eta(1)-H(2)O)(eta(1)-OAc)(TPA)(2)](3+) (TPA = tris[(2-pyridyl)methyl]amine), 1, formed in situ at pH 4.2 from [Fe(2)O(&mgr;-OAc)(TPA)(2)](3+), 2, was embedded in an amorphous silicate surface modified by a combination of hydrophilic poly(ethylene oxide) and hydrophobic poly(propylene oxide). The resulting catalytic assembly was found to be a biomimetic model for the MMO active site within a hydrophobic macroenvironment, allowing alkane functionalization with tert-butyl hydroperoxide (TBHP)/O(2) in an aqueous reaction medium (pH 4.2). For example, cyclohexane was oxidized to a mixture of cyclohexanone, cyclohexanol, and cyclohexyl-tert-butyl peroxide, in a ratio of approximately 3:1:2. The balance between poly(ethylene oxide) and poly(propylene oxide), tethered on the silica surface, was crucial for maximizing the catalytic activity. The silica-based catalytic assembly showed reactivity somewhat higher in comparison to an aqueous micelle system utilizing the surfactant, cetyltrimethylammonium hydrogen sulfate at its critical micelle concentration, in which functionalization of cyclohexane with TBHP/O(2) in the presence of 1 was also studied at pH 4.2 and was found to provide similar products: cyclohexanol, cyclohexanone, and cyclohexyl-tert-butyl peroxide, in a ratio of approximately 2:3:1. Moreover, the mechanism for both the silica-based catalytic assembly and the aqueous micelle system was found to occur via the Haber-Weiss process, in which redox chemistry between 1 and TBHP provides both the t-BuO(*)() and t-BuOO(*)()( )()radicals. The t-BuO(*)()( )()radical initiates the C-H functionalization reaction to form the carbon radical, followed by O(2) trapping, to provide cyclohexyl hydroperoxide, which produces the cyclohexanol and cyclohexanone in the presence of 1, whereas the coupling product emanates from t-BuOO(*)() and cyclohexyl radicals. A discussion concerning both approaches for

  9. Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly

    PubMed Central

    1994-01-01

    The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level. PMID:7806568

  10. Dissimilar effects of phorbol ester and diacylglycerol derivative on protein kinase activity in the monoblastoid U937 cell.

    PubMed

    Ways, D K; Dodd, R C; Earp, H S

    1987-07-01

    Mechanism, in addition to protein kinase C activation may mediate 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated differentiation of leukemic cells. We compared the effect of pretreating intact monoblastoid U937 cells with TPA or the diacylglycerol derivative, 1-oleoyl-2-acetylglycerol (OAG), by studying the protein kinase C dependent and independent histone phosphotransferase activity, the phosphorylation of endogenous substrates, and the ability to stimulate differentiation. In cellular fractions derived from cells treated with TPA or OAG, cytosolic protein kinase C activity decreased. In the detergent extracted particulate fraction, TPA produced a time and dose dependent decrease in protein kinase C activity. In contrast, OAG increased particulate protein kinase C activity. In addition, the particulate fraction derived from cells treated with TPA exhibited increased phosphatidyl serine and diolein independent histone phosphotransferase activity as well as an increase in the phosphorylation of two endogenous substrates with molecular weights of 120,000 and 80,000. OAG did not mimic these effects. When exposed to 32P-labeled intact cells, OAG and TPA stimulated phosphorylation of three substrates. Thus, the inability of OAG to mimic the effects of TPA was not due to lack of protein kinase C activation. TPA, but not OAG, stimulated differentiation of the U937 cell to a monocyte-like cell. These data demonstrate that TPA and OAG have dissimilar effects on protein kinase activity and differentiation in the U937 monoblastoid cell.

  11. Potentiation of phorbol ester-induced coronary vasoconstriction in dogs following endothelium disruption

    SciTech Connect

    Roberts, R.B.; Ku, D.D.

    1986-03-05

    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 (1..mu..M), cyproheptadine (1..mu..H) and ibuprofen (1..mu..g/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/sub 0/) or addition of 1..mu..M nimodipine after TPA(30nM) resulted in only 32 +/- 4% and 25 +/- 3% reversal of TPA contraction, respectively. Addition of amiloride (10..mu..M 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/sub 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.

  12. Pro-apoptotic NOXA is implicated in atmospheric-pressure plasma-induced melanoma cell death

    NASA Astrophysics Data System (ADS)

    Ishaq, M.; Bazaka, K.; Ostrikov, K.

    2015-11-01

    Atmospheric-pressure plasma (APP) has been successfully used to treat several types of cancers in vivo and in vitro, with the effect being primarily attributed to the generation of reactive oxygen species (ROS). However, the mechanisms by which APP induces apoptosis in cancer cells require further elucidation. In this study, the effects of APP on the expression of 500 genes in melanoma Mel007 cancer cells were examined. Pro-apoptotic phorbol-12-myristate-13-acetate-induced protein (PMAIP1), also known as NOXA, was highly expressed as a result of APP treatment in a dose-dependent manner. Blocking of ROS using scavenger NAC or silencing of NOXA gene by RNA interference inhibited the APP-induced NOXA genes upregulation and impaired caspases 3/7 mediated apoptosis, confirming the important role plasma-generated ROS species and pro-apoptotic NOXA play in APP-induced cancer cell death.

  13. Requirement for tyrosine phosphorylation in lipopolysaccharide-induced murine B-cell proliferation.

    PubMed Central

    Dearden-Badet, M T; Revillard, J P

    1993-01-01

    Bacterial lipopolysaccharide (LPS) induces a strong B-cell proliferative response with subsequent differentiation, through a complex signal transduction pathway. This process is known to be mediated through protein kinase C (PKC) translocation without Ca2+ mobilization. Here, we show that B-cell proliferative responses induced by five different LPS preparations, as well as by F(ab')2 anti-IgM antibodies, are inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A. In contrast, B-cell proliferation induced by the combination of phorbol 12-myristate 13-acetate (PMA) plus ionomycin was not influenced by treatment with either herbimycin A or genistein. These data indicate that tyrosine phosphorylation is required to initiate B-cell proliferation by LPS. PMID:8307617

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

    SciTech Connect

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

    2008-07-11

    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.

  15. Post-transcriptional Regulation of Meprin α by the RNA-binding Proteins Hu Antigen R (HuR) and Tristetraprolin (TTP)*

    PubMed Central

    Roff, Alanna N.; Panganiban, Ronaldo P.; Bond, Judith S.; Ishmael, Faoud T.

    2013-01-01

    Meprins are multimeric proteases that are implicated in inflammatory bowel disease by both genetic association studies and functional studies in knock-out mice. Patients with inflammatory bowel disease show decreased colonic expression of meprin α, although regulation of expression, particularly under inflammatory stimuli, has not been studied. The studies herein demonstrate that the human meprin α transcript is bound and stabilized by Hu antigen R at baseline, and that treatment with the inflammatory stimulus phorbol 12-myristate 13-acetate downregulates meprin α expression by inducing tristetraprolin. The enhanced binding of tristetraprolin to the MEP1A 3′-UTR results in destabilization of the transcript and occurs at a discrete site from Hu antigen R. This is the first report to describe a mechanism for post-transcriptional regulation of meprin α and will help clarify the role of meprins in the inflammatory response and disease. PMID:23269677

  16. Lymphocytes possess an electrogenic H(+)-transporting pathway in their plasma membrane.

    PubMed Central

    Káldi, K; Szászi, K; Suszták, K; Kapus, A; Ligeti, E

    1994-01-01

    The existence of an electrogenic H(+)-transporting pathway similar to that described in the plasma membrane of granulocytes and macrophages is reported in pig peripheral lymphocytes. The function of the H(+)-transport pathway can only be detected when free movement of charge-compensating cations is allowed. H+ transport is stimulated by arachidonic acid and various unsaturated fatty acids, and inhibited by bivalent cations, with the following sequence of efficiency: Zn2+ > Cd2+ = Co2+ = Ni2+ > Mn2+ > Ba2+ = Ca2+ = Mg2+. The transport pathway is activated by intracellular acidification and by NN'-dicyclohexylcarbodiimide, but it is not influenced by phorbol 12-myristate 13-acetate. As pig peripheral lymphocytes are not able to produce O2-., it is suggested that the operation of the electrogenic H+ conductance does not require the assembly of a functional NADPH oxidase. Images Figure 1 PMID:7519007

  17. [Chemiluminescence of the polymorphonuclear leukocytes-luminol system in the presence of biogenic chloramines].

    PubMed

    Murina, M A; Belakina, N S; Roshchupkin, D I

    2004-01-01

    It was demonstrated that N-chlorphenylalanine and other chloramines strengthen sharply chemiluminescence in the polymorphonuclear leukocytes (PML)-luminol system without special activation of cells. The intensity of chemiluminescence is higher than the intensity of luminol solution emission induced by N-chlorphenylalanine. But it was nearly equal to chemiluminescence intensity of a mixture of luminol, N-chlorphenylalanine and 20-30 nM H2O2. The increase in chemiluminescence in the PML-luminol system in the presence of N-chlorphenylalanine is not related to PML activation but is the result of direct oxidation of luminol by N-chlorphenylalanine. Chloramine derivatives of amino acids and taurine at final concentrations of 0.01-0.1 mM do not suppress luminol chemiluminescence in suspension of PML stimulated by phorbol-12-myristate-13-acetate. At the same time, hypochlorite inhibits sharply luminol emission induced by stimulated cells.

  18. Critical role of s465 in protein kinase C-increased rat glutamate transporter type 3 activity.

    PubMed

    Baik, Hee Jung; Huang, Yueming; Washington, Jacqueline M; Zuo, Zhiyi

    2009-01-01

    Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.

  19. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts

    SciTech Connect

    Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.

    1986-05-29

    Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

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

    SciTech Connect

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

    1989-04-01

    The actions of somatostatin and of the phorbol ester 4{beta}-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and {sup 86}Rb{sup +} flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K{sup +} 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 {sup 86}Rb{sup +} 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{sup +} channels are discussed.

  1. An improved AhR reporter gene assay for analyzing dioxins in soil, sediment and fish.

    PubMed

    Chao, How-Ran; Wang, Ya-Fan; Wang, Yao-Nan; Lin, Ding-Yan; Gou, Yan-You; Chen, Chien-Yu; Chen, Kuan-Chung; Wu, Wen-Kai; Chiang, Bao-An; Huang, Yu-Ting; Hsieh, Lien-Te; Yeh, Kuei-Jyum C; Tsou, Tsui-Chun

    2012-10-01

    Our goal was to develop a fast-screening bioassay to determine dioxin levels in the environmental and biological samples from dioxin-contaminated areas. Our original dioxin-responsive-element (DRE)-driven luciferase bioassay (using Huh7-DRE-Luc cells) was modified by reducing the incubation temperature of the cell culture from 37 to 35°C and by adding phorbol-12-myristate-13-acetate, and the modified bioassay was used to examine samples from soil, sediment, and fish. The results of this bioassay were shown to be significantly related to those of the high-resolution gas chromatography/high-resolution mass spectrometry assay of dioxins. The correlative equation was: log (PCDD/Fs I-TEQs) = 1.19 × log (BEQs) - 1.15 with R(2) = 0.95 (p < 0.001).

  2. Macelignan inhibits histamine release and inflammatory mediator production in activated rat basophilic leukemia mast cells.

    PubMed

    Han, Young Sun; Kim, Myung-Suk; Hwang, Jae-Kwan

    2012-10-01

    Type I allergy is characterized by the release of granule-associated mediators, lipid-derived substances, cytokines, and chemokines by activated mast cells. To evaluate the anti-allergic effects of macelignan isolated from Myristica fragrans Houtt., we determined its ability to inhibit calcium (Ca(2+)) influx, degranulation, and inflammatory mediator production in RBL-2 H3 cells stimulated with A23187 and phorbol 12-myristate 13-acetate. Macelignan inhibited Ca(2+) influx and the secretion of β-hexosaminidase, histamine, prostaglandin E(2), and leukotriene C(4); decreased mRNA levels of cyclooxygenase-2, 5-lipoxygenase, interleukin-4 (IL-4), IL-13, and tumor necrosis factor-α; and attenuated phosphorylation of Akt and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. These results indicate the potential of macelignan as a type I allergy treatment.

  3. A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.

    PubMed

    Jeffery, Unity; Gray, Robert D; LeVine, Dana N

    2016-11-21

    Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

  4. Modulation of human c-mpl gene expression by thrombopoietin through protein kinase C.

    PubMed

    Sunohara, M; Morikawa, S; Sato, T; Sato, I; Sato, T; Fuse, A

    2003-01-01

    The c-Mpl, thrombopoietin (TPO) receptor specificially controls megakaryocytic growth and differentiation. TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter in the human megakaryoblastic cell line CMK. The maximal promoter activity of c-mpl was obtained 24 hr after pretreatment with TPO for 3 hr and then declined with time. This increase was completely abolished by protein kinase C (PKC) inhibitors (GF109203, calphostin C and H7). Phorbol 12-myristate 13-acetate (PMA) treatment led to an increase in c-mpl promoter activity. These results demonstrate that the promoter activity of c-mpl is modulated by transcription through a PKC-dependent pathway.

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

    SciTech Connect

    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.

  6. Anti-inflammatory activity of the oriental herb medicine, Arisaema cum Bile, in LPS-induced PMA-differentiated THP-1 cells.

    PubMed

    Ahn, Chang-Bum; Je, Jae-Young

    2012-06-01

    Arisaema cum Bile is widely used as a folk medicine in Korea. However, the systematic biological properties of Arisaema cum Bile have seldom been addressed. In this study, we evaluated the anti-inflammatory activity of Arisaema cum Bile extract on lipopolysaccharide (LPS)-induced inflammation in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages. The Arisaema cum Bile extract markedly inhibited the production of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, and also suppressed the mRNA and protein expressions of these cytokines. Furthermore, the Arisaema cum Bile extract also inhibited LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and gene expressions in PMA-differentiaed THP-1 macrophages. These results suggest that Arisaema cum Bile extract may have potential for development into an effective anti-inflammatory agent, and/or as an ingredient of functional foods.

  7. Functional heterologous gap junctions in Fundulus ovarian follicles maintain meiotic arrest and permit hydration during oocyte maturation.

    PubMed

    Cerdá, J L; Petrino, T R; Wallace, R A

    1993-11-01

    The physiological significance of heterologous gap junctions between granulosa cells and the oocyte was investigated in late vitellogenic ovarian follicles of the teleost Fundulus heteroclitus. Lucifer Yellow injected into the oocyte readily passed to the overlying granulosa cells, demonstrating effective dye-coupling. Passage of the fluorescent dye, and hence intercellular communication, was inhibited both by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and by 1-octanol, known uncouplers of gap junctions in a variety of invertebrate and vertebrate cell types. Octanol alone also initiated resumption of meiosis in follicle-enclosed oocytes, indicating that granulosa cells normally maintain meiotic arrest, as apparently occurs in mammalian and amphibian follicles. Both PMA and octanol also consistently inhibited the hydration process that normally accompanies meiotic maturation. These results support a previously suggested hypothesis that K+, which is the primary osmotic effector for oocyte hydration, is translocated via gap junction from granulosa cells to the maturing oocyte.

  8. Non-small cell lung cancer-derived soluble mediators enhance apoptosis in activated T lymphocytes through an I kappa B kinase-dependent mechanism.

    PubMed

    Batra, Raj K; Lin, Ying; Sharma, Sherven; Dohadwala, Mariam; Luo, Jie; Pold, Mehis; Dubinett, Steven M

    2003-02-01

    T lymphocyte survival is critical for the development and maintenance of an effective host antitumor immune response; however, the tumor environment can negatively impact T-cell survival. Lymphocytes exposed to tumor supernatants (TSNs) were evaluated for apoptosis after mitogen stimulation. TSN was observed to significantly enhance phorbol 12-myristate 13-acetate/ionomycin- and anti-CD3-stimulated lymphocyte apoptosis. Enhanced lymphocyte apoptosis was associated with an impairment of nuclear factor kappa B nuclear translocation and diminished I kappa B alpha degradation. In lymphocytes stimulated after exposure to TSNs, cytoplasmic I kappa B alpha persisted as a result of alterations in I kappa B kinase (IKK) activity. Accordingly, although there were no apparent differences in IKK component concentrations, lymphocytes preexposed to TSNs exhibited markedly reduced IKK activity. We conclude that non-small cell lung cancer-derived soluble factors promote apoptosis in activated lymphocytes by an IKK-dependent pathway.

  9. Proteolysis of synaptobrevin, syntaxin, and SNAP-25 in alveolar epithelial type II cells.

    PubMed

    Zimmerman, U J; Malek, S K; Liu, L; Li, H L

    1999-10-01

    Synaptobrevin-2, syntaxin-1, and SNAP-25 were identified in rat alveolar epithelial type II cells by Western blot analysis. Synaptobrevin-2 was localized in the lamellar bodies, and syntaxin-1 and SNAP-25 were found in 0.4% Nonidet P40-soluble and -insoluble fractions, respectively, of the type II cells. When the isolated type II cells were stimulated for secretion with calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, these proteins were found to have been proteolyzed. Preincubation of cells with calpain inhibitor II (N-acetylleucylleucylmethionine), however, prevented the proteolysis. Treatment of the cell lysate with exogenous calpain resulted in a time-dependent decrease of these proteins. The data suggest that synaptobrevin, syntaxin, and SNAP-25 are subject to proteolytic modification by activated calpain in intact type II cells stimulated for secretion.

  10. Activation of protein kinase C inhibits potassium currents in cultured endothelial cells.

    PubMed

    Zhang, H; Weir, B; Daniel, E E

    1995-04-01

    The effect of protein kinase C on potassium channels in cultured endothelial cells was investigated by using whole-cell patch-clamp techniques. Activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), but not phorbol 12-monomyristate (PMM), an inactive analogue of phorbol esters, depressed an outward calcium-dependent potassium current. The inhibitory actions of PMA and PDBu could be reversed by the kinase inhibitor H-7. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum calcium pump, and LP-805, a novel vasodilator which also releases endothelium-derived relaxing factors, activated the outward calcium-dependent potassium conductance. PMA and PDBu, but not PMM, reduced the outward conductance induced by cyclopiazonic acid and LP-805. These effects of PMA and PDBu on potassium currents may be mediated either by phosphorylation of ion channels, or by decreasing intracellular calcium concentration.

  11. ADAM10 Is the Major Sheddase Responsible for the Release of Membrane-associated Meprin A*

    PubMed Central

    Herzog, Christian; Haun, Randy S.; Ludwig, Andreas; Shah, Sudhir V.; Kaushal, Gur P.

    2014-01-01

    Meprin A, composed of α and β subunits, is a membrane-bound metalloproteinase in renal proximal tubules. Meprin A plays an important role in tubular epithelial cell injury during acute kidney injury (AKI). The present study demonstrated that during ischemia-reperfusion-induced AKI, meprin A was shed from proximal tubule membranes, as evident from its redistribution toward the basolateral side, proteolytic processing in the membranes, and excretion in the urine. To identify the proteolytic enzyme responsible for shedding of meprin A, we generated stable HEK cell lines expressing meprin β alone and both meprin α and meprin β for the expression of meprin A. Phorbol 12-myristate 13-acetate and ionomycin stimulated ectodomain shedding of meprin β and meprin A. Among the inhibitors of various proteases, the broad spectrum inhibitor of the ADAM family of proteases, tumor necrosis factor-α protease inhibitor (TAPI-1), was most effective in preventing constitutive, phorbol 12-myristate 13-acetate-, and ionomycin-stimulated shedding of meprin β and meprin A in the medium of both transfectants. The use of differential inhibitors for ADAM10 and ADAM17 indicated that ADAM10 inhibition is sufficient to block shedding. In agreement with these results, small interfering RNA to ADAM10 but not to ADAM9 or ADAM17 inhibited meprin β and meprin A shedding. Furthermore, overexpression of ADAM10 resulted in enhanced shedding of meprin β from both transfectants. Our studies demonstrate that ADAM10 is the major ADAM metalloproteinase responsible for the constitutive and stimulated shedding of meprin β and meprin A. These studies further suggest that inhibiting ADAM 10 activity could be of therapeutic benefit in AKI. PMID:24662289

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

    PubMed

    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.

  13. Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M.

    PubMed Central

    Malik, N; Kallestad, J C; Gunderson, N L; Austin, S D; Neubauer, M G; Ochs, V; Marquardt, H; Zarling, J M; Shoyab, M; Wei, C M

    1989-01-01

    Oncostatin M is a polypeptide of Mr approximately 28,000 that acts as a growth regulator for many cultured mammalian cells. We report the cDNA and genomic cloning, sequence analysis, and functional expression in heterologous cells of oncostatin M. cDNA clones were isolated from mRNA of U937 cells that had been induced to differentiate into macrophagelike cells by treatment with phorbol 12-myristate 13-acetate, and a genomic clone was also isolated from human brain DNA. Sequence analysis of these clones established the 1,814-base-pair cDNA sequence as well as exon boundaries. This sequence predicted that oncostatin M is synthesized as a 252-amino-acid polypeptide, with a 25-residue hydrophobic sequence resembling a signal peptide at the N terminus. The predicted oncostatin M amino acid sequence shared no homology with other known proteins, but the sequence of the 3' noncoding region of the cDNA contained an A + T-rich stretch with sequence motifs found in the 3' untranslated regions of many cytokine and lymphokine cDNAs. Oncostatin M mRNA of approximately 2 kilobase pairs was detected in phorbol 12-myristate 13-acetate-treated U937 cells and in activated human T cells. Transfection of cDNA encoding the oncostatin M precursor into COS cells resulted in the secretion of proteins with the structural and functional properties of oncostatin M. The unique amino acid sequence, expression by lymphoid cells, and growth-regulatory activities of oncostatin M suggest that it is a novel cytokine. Images PMID:2779549

  14. Serotonin-induced muscle contraction in rat stomach fundus is mediated by a G alpha z-like guanine nucleotide binding protein.

    PubMed

    Wang, H Y; Eberle-Wang, K; Simansky, K J; Friedman, E

    1993-11-01

    Serotonin (5-HT) potently contracts the fundus of the rat stomach; however, the associated transduction pathway has not been described fully. Experiments were performed in an attempt to gain insight into the coupling mechanism associated with this fundal 5-HT receptor. 5-HT-stimulated [35S]GTP gamma S binding to a protein which was recognized by anti-G alpha Z antiserum in a Mg(++)-dependent fashion. 5-HT increased [35S]GTP gamma S binding in the fundus, but not in the corpus of the rat stomach. 5-HT also enhanced the binding of [alpha-32P]GTP to the fundal protein and increased the hydrolysis of GTP to GDP in fundal membranes. The fundal protein which binds GTP is 25 to 29 kDa in size whereas the brain G alpha Z protein which is recognized by the anti-G alpha Z antibody is a 41 kDa protein. Mixing experiments revealed that the fundal guanine nucleotide binding protein does not appear to be a proteolytic product of the 41 kDa G alpha Z protein. Activating protein kinase C with phorbol-12-myristate, 13-acetate induced a concentration-dependent, noncompetitive inhibition of [35S]GTP gamma S binding to the fundal protein, and of 5-HT-induced contraction of fundal strips. Phorbol-12-myristate, 13-acetate did not alter carbachol- or KCl-mediated fundus contraction. Furthermore, the activation of [35S]GTP gamma S binding by serotonergic agonists and its inhibition by pharmacological antagonists corresponded to the known actions of these agents on contraction of fundal muscle. The results provide evidence that the 5-HT receptor in the rat stomach fundus is coupled directly or indirectly to a G alpha z-like protein which may mediate 5-HT-induced contraction in this tissue.

  15. Differentiation of human pituitary adenomas determines the pattern of chromogranin/secretogranin messenger ribonucleic acid expression.

    PubMed

    Jin, L; Chandler, W F; Smart, J B; England, B G; Lloyd, R V

    1993-03-01

    The distribution of chromogranin/secretogranin (Cg/Sg) mRNAs, determined by Northern and in situ hybridization, was analyzed in 14 cultured pituitary adenomas characterized by immunohistochemistry and hormone secretion in a defined medium in vitro. There were 5 functional GH adenomas, 1 silent GH adenoma, 7 null cell adenomas, and 1 oncocytoma. The null cell adenomas, oncocytoma, and silent GH adenomas were also analyzed by electron microscopy. Most null cell adenomas and the oncocytoma secreted FSH and LH into the culture medium. GH adenomas, which are examples of well differentiated tumors based on morphological examination, expressed significantly more SgIII mRNA compared to the null cell adenomas and oncocytoma (70 +/- 6% vs. 22 +/- 5%; P < 0.001). GH adenomas also expressed significantly less CgA mRNA compared to the less well differentiated null cell adenomas and oncocytoma (27 +/- 6% vs. 67 +/- 4%; P < 0.001), which could be considered less well differentiated based on ultrastructural morphological features. After treatment with phorbol 12-myristate 13-acetate (10(-7) M) for 7 days, there was an increase in the mRNA for CgB and SgII mRNAs in GH and null cell tumors, while dexamethasone treatment for 7 days increased CgA mRNA in GH and null cell adenomas. GnRH treatment for 7 days increased CgB mRNA in null cell adenomas. Phorbol 12-myristate 13-acetate also decreased the percentage of immunoreactive GH cells and GHm RNA, determined by in situ and Northern hybridization analyses. These results indicate that pituitary adenomas have a distinct pattern of Cg/Sg mRNA expression, which appears to be related to the degree of morphological differentiation of these neoplasms, and suggest that the effects of secretagogues on various Cg/Sg mRNA levels may be related to the stimulation of hormone secretion.

  16. Induction of c-fos and c-myc mRNA by epidermal growth factor or calcium ionophore is cAMP dependent.

    PubMed Central

    Ran, W; Dean, M; Levine, R A; Henkle, C; Campisi, J

    1986-01-01

    Phorbol esters activate protein kinase C and induce expression of the c-fos and c-myc protooncogenes in density-arrested BALB/c 3T3 (A31) cells; in contrast, epidermal growth factor (EGF) does not activate protein kinase C and is a poor inducer of c-fos and c-myc in these confluent cells. We show that, when A31 cells were subconfluent and made quiescent by serum deprivation, the phorbol ester phorbol 12-myristate 13-acetate induced c-fos and c-myc mRNA poorly, whereas EGF was a better inducer. Another platelet-derived growth factor-inducible gene, JE, did not show this differential regulation by phorbol 12-myristate 13-acetate and EGF. The ability of EGF to induce protooncogene mRNA was associated with elevated levels of intracellular cAMP. First, serum-deprived cells maintained cAMP at about 2-fold higher level than density-arrested cells. Second, induction was greatly enhanced by cholera toxin and 3-isobutyl-1-methylxanthine, which increased intracellular cAMP 3- to 10-fold. The calcium ionophore A23187 mimicked EGF in that it elevated c-fos and c-myc mRNA when administered with cholera toxin and isobutylmethylxanthine. Neither cholera toxin and isobutyl-methylxanthine nor A23187 appreciably induced these mRNAs when used alone. Our results suggest that c-fos and c-myc expression can be regulated by an EGF-directed pathway that utilizes calcium and cAMP as cooperating cytoplasmic messengers. Images PMID:2430281

  17. Selective incorporation of ( sup 15 S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: Agonist-induced deacylation and transformation of stored hydroxyeicosanoids

    SciTech Connect

    Brezinski, M.E.; Serhan, C.N. )

    1990-08-01

    The uptake and mobilization of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a major product of arachidonic acid metabolism, was examined with human neutrophils. Upon exposure to labeled 15-HETE, PMNs rapidly (15 sec to 20 min) incorporated approximately 20% of the label into phosphatidylinositol, while less than 4% was associated with other phospholipid classes and neutral lipids. This pattern was distinct from that of either labeled arachidonate or labeled(5S)-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid (5-HETE), which within 20 min were predominantly associated with triglycerides and phosphatidylcholine. After reversed-phase HPLC, greater than 98% of the label in phosphatidylinositol, isolated from PMNs, was released with phospholipase A2. Upon exposure to either chemotactic peptide (FMLP), phorbol 12-myristate 13-acetate, or an ionophore (A23187), 15-HETE-labeled PMNs released 15-HETE from phosphatidylinositol and displayed an impaired ability to generate leukotriene B4 (LTB4), 20-OH-LTB4, and 20-COOH-LTB4. Deacylated (3H)15-HETE was converted to (5S,15S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid (5,15-DHETE), lipoxin A4, and lipoxin B4, each carrying 3H label. PMNs labeled with 5-HETE also released and transformed this HETE when stimulated. However, the profile of labeled products differed between PMNs with either esterified 15-HETE or 5-HETE. When activated, 5-HETE-labeled PMNs generated both 5,20-DHETE and 5,15-DHETE but not labeled lipoxins. Threshold aggregation induced by FMLP with 15-HETE-labeled PMNs was inhibited, while the threshold response was relatively unimpaired with either A23187 or phorbol 12-myristate 13-acetate-induced aggregation. Results indicate that 15-HETE is esterified into phosphatidylinositol of PMNs, which can be mobilized and transformed upon exposure of the cells to a second signal.

  18. Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms

    PubMed Central

    Bhattacharyya, Samarjit; Puri, Sapna; Miledi, Ricardo; Panicker, Mitradas M.

    2002-01-01

    Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT2 receptor subtypes increase IP3 levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT2A-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT2A receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT2A receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT2A receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT2A receptors are recycled. PMID:12388782

  19. Insulin reverses the growth retardation effect of phorbol ester in chicken embryos during organogenesis

    SciTech Connect

    Girbau, M.; Bassas, L.; Roth, J.; de Pablo, F. )

    1989-01-01

    The tumor promoting phorbol esters can affect early embryonic development by causing interference with the normal pathways of cellular growth and differentiation. The present study was designed to: (a) define a time in organogenesis when a vertebrate embryo model, the chicken, was sensitive to the phorbol ester 12-0-tetradecanoil-13-acetate (TPA), and (b) attempt a rescue of the embryos disturbed by TPA with simultaneous addition of insulin. In embryos treated at days 2 and 3 of development, TPA caused dose-dependent mortality. Survivors were biochemically retarded as indicated by their decreased weight, protein, DNA, RNA, total creatine kinase, triglycerides, phospholipids and cholesterol contents. When intermediated doses of TPA were applied together with insulin the embryonic growth disturbance was largely antagonized. These data, generated with an in vivo whole embryo, support the strong link between the mode of action of insulin and signal transduction mechanisms typical of phorbol esters.

  20. Terminal differentiation-resistant epidermal cells in mice undergoing two-stage carcinogenesis.

    PubMed

    Miller, D R; Viaje, A; Aldaz, C M; Conti, C J; Slaga, T J

    1987-04-01

    We have used an in vivo-in vitro approach to investigate the cellular aspects of two-stage skin carcinogenesis. Female SENCAR mice initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were promoted twice weekly with 12-O-tetradecanoylphorbol-13-acetate (TPA). Epidermal cultures from untreated or TPA-treated mice had few focus-forming cells resistant to calcium-induced terminal differentiation. Cultures from mice treated with MNNG alone formed numerous foci. Brief promotion (four TPA treatments) of MNNG-treated mice produced fewer but statistically larger foci, suggesting that TPA was selecting against more slowly growing cells. MNNG plus TPA-treated mice with very early papillomas produced more and larger foci than those due to MNNG treatment alone, suggesting that the papillomas may have comprised calcium-resistant cells. These cells may indeed be initiated cells since a permanent cell line arising after MNNG plus brief TPA treatment eventually formed histological papillomas in vivo. If calcium-resistant cells are initiated, then there were many more initiated cells in the skin (with or without TPA treatment) than papillomas expected, implying that either some initiated cells never formed papillomas, or that a significant accumulation of initiated cells had already occurred in the skin within 2 weeks of MNNG treatment. Subsequent TPA promotion of these cells apparently produced a toxic response that passively selected for more rapidly growing initiated cells, which eventually accumulated into papillomas.

  1. Inhibitory Effects of Gymnema (Gymnema sylvestre) Leaves on Tumour Promotion in Two-Stage Mouse Skin Carcinogenesis.

    PubMed

    Yasukawa, Ken; Okuda, Sakiko; Nobushi, Yasuhito

    2014-01-01

    Ethanol extracts of gymnema (Gymnema sylvestre) leaves exhibited marked antitumour-promoting activity in an in vivo two-stage carcinogenesis test in mice using 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. From the active fraction of the ethanol extract of the gymnema leaves, three triterpenoids were isolated and identified. These compounds were evaluated for their inhibitory effects on TPA-induced inflammation (1 µg/ear) in mice. The tested compounds showed marked anti-inflammatory effects, with a 50% inhibitory dose of 50-555 nmol/ear.

  2. Inhibitory Effects of Gymnema (Gymnema sylvestre) Leaves on Tumour Promotion in Two-Stage Mouse Skin Carcinogenesis

    PubMed Central

    Yasukawa, Ken; Okuda, Sakiko; Nobushi, Yasuhito

    2014-01-01

    Ethanol extracts of gymnema (Gymnema sylvestre) leaves exhibited marked antitumour-promoting activity in an in vivo two-stage carcinogenesis test in mice using 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. From the active fraction of the ethanol extract of the gymnema leaves, three triterpenoids were isolated and identified. These compounds were evaluated for their inhibitory effects on TPA-induced inflammation (1 µg/ear) in mice. The tested compounds showed marked anti-inflammatory effects, with a 50% inhibitory dose of 50–555 nmol/ear. PMID:24734106

  3. Discovering a new analogue of thalidomide which may be used as a potent modulator of TNF-alpha production.

    PubMed

    Fernández Braña, Miguel; Acero, Nuria; Añorbe, Loreto; Muñoz Mingarro, Dolores; Llinares, Francisco; Domínguez, Gema

    2009-09-01

    A new series of imide derivatives related to thalidomide were synthesized and evaluated as modulators of TNF-alpha production. These derivatives enhance TNF-alpha production using human leukemia HL-60 cells induced with 12-O-tetradecanoylphorbol 13-acetate (TPA), while inhibiting TNF-alpha production induced with okadaic acid (OA) in the same cell line. The diphenylmaleimide derivative 2f, was found to be the most active product, producing a strong modulation of the cytokine level.

  4. 12-tetradecanoyl-phorbol-13-acetate (PMA) produces injury to isolated rat lungs in the presence and absence of perfused neutrophils

    SciTech Connect

    Carpenter, L.J.; Roth, R.A.

    1986-03-01

    PMA produced injury to isolated, perfused rat lungs when eutrophils were added to or omitted from the buffer/albumin perfusion medium. When a high dose of PMA (57 ng/ml) was added to medium devoid of added neutrophils, perfusion pressure and lung weight increased. Together, superoxide dismutase (500 U/ml) and catalase (400 U/ml) had no effect on the increases in lung weight or perfusion pressure. However, papaverine (0.5 mM) prevented both the increase in perfusion pressure and fluid accumulation. When a concentration of PMA (14 ng/ml) that did not by itself cause lungs to accumulate fluid was added to perfusion medium containing neutrophils (1 x 10/sup 8/), perfusion pressures increased and lungs accumulated fluid. This concentration of PMA stimulated neutrophils (1 x 10/sup 8/) to release superoxide. Addition of superoxide dismutase (500 U/ml) and catalase (400 U/ml) to this medium prevented the increase in lung weight, but not the increase in perfusion pressure. Papaverine (0.5 mM) attenuated the increase in perfusion pressure and prevented fluid accumulation in these lungs. In summary, high concentrations of PMA produce lung injury which is independent of oxygen radicals; at lower concentrations it produces injury which is neutrophil-dependent and mediated by oxygen radicals.

  5. Tumor promoting phorbol diesters: substrates for diacylglycerol lipase

    SciTech Connect

    Cabot, M.C.

    1984-08-30

    Enzyme activity in rat serum was examined utilizing the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and various glycerolipids as substrates. The serum activity was specific for hydrolysis of the long chain tetradecanoate moiety of TPA, hydrolyzed mono- and diacylglycerols, but was not effective against triacylglycerols, cholesterylesters, or phospholipids. Heating the enzyme preparation at 56/sup 0/C for 1 min was dually effective in reducing the hydrolysis of both TPA and dioleoylglycerol by 83-86% of control levels. The potent diacylglycerol lipase inhibitor, RHC 80267, inhibited the hydrolysis of TPA in the 0.2-1.0 ..mu..M range and was also a potent blocker of monoacyl- and diacylglycerol hydrolysis. In substrate competition studies, exogenous unlabeled TPA was added to the (/sup 14/C)dioleoylglycerol-containing reaction mixture, however, this produced an approximate 3-fold stimulation of (/sup 14/)dioleoylglycerol hydrolysis. Although we have not established whether the hydrolysis of TPA and diacylglycerol is the work of one enzyme, the effectiveness of the specific lipase inhibitor, RHC 80267, demonstrates that diacylglycerol lipase can utilize TPA as substrate, a finding never before documented. This point is of interest in light of the theory that phorbol esters act by mimicry of the natural lipid mediator, diacylglycerols. 44 references, 3 figures, 1 table.

  6. Induction of terminal differentiation-resistant epidermal cells in mouse skin and in papillomas by different initiators during two-stage carcinogenesis.

    PubMed

    Miller, D R; Viaje, A; Rotstein, J; Aldaz, C M; Conti, C J; Slaga, T J

    1989-01-15

    Carcinogen treatment of normal mouse epidermal cells causes some cells, if cultured under the appropriate conditions, to continue to proliferate instead of terminally differentiate, forming foci at 37 degrees C in medium with a calcium level above 0.1 mM. We have examined these Calcium (Ca)-resistant cells formed in the skin of SENCAR mice after treatment with the carcinogen initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although in our previous studies TPA promotion initially increased the size but reduced the number of foci caused by the carcinogen initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), TPA promotion of DMBA-treated mice increased the size but had no effect on the number of foci. Papillomas resulting from DMBA plus TPA treatment contained many rapidly growing Ca-resistant cells, corroborating our earlier results with MNNG. Permanent cell lines prepared from papilloma-derived foci formed squamous cell carcinomas in nude mice after relatively short periods in culture. These data provide further evidence that Ca-resistant cells may be papilloma (and perhaps carcinoma) precursors in vivo. In addition, since TPA tends to reduce the number of early Ca-resistant cells caused by MNNG but not by DMBA, this may at least partially explain why treatment with DMBA plus TPA is much more effective in producing papillomas in SENCAR mice than is treatment with MNNG plus TPA.

  7. Dissociation of mitogenesis and late-stage promotion of tumor cell phenotype by phorbol esters: mitogen-resistant variants are sensitive to promotion.

    PubMed Central

    Colburn, N H; Wendel, E J; Abruzzo, G

    1981-01-01

    The JB-6 mouse epidermal cell line has been used as a model system for studying the mechanism of late-stage promoter-dependent preneoplastic progression. The studies reported here are concerned with determining whether there is a requirement for mitogenic stimulation in promotion of anchorage independence and tumorigenicity in JB-6 cells. Such a requirement would predict that variants selected for 12-O-tetradecanoylphorbol 13-acetate (TPA) mitogen resistance would also be promotion resistant. Promotion-responsive cell lines have been selected for resistance to TPA-induced mitogenic stimulation at plateau density by cotreatment with colchicine and removal of mitogen-responsive colchicine-detached cells. The selected TPA-resistant population of cells showed a mitogenic response that was diminished by a factor of four but showed no diminution in the promotion-of-anchorage-independence response to TPA. Mitogen-resistant clonal lines derived from the selected population fell into three phenotypic classes when assayed in soft agar: (i) anchorage-independent transformants; (ii) variants resistant to promotion of anchorage independence by TPA; and (iii) variants sensitive to promotion by TPA. The existence of the latter class (i.e., the mitogen-resistant promotable variants) indicates a lack of obligatory causal relationship between TPa-induced mitogenesis and late-stage promotion and, thereby suggests that the two events can be dissociated. Images PMID:6947266

  8. Is Intravenous Heparin a Contraindication for TPA in Ischemic Stroke?

    PubMed Central

    Deol, Nisha; Tolly, Renee; Manocha, Rohan; Naseer, Maliha

    2017-01-01

    There are approximately 2 million cardiac catheterizations that occur every year in the United States and with an aging population this number continues to rise. Adverse events due to this procedure occur at low rates and include stroke, arrhythmia, and myocardial infarctions. Due to the high volume of procedures there are a growing number of adverse events. Stroke after cardiac catheterization (SCC) has an incidence between 0.27 and 0.5% and is one of the most debilitating complications leading to high rates of mortality and morbidity. Given the relatively uncommon clinical setting of stroke after cardiac catheterization, treatment protocols regarding the use of IV or IA thrombolysis have not been adequately developed. Herein, we describe a case of a 39-year-old male who developed a stroke following a cardiac catheterization where IV thrombolysis was utilized although the patient was on heparin prior to cardiac catheterization. PMID:28261510

  9. Stroke, tPA, and Physician Decision-Making

    MedlinePlus

    ... uncer- tainty of diagnostic imaging procedures influenced their decision-making, but this study was unable to identify the source for physician uncertainty. All respondents reported having witnessed the “ Lazarus effect ” during their career, which is charac- terized by rapid recovery after ...

  10. SecureCore Software Architecture: Trusted Path Application (TPA) Requirements

    DTIC Science & Technology

    2007-12-01

    Cynthia E. Irvine Timothy E. Levin Thuy D. Nguyen Timothy M. Vidas December 2007...Timothy M. Vidas Research Associate Reviewed by: Released by: ______________________________ _________________ Peter...Levin, Cynthia E. Irvine, and Thuy D. Nguyen, Timothy M. Vidas 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES

  11. Tumor promoters alter the temporal program of adenovirus replication in human cells.

    PubMed Central

    Fisher, P B; Young, C S; Weinstein, I B; Carter, T H

    1981-01-01

    In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences. Images PMID:6965103

  12. Tumor promoters alter the temporal program of adenovirus replication in human cells.

    PubMed

    Fisher, P B; Young, C S; Weinstein, I B; Carter, T H

    1981-04-01

    In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

  13. Origin of tumor-promoter released fibronectin in fibroblasts

    SciTech Connect

    Burrous, B.A.; Wolf, G.

    1986-05-01

    Previous work from the laboratory showed that the chemical tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated release of the cell surface glycoprotein, fibronectin (FN) from human lung fibroblasts (HLF), leading to depletion of cell surface FN, while FN synthesis is not altered by TPA. To further investigate the mechanism(s) by which TPA stimulates FN release, two types of experiments were performed. In the first, HLF were pulsed with /sup 35/S-methionine-labeled medium with or without TPA. In the second, cell-surface proteins were labeled by iodination (/sup 125/I) and then incubated in unlabeled medium with or without TPA. In both cases, the fate of labeled FN was followed over 12 hr. The /sup 35/S-meth-labeled HLF showed a rapid loss of labeled FN, first into a small, highly-labeled pool of cell surface FN (1 hr), later into the medium (4 hr or longer). Specific activities showed that this small pool in the cell surface turned over rapidly. TPA treatment resulted in more rapid movement of /sup 35/S-meth pulse-labeled FN to the cell surface and into the medium than in control cells. TPA thus affected the fate of intracellular FN. TPA treatment of HLF also resulted in more rapid removal of /sup 125/I-cell surface-labeled FN into the medium than in control cells. Thus, TPA affects the fate of preexisting cell surface FN in HLF. From these results, they hypothesize that TPA has two separate effects: it stimulates depletion of preexisting intracellular FN during the first hr of treatment, and it stimulates release of preexisting cell surface FN over all treatment times.

  14. Studies on the mechanism of skin tumor promotion: Evidence for several stages in promotion

    PubMed Central

    Slaga, T. J.; Fischer, S. M.; Nelson, K.; Gleason, G. L.

    1980-01-01

    The effects of nonpromoting and weakly promoting diterpenes on skin tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) were investigated. When phorbol and phorbol 12,13-diacetate (both nonpromoting) were given simultaneously with TPA after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female mice, they had no effect on TPA promotion. However, the nonpromoter 4-O-methyl-TPA and the weak promoter mezerein were found to inhibit TPA promotion in a dose-dependent manner when given simultaneously with TPA. Because mezerein was found to be an effective inhibitor of TPA promotion when given simultaneously and because it induces many biological responses similar to those to TPA, the capacity of mezerein to act as an incomplete promoter in a two-stage promotion protocol was also investigated. Twice-weekly applications of 1,2, or 5 μg of TPA for 2 weeks after DMBA initiation produced 0, 0, and 0.5 papilloma per mouse, respectively, at 20 weeks. When the twice-weekly applications of TPA for 2 weeks were followed by twice-weekly treatments with 2 μg of mezerein for 18 weeks, the number of papillomas per mouse was 2.2, 3.5, and 9.0, respectively. Twice-weekly applications of 2 μg of TPA for 2 weeks followed by twice-weekly treatments with 1, 2, or 4 μg of mezerein for 18 weeks produced 2.1, 3.5, and 6.8 papillomas per mouse, respectively, in DMBA-treated mice. Twice-weekly doses as high as 40 μg of 4-O-methyl-TPA were not effective in producing tumors when given after a limited treatment with TPA; however, 4-O-methyl-TPA had weak activity as a first-stage promoter. The results suggest that although mezerein by itself is a weak promoter and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen. PMID:6774342

  15. Protein kinase C and the antiviral effect of human interferon.

    PubMed

    Cernescu, C; Constantinescu, S N; Baltă, F; Popescu, L M; Cajal, N

    1989-01-01

    Protein kinase C (PKC) inhibitors: Hidaka's compounds H-7 (10 microM) and H-8 (20 microM), palmitoyl-carnitine (10 microM) and phloretin (50 microM), did not modify the antiviral effect of human natural or recombinant interferon alpha and of natural interferon beta. The tumor promoter 12-o-tetradecanoyl-phorbol-13-acetate (TPA) (200 nM), known as activator of PKC induced an antiviral state when tested on human embryo fibroblasts challenged with the vesicular stomatitis virus. The battery of PKC inhibitors used inhibited the antiviral effect induced by TPA. Palmitoyl-carnitine (10 microM) exerted a toxic effect that was reversed by interferon treatment (2,000 IU/ml interferon alpha). These results suggest that PKC, possibly activated by interferon-receptor interaction, is not essential for inducing the antiviral effect of interferon, but, probably, mediates the antiviral effect of TPA.

  16. Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes

    PubMed Central

    1985-01-01

    We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O- tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation. PMID:3156868

  17. Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents.

    PubMed

    Nakagawa-Goto, Kyoko; Yamada, Koji; Taniguchi, Masahiko; Tokuda, Harukuni; Lee, Kuo-Hsiung

    2009-07-01

    C-3 esterifications of betulinic acid (BA, 1) and its A-ring homolog, ceanothic acid (CA, 2), were carried out to provide sixteen terpenoids, 4-19, including nine new compounds (4-12). All synthesized compounds were evaluated in an in vitro antitumor-promoting assay using the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among them, compounds 4-6, 11-14, 16, and 17 displayed remarkable inhibitory effects of EBV-EA activation. BA analog 6, which contains a prenyl-like group, showed the most potent inhibitory effect (100%, 76%, 37%, and 11% inhibition of EBA activation at 1000, 500, 100, and 10mol ratio/TPA, respectively, with IC(50) value of 285mol ratio/32pmol TPA). Compound 6 merits further development as a cancer preventive agent.

  18. A-ring modified betulinic acid derivatives as potent cancer preventive agents.

    PubMed

    Hung, Hsin-Yi; Nakagawa-Goto, Kyoko; Tokuda, Harukuni; Iida, Akira; Suzuki, Nobutaka; Bori, Ibrahim D; Qian, Keduo; Lee, Kuo-Hsiung

    2014-02-01

    Ten new 3,4-seco betulinic acid (BA) derivatives were designed and synthesized. Among them, compounds 7-15 exhibited enhanced chemopreventive ability in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus early antigen (EBV-EA) activation assay in Raji cells. Specifically, analogs with a free C-28 carboxylic acid, including 7, 8, 11, and 13, inhibited EBV-EA activation significantly. The most potent compound 8 displayed 100% inhibition at 1×10(3) mol ratio/TPA and 73.4%, 35.9%, and 8.4% inhibition at 5×10(2), 1×10(2), and 1×10 mol ratio/TPA, respectively, comparable with curcumin at high concentration and better than curcumin at low concentration. The potent chemopreventive activity of novel seco A-ring BAs (8 and 11) was further confirmed in an in vivo mouse skin carcinogenesis assay.

  19. Effect of phorbol esters on iron uptake in human hematopoietic cell lines

    SciTech Connect

    Testa, U.; Titeux, M.; Louache, F.; Thomopoulos, P.; Rochant, H.

    1984-11-01

    We have investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on iron uptake into human hematopoietic cell lines K562, U937, and HL-60. TPA inhibited both cell growth and iron uptake by these cell lines. This effect was rapid, which is typical of phorbol esters which are biologically active, and it occurred at very low concentrations of TPA. This effect of TPA was dependent upon an inhibition of the transferrin-binding capacity as estimated on intact cells. However, experiments with transferrin binding on cell samples dissolved in 1% Triton X-100 showed that TPA-treated cells exhibited a transferrin-binding capacity similar to that of control cells. On the basis of this result, it is suggested that TPA modified a part of transferrin receptors present in the cells; as a result of this modification, these receptors became unavailable for binding transferrin, but they remained physically present in the cell. Other compounds capable of inducing the differentiation of leukemic cells, such as dimethyl sulfoxide, butyrate, retinoic acid, and 1 alpha,25-dihydroxy-vitamin D3, did not acutely inhibit iron uptake. We also investigated the effect of TPA on transferrin receptors in a cellular system in which phorbol esters stimulate cell proliferation. At 16 X 10(-9) M, TPA markedly stimulated the proliferation of T-lymphocytes. However, in spite of this marked stimulation of cell proliferation, TPA-stimulated lymphocytes exhibited a transferrin-binding capacity much inferior to cells stimulated by other mitogens, such as phytohemagglutinin.

  20. Multiple effects of phorbol esters on hormone-sensitive adenylate cyclase activity in S49 lymphoma cells

    SciTech Connect

    Bell, J.D.; Brunton, L.L.

    1987-06-01

    In S49 lymphoma cells, 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhances adenylate cyclase activity and doubles cAMP accumulation in response to ..beta..-adrenergic stimulation at 37/sup 0/C, putatively via the action of protein kinase C. at 27/sup 0/C, TPA has the opposite effect, inhibiting cAMP production in response to isoproterenol by approx. 25%. TPA also inhibits the response to prostaglandin E/sub 1/ (PGE/sub 1/), another stimulant of hormone-sensitive adenylate cyclase in these cells, by 30% at 37/sup 0/C and almost 50% at 27/sup 0/C. In contrast, TPA enhances responses to forskolin and cholera toxin at both 27 and 37/sup 0/C. In membranes from cells treated with TPA, PGE/sub 1/-stimulated adenylate cyclase activity is inhibited by 50%, whereas the catalytic activity stimulated by NaF or forskolin is enhanced. TPA reduces the potency of both PGE/sub 1/ and isoproterenol for cAMP generation by 50%. TPA causes a similar decrease in ..beta..-adrenergic agonist affinity with no reduction in the density of either antagonist of agonist binding sites in wild type cells and in cells lacking the ..cap alpha..-subunit of the stimulatory transducer protein (G/sub s/) (cyc/sup -/) or lacking functional receptor G/sub s/ coupling (UNC). Therefore, TPA has at least three functionally distinct effects on hormone-sensitive adenylate cyclase in S49 cells. The authors conclude that multiple and opposing effects of TPA on hormone-sensitive adenylate cyclase occur simultaneously within the same cell, affecting the responses to several agonists differently. In addition, the data offer a mechanism by which a cell can achieve heterogeneous efficacies to hormones that activate adenylate cyclase.

  1. Interaction between human peripheral blood monocytes and tumor promoters: Effect on growth differentiation and function in vitro

    SciTech Connect

    Keisari, Y.; Bucana, C.; Markovich, S.; Campbell, D.E. )

    1990-08-01

    Studies on the differentiation and activation of human monocytes in tissue cultures have usually been limited by the deterioration of human monocytes and macrophages in long-term cultures. In this study, we attempted to establish long-term human monocyte/macrophage cultures using the phorbol ester 12-0 tetradecanoyl-phorbol-13-acetate (TPA), and we studied the morphology, function, and biochemical properties of such treated human blood monocytes. Enriched suspensions of monocytes were obtained using Ficoll-Hypaque gradient and cultured in the absence or presence of various concentrations of TPA. Samples were removed at different times and processed for scanning electron microscopy. Parallel samples were examined for numbers of adherent cells, phagocytosis, oxidative burst, beta-galactosidase assays, and lectin-mediated erythrolysis. TPA-treated monocytes survived in larger numbers in culture for up to 7 weeks and were more pleomorphic and exhibited higher beta-galactosidase activities after 14 days in culture than untreated monocytes. TPA-treated cells and untreated cells in long-term cultures showed a decrease in their oxidative burst activity while their phagocytic activity was not affected, and the TPA treatment augmented the lysis of wheat germ agglutinin-opsonized erythrocytes by the cultured monocytes. TPA treatment of adherent human monocytes resulted in cell cultures with increased numbers of viable and functionally adherent cells for extended periods of time and does not seem to interfere with the differentiation and maturation of the cells in culture.

  2. Differential effects of fibroblast growth factor and tumor promoters on the initiation and maintenance of adipocyte differentiation

    PubMed Central

    1989-01-01

    Fibroblast growth factor (FGF) has been shown to inhibit the differentiation of myogenic and adipogenic cell lines without inducing a proliferative response. We have previously shown that agents capable of activating protein kinase C (PKC), such as FGF and the phorbol ester tetradecanoyl phorbol-13-acetate (TPA), inhibit the differentiation of the adipogenic cell line TA1, as measured by the rapid loss of adipocyte-specific RNAs. We report here that the treatment of fully differentiated TA1 adipocytes with FGF at 10 ng/ml induces the reversal of adipocyte differentiation, even in cells where PKC activity has been down-regulated by TPA pretreatment. In contrast, TPA or lower concentrations of FGF (1 ng/ml), both effective inducers of c-fos RNA in adipocytes, fail to reverse adipocyte differentiation. The adipocytes, however, will extinguish differentiation-specific functions in response to TPA by the addition of a calcium ionophore. Therefore, we propose that there are two FGF-sensitive pathways in TA1 cells: one responsible for the initiation of differentiation (TPA sensitive) and another required for maintenance of the adipose phenotype (TPA insensitive). These results suggest that activation of two distinct signaling pathways--one PKC and calcium dependent, the other FGF activated but PKC independent--are capable of inhibiting the biochemical events responsible for the maintenance of adipocyte differentiation. PMID:2507555

  3. Involvement of the antioxidative property of morusin in blocking phorbol ester-induced malignant transformation of JB6 P(+) mouse epidermal cells.

    PubMed

    Cheng, Pai-Shan; Hu, Chao-Chin; Wang, Chau-Jong; Lee, Yean-Jang; Chung, Wei-Chia; Tseng, Tsui-Hwa

    2017-02-25

    Chemoprevention has been acknowledged as an important and practical strategy for managing cancer. We have previously synthesized morusin, a prenylated flavonoid that exhibits anti-cancer progression activity. In the present study, we evaluated the anti-cancer promotion potential of morusin by using the mouse epidermal JB6 P(+) cell model. Extensive evidence shows that tumor promotion by phorbol esters is due to the stimulation of reactive oxygen species (ROS). Therefore, the effect of morusin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ROS production was assessed. Noncytotoxic concentrations of morusin were found to dose-dependently reduce TPA-induced ROS production. Moreover, morusin inhibited TPA-induced activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) activation, which can mediate cell proliferation and malignant transformation. Furthermore, morusin inhibited the TPA upregulation of cyclooxygenase 2 (COX-2), which may be regulated by AP-1 and NF-κB. In addition, noncytotoxic concentrations of morusin reduced the TPA-promoted cell growth of JB6 P(+) cells and inhibited TPA-induced malignant properties, such as cytoskeletal rearrangement and cell migration of JB6 P(+) cells. Similar to the effects of glutathione (GSH) pretreatment, morusin inhibited TPA-induced expression of N-cadeherin and vimentin, which are malignant cell surface proteins. Finally, morusin treatment dose-dependently suppressed the TPA-induced anchorage-independent cell transformation of JB6 P(+) cells. In conclusion, our results evidence that morusin possesses anti-cancer promotion potential because of its antioxidant property, which mediates multiple transformation-associated gene expression.

  4. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

    PubMed

    Takayama, S; White, M F; Kahn, C R

    1988-03-05

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

  5. Fibronectin Fragment Activation of Proline-rich Tyrosine Kinase PYK2 Mediates Integrin Signals Regulating Collagenase-3 Expression by Human Chondrocytes through a Protein Kinase C-dependent Pathway*

    PubMed Central

    Loeser, Richard F.; Forsyth, Christopher B.; Samarel, Allen M.; Im, Hee-Jeong

    2010-01-01

    Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the α5β1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the α5β1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCδ inhibitor rottlerin. Furthermore, PKCδ translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after co-transfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves

  6. Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic β cells.

    PubMed

    Santo-Domingo, Jaime; Chareyron, Isabelle; Dayon, Loïc; Núñez Galindo, Antonio; Cominetti, Ornella; Pilar Giner Giménez, María; De Marchi, Umberto; Canto, Carles; Kussmann, Martin; Wiederkehr, Andreas

    2017-03-01

    Mitochondria play a central role in pancreatic β-cell nutrient sensing by coupling their metabolism to plasma membrane excitability and insulin granule exocytosis. Whether non-nutrient secretagogues stimulate mitochondria as part of the molecular mechanism to promote insulin secretion is not known. Here, we show that PKC signaling, which is employed by many non-nutrient secretagogues, augments mitochondrial respiration in INS-1E (rat insulinoma cell line clone 1E) and human pancreatic β cells. The phorbol ester, phorbol 12-myristate 13-acetate, accelerates mitochondrial respiration at both resting and stimulatory glucose concentrations. A range of inhibitors of novel PKC isoforms prevent phorbol ester-induced respiration. Respiratory response was blocked by oligomycin that demonstrated PKC-dependent acceleration of mitochondrial ATP synthesis. Enhanced respiration was observed even when glycolysis was bypassed or fatty acid transport was blocked, which suggested that PKC regulates mitochondrial processes rather than upstream catabolic fluxes. A phosphoproteome study of phorbol ester-stimulated INS-1E cells maintained under resting (2.5 mM) glucose revealed a large number of phosphorylation sites that were altered during short-term activation of PKC signaling. The data set was enriched for proteins that are involved in gene expression, cytoskeleton remodeling, secretory vesicle transport, and exocytosis. Interactome analysis identified PKC, C-Raf, and ERK1/2 as the central phosphointeraction cluster. Prevention of ERK1/2 signaling by using a MEK1 inhibitor caused a marked decreased in phorbol 12-myristate 13-acetate-induced mitochondrial respiration. ERK1/2 signaling module therefore links PKC activation to downstream mitochondrial activation. We conclude that non-nutrient secretagogues act, in part, via PKC and downstream ERK1/2 signaling to stimulate mitochondrial energy production to compensate for energy expenditure that is linked to β-cell activation

  7. Effect of acute and chronic excesses of dietary nitrogen on blood neutrophil functions in cattle.

    PubMed

    Raboisson, D; Caubet, C; Tasca, C; De Marchi, L; Ferraton, J M; Gannac, S; Millet, A; Enjalbert, F; Schelcher, F; Foucras, G

    2014-12-01

    phorbol 12-myristate 13-acetate was not modified, in contrast to OZ stimulation. Decreased ROS production during chronic EDN probably involves the early events leading to ROS production, as OZ acts through membrane receptors and phorbol 12-myristate 13-acetate directly activates protein kinase C. This is the first study to provide evidence that the modifications of neutrophil functions produced by excess nitrogen depend on the intensity and duration of the excess. Further studies, including epidemiological studies during risk periods, are needed to resolve the issues linked to EDN.

  8. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons.

    PubMed

    Cabell, L; Audesirk, G

    1993-06-01

    A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every

  9. Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids.

    PubMed Central

    Craven, P A; Pfanstiel, J; DeRubertis, F R

    1987-01-01

    Deoxycholate (DOC), chenodeoxycholate, 12-O-tetradecanoyl phorbol-13-acetate (TPA), or 1-oleoyl-2-acetyl-glycerol (OAG) activated colonic epithelial protein kinase C as reflected by translocation from the soluble to the particulate cell fraction. Activation of protein kinase C was correlated with stimulation of enhanced proliferative activity of colonic mucosa and reactive oxygen production. TPA and OAG, but not DOC, directly activated soluble protein kinase C in vitro. However, DOC rapidly increased labeled inositol phosphate and diacylglycerol accumulation in colonic epithelial cells. Retinoic acid inhibited protein kinase C activity and suppressed DOC-, TPA-, and OAG-induced increases in reactive oxygen production. The results support a role for protein kinase C in the stimulation of colonic epithelial proliferative activity and reactive oxygen production induced by bile acids, TPA and OAG. In contrast to TPA and OAG, which activate protein kinase C directly, bile acids appear to activate protein kinase C indirectly by increasing the diacylglycerol content of colonic epithelium. PMID:3027128

  10. Phorbol ester-inducible T-cell-specific expression of variant mouse mammary tumor virus long terminal repeats

    SciTech Connect

    Theunissen, H.J.M.; Paardekooper, M.; Maduro, L.J.; Michalides, R.J.A.M.; Nusse, R. )

    1989-08-01

    Acquired proviruses of mouse mammary tumor virus (MMTV) in T-cell leukemias of male GR mice have rearrangements in the U3 region of their long terminal repeats (LTR). In contrast to the endogenous nonrearranged MMTV proviruses, these mutated copies are highly expressed in leukemic T cells. To investigate whether the sequence alterations in the LTR are responsible for the high expression of rearranged MMTV proviruses, the authors made constructs in which normal and variant LTRs drive the bacterial reporter gene chloramphenicol acetyltransferase (CAT). Two different rearranged LTRs were used, one containing a 420-base-pair (bp) deletion (L13) and another carrying a 456-bp deletion plus an 82-bp insertion (L42). These constructs were transfected into murine (GRSL) and human (MOLT-4) T-cell lines that either had or had not been treated with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)). In GRSL cells, the L13-LTR-CAT construct showed transcriptional activity that was further enhanced by TPA. In MOLT-4 cells, both variant LTRs were active, but only after stimulation with TPA. In contrast, normal(N)-LTR-CAT constructs were not expressed, irrespective of TPA addition. They conclude that the LTR rearrangements generate TPA responsiveness and contribute to T-cell-specific expression of MMTV variants.

  11. Effect of tannic acid, resveratrol and its derivatives, on oxidative damage and apoptosis in human neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Ignatowicz, Ewa; Krajka-Kuźniak, Violetta; Baer-Dubowska, Wanda

    2015-10-01

    In this study we compared the antioxidant and DNA protective activity of tannic acid and stilbene derivatives, resveratrol, 3,5,4(')-trimethoxystilbene (TMS) and pterostilbene in human neutrophils stimulated to oxidative burst by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in relation to apoptosis induction. All polyphenols within the concentration range 1-100 μM reduced the intracellular ROS and H2O2 production in the TPA-stimulated cells. Tannic acid was the most effective polyphenol in protection against DNA damage induced by TPA. In the resting neutrophils resveratrol and to lesser extent other polyphenols increased DNA damage and increased the level of p53. Pretreatment of the TPA-stimulated cells with tannic acid or stilbenes led to the induction of apoptosis. The most significant effect was observed as a result of treatment with TMS and resveratrol. These compounds appeared the most effective inducers of p53 in the TPA-challenged neutrophils, what may suggest that pro-apoptotic activity of these stilbenes might be related to p53 activation. Overall, the results of our present study demonstrate that tannic acid and stilbenes modulate the ROS production, ultimately leading to cell apoptosis in human neutrophils stimulated to oxidative burst. In resting neutrophils they exhibit pro-oxidant activity, which is accompanied by p53 induction.

  12. Interactions of promonocytic U937 cells with proteins of the extracellular matrix.

    PubMed Central

    Pucillo, C E; Colombatti, A; Vitale, M; Salzano, S; Rossi, G; Formisano, S

    1993-01-01

    Monocyte interaction with proteins of the extracellular matrix (ECM) is regulated by expression of specific cell-surface receptors. 12-O-tetradecanoyl phorbol-13-acetate (TPA) has been shown to induce the promonocytic cell line U937 to a more differentiated monocyte-like state. In this study we have analysed the attachment of U937 cells to ECM proteins and the effects of treatment with TPA on this process. Non-induced U937 cells attach to fibronectin- and Matrigel-coated surfaces without TPA stimulation, but TPA further increases adherence to these substrates as measured by an enhanced binding and by the lower concentration of proteins needed in the substrate to achieve 50% of maximal cell adhesion. Attachment to type I collagen was seen only with activated U937 cells, whereas no measurable attachment to bovine serum albumin, vitronectin, and type IV collagen was detected. TPA-activated U937 cells showed a two-fold increase in the expression of the RGD-dependent integrin receptors alpha 3 and alpha 5, and a reduction in the expression of alpha 4, another fibronectin-specific receptor, whereas the common beta 1 chain was unchanged. Attachment of U937 cells to fibronectin was primarily mediated by the alpha 3 and alpha 5 integrins, as revealed by the ability of GRGDS peptides to inhibit attachment, whereas the CS-1 peptide, containing the alpha 4 binding site, was largely ineffective in blocking attachment. PMID:8262552

  13. The preventive role of breadfruit against inflammation-associated epithelial carcinogenesis in mice.

    PubMed

    Lin, Jer-An; Chen, Hsiang-Chi; Yen, Gow-Chin

    2014-01-01

    Artocarpus communis has been identified as a rich source of flavonoids and has been gaining attention for its potential chemopreventive abilities. In this study, methanol extracts from the fruit of A. communis (MEFA) and leaf of A. communis (MELA) were prepared, and their effects on inflammation-associated skin tumorigenesis were assessed using mouse models, including 12-O-tetradecanoylphorbol-13-acetate (TPA) induced cutaneous inflammation as well as 7,12-dimethylbenz[α]anthracene (DMBA) initiated and TPA-promoted skin tumorigenesis. According to the results, both MEFA and MELA decreased the intensity of leukocyte infiltration in mouse dorsal skin and cutaneous edema induced by TPA, which appeared to be mediated by inhibition of proinflammatory genes (inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), IL-1β, and IL-6) and proinflammatory mediators (TNF-α, IL-1β, and Prostaglandin E2 ). In addition, topical application with MEFA or MELA effectively attenuated tumor incidence, multiplicity, volume, malignancy as well as angiogenesis of TPA-stimulated skin tumor promotion in DMBA-initiated mice. Notably, immunohistochemical stain showed that MEFA and MELA attenuated COX-2 expression of both skin and tumor tissues in different animal tests, which may be closely related to the suppression of nuclear factor kappa B/activator protein signaling networks. These findings first demonstrate that flavonoid-rich A. communis may exert potent anti-inflammatory activity through modulation of COX-2 in TPA-activated skin and tumor tissues.

  14. SENCAR mouse skin tumorigenesis model versus other strains and stocks of mice.

    PubMed Central

    Slaga, T J

    1986-01-01

    The SENCAR mouse stock was selectively bred for eight generations for sensitivity to skin tumor induction by the two-stage tumorigenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. The SENCAR mouse was derived by crossing Charles River CD-1 mice with skin-tumor-sensitive mice (STS). The SENCAR mice are much more sensitive to both DMBA tumor initiation and TPA tumor promotion than CD-1, BALB/c, and DBA/2 mice. An even greater difference in the sensitivity to two-stage skin tumorigenesis is apparent between SENCAR and C57BL/6 mice when using DMBA-TPA treatment. However, the SENCAR and C57BL/6 mice have a similar tumor response to DMBA-benzoyl peroxide treatment, suggesting that TPA is not an effective promoter in C57BL/6 mice. The DBA/2 mice respond in a similar manner to the SENCAR mice when using N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-TPA treatment. The SENCAR mouse model provides a good dose-response relationship for many carcinogens used as tumor initiators and for many compounds used as tumor promoter. When compared to other stocks and strains of mice, the SENCAR mouse has one of the largest data bases for carcinogens and promoters. PMID:3096709

  15. IDH1 Is Downregulated during Early Skin Tumorigenesis Which Can Be Inhibited by Overexpression of MnSOD

    PubMed Central

    Robbins, Delira; Wittwer, Jennifer A.; Codarin, Sarah; Circu, Magdalena L.; Aw, Tak Yee; Huang, Ting-Ting; VanRemmen, Holly; Richardson, Arlan; Wang, David B.; Witt, Stephan N.; Klein, Ronald L.; Zhao, Yunfeng

    2012-01-01

    SUMMARY Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme which converts isocitrate to alpha-ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development, are unclear. We utilized a skin cell transformation model, and, mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with DMBA/TPA also showed decreases in IDH1 expression and activity. In non-promotable JB6 P− cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed TPA-induced cell transformation. Finally, manganese superoxide dismutase (MnSOD) overexpression suppressed tumor promoter-induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha-ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress may contribute to IDH1 inactivation, because MnSOD, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity. PMID:22533343

  16. Tumor promotion by depleting cells of protein kinase C delta.

    PubMed Central

    Lu, Z; Hornia, A; Jiang, Y W; Zang, Q; Ohno, S; Foster, D A

    1997-01-01

    Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCdelta isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCdelta as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCdelta mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCdelta, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCdelta, which has an apparent tumor suppressor function. PMID:9154841

  17. Inhibition of ERK Oscillations by Ionizing Radiation and Reactive Oxygen Species

    SciTech Connect

    Shankaran, Harish; Chrisler, William B; Sontag, Ryan L; Weber, Thomas J

    2010-12-28

    The shuttling of activated protein kinases between the cytoplasm and nucleus is an essential feature of normal growth factor signaling cascades. Here we demonstrate that transforming growth factor alpha (TGFα) induces oscillations in extracellular signal regulated kinase (ERK) cytoplasmic-nuclear translocations in human keratinocytes. TGFα-dependent ERK oscillations mediated through the epidermal growth factor receptor (EGFR) are inhibited by low dose X-irradiation (10 cGy) and low concentrations of hydrogen peroxide (0.32–3.26 µM H2O2) used as a model reactive oxygen species (ROS). A fluorescent indicator dye (H2-DCFDA) was used to measure cellular ROS levels following X-irradiation, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and H2O2. X-irradiation did not generate significant ROS production while 0.32 µM H2O2 and TPA induced significant increases in ROS levels with H2O2 > TPA. TPA alone induced transactivation of the EGFR but did not induce ERK oscillations. TPA as a cotreatment did not inhibit TGFα-stimulated ERK oscillations but qualitatively altered TGFα-dependent ERK oscillation characteristics (amplitude, time-period). Collectively, these observations demonstrate that TGFα-induced ERK oscillations are inhibited by ionizing radiation/ROS and perturbed by epigenetic carcinogen in human keratinocytes. © 2010 Wiley-Liss, Inc.

  18. Crosstalk between Wnt signaling and Phorbol ester-mediated PKC signaling in MCF-7 human breast cancer cells.

    PubMed

    Kim, Soyoung; Chun, So-Young; Kwon, Yun-Suk; Nam, Kyung-Soo

    2016-02-01

    Although many studies have implicated the crosstalk between the Wnt and PKC signaling pathways in tumor initiation and progression, the molecular roles of PKC isoforms in the Wnt signaling pathway remain poorly understood. In this study, we explored the contribution of PKC isoforms to canonical and noncanonical Wnt signaling pathway in mediating cell migration and an epithelial-mesenchymal transition (EMT). When MCF-7 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 3 weeks, the effect of TPA on Wnt signaling pathway was dramatically different depending on the exposure time. The short term exposure (3 days) of MCF-7 cells to TPA exhibited significant induction of Wnt5a expression, along with the enhanced expression of PKC-α, to promote cell migration, which suggested that activation of noncanonical Wnt signaling pathway is associated with PKC-α. However, the chronic exposure (3 weeks) of cells to TPA completely suppressed Wnt5a expression and the expression of PKC-η and PKC-δ, whereas the expression of Wnt3a and PKC-θ were up-regulated to activate the canonical Wnt signaling pathway. Moreover, the loss of epithelial markers, including E-cadherin and GATA-3, suggested that chronic exposure of TPA stimulates EMT. Taken together, our data suggest that PKC-θ positively regulates the canonical Wnt signaling pathway, and that PKC-η and PKC-δ negatively modulate this signaling pathway.

  19. Basic fibroblast growth factor (bFGF) acts intracellularly to cause the transdifferentiation of avian neural crest-derived Schwann cell precursors into melanocytes.

    PubMed

    Sherman, L; Stocker, K M; Morrison, R; Ciment, G

    1993-08-01

    We previously found that cultured neural crest-derived cells from embryonic quail peripheral nerves, which consist mostly of Schwann cell precursors, gave rise to melanocytes following treatment with basic fibroblast growth factor (bFGF) or 12-O-tetradecanoyl phorbol-13-acetate (TPA). Here, we show that antisense deoxyoligonucleotides targeted against two regions of the bFGF mRNA transcript blocked this TPA-induced transdifferentiation of Schwann cell precursors. Neither sense nor scrambled antisense control oligonucleotides had any effect in this regard. TPA increased bFGF protein expression in cell lysates but not in conditioned media from these cultures, and this expression was localized to the nucleus and cytoplasm. Furthermore, bFGF-neutralizing antibodies and inositol-hexakisphosphate (InsP6) both inhibited pigmentation caused by exogenous bFGF, but had no affect on TPA-induced melanogenesis, suggesting that bFGF is not released by these cells. These data indicate that bFGF is necessary for the TPA-induced transdifferentiation of Schwann cell precursors into melanocytes and that bFGF acts via an intracrine mechanism.

  20. Immune phenotype and some enzyme patterns in phorbol ester-induced chronic lymphocytic leukemia cells.

    PubMed

    Babusíková, O; Mesárosová, A; Kusenda, J; Koníková, E; Klobusická, M; Hrivnáková, A

    1995-01-01

    Leukemic cells from 10 patients with B-chronic lymphocytic leukemia (B-CLL) were isolated and cultured in the presence of 12-0-tetradecanoylphorbol 13-acetate (TPA) at a concentration of 8 x 10(-7) mol for 72 hours. Cells were analyzed before cultivation and after 72 h of cultivation with and without TPA for changes in surface membrane (Sm) and cytoplasmic (cyt) markers expression, presence of receptor for mouse rosette forming cells (MRFC) and some enzyme profiles. All B-CLL cases studied showed typical B-cell phenotype. TPA treatment induced hairy cell leukemia (HCL) characteristics, given by the membrane CD22 and CD25 expression and TRAP positivity in the majority of the cases tested. Cells had hairy cell-like morphology with more intensive cytoplasmic immunoglobulin (CIg) fluorescence staining, absent receptor for MRFC and increased activity of purine nucleosidephosphorylase. In common these changes indicate that TPA can induce hairy cell characteristics on B-CLL cells in vitro suggesting the more mature differentiation stage of HCL compared with CLL. Furthermore, we originally demonstrated that the CD22, present in the cell membrane after TPA, could be detected in the majority of unaffected B-CLL cells in their cytoplasm. From the technical point of view some intracellular CD markers and Igs of B-CLL cells in viable cells in suspension assayed by flow cytometry are described in this study.

  1. Effect of phorbol esters on mitochondrial actions of glucagon

    SciTech Connect

    Cardellach, F.; Moehren, G.; Hoek, J.B.

    1987-05-01

    Glucagon generates different second messenger signals in liver. It increases cAMP levels and elevates cytosolic Ca/sup 2 +/ levels by degradation of polyphosphoinositides. The phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) inhibits glucagon-induced calcium mobilization, but not cAMP formation. TPA can thus be used to assess the role of Ca/sup 2 +/ and cAMP in the activation of mitochondrial processes. In isolated hepatocytes, glucagon increased the steady state NAD(P)H level, probably by activating mitochondrial Ca/sup 2 +/ dependent dehydrogenases. TPA inhibited the glucagon-induced NAD(P) reduction without affecting phosphorylase activation. The effects of glucagon and TPA on mitochondrial respiratory activity and calcium retention were tested after isolation of the mitochondria from perfused livers. Electron transport rates were increased by 15-25% and calcium retention time was increased four-fold after glucagon treatment. When livers were pretreated with TPA, glucagon had no effect on electron transport activity, but calcium retention was increased by the same factor. The results suggest that glucagon-induced calcium mobilization is required for the stimulation of the respiratory activity but not for the increased capacity to retain a calcium overload in the mitochondria.

  2. SENCAR mouse skin tumorigenesis model versus other strains and stocks of mice

    SciTech Connect

    Slaga, T.J.

    1986-09-01

    The SENCAR mouse stock was selectively bred for eight generations for sensitivity to skin tumor induction by the two-stage tumorigenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. The SENCAR mouse was derived by crossing Charles River CD-1 mice with skin-tumor-sensitive mice (STS). The SENCAR mice are much more sensitive to both DMBA tumor initiation and TPA tumor promotion than CD-1, BALB/c, and DBA/2 mice. An even greater difference in the sensitivity to two-stage skin tumorigenesis is apparent between SENCAR and C57BL/6 mice when using DMBA-TPA treatment. However, the SENCAR and C57BL/6 mice have a similar tumor response to DMBA-benzoyl peroxide treatment, suggesting that TPA is not an effective promoter in C57BL/6 mice. The DBA/2 mice respond in a similar manner to the SENCAR mice when using N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-TPA treatment. The SENCAR mouse model provides a good dose-response relationship for many carcinogens used as tumor initiators and for many compounds used as tumor promoter. When compared to other stocks and strains of mice, the SENCAR mouse has one of the largest data bases for carcinogens and promoters.

  3. Sapinmusaponins F-J, bioactive tirucallane-type saponins from the galls of Sapindus mukorossi.

    PubMed

    Huang, Hui-Chi; Tsai, Wei-Jern; Morris-Natschke, Susan L; Tokuda, Harukuni; Lee, Kuo-Hsiung; Wu, Yang-Chang; Kuo, Yao-Haur

    2006-05-01

    Five new tirucallane-type saponins, sapinmusaponins F-J (1-5), were isolated from the galls of Sapindus mukorossi. The structures of these saponins were elucidated on the basis of spectroscopic analysis including 1D and 2D NMR techniques ((1)H-(1)H COSY, HMQC, HMBC, TOCSY, and NOESY). Compounds 1-5 showed anti-platelet-aggregation effects, but no obvious cytotoxic activity for platelets as assayed by lactate dehydrogenase (LDH) leakage. Compounds 1-5 also showed moderate activity in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) activation assay.

  4. Water Quality Criteria for Colored Smokes: 1,4-Diamino-2,3- Dihydroanthraquinone

    DTIC Science & Technology

    1988-01-01

    fol- lowing: analytical and preparative scale thin- layer chromatography (TLC), visible, ultraviolet, and fluorescence spectrophotometry, capillary...methylaminoanthraquinone) NMR Nuclear Magnetic Resonance TLC Thin- Layer Chromatography TPA 12-O-Tetradecanoylphorbol-13-acetate USEPA United States Environmental...column 9 "r4 0-C Ř. 00 f-4 cc r4 r% 0% H 0 hrq4 rNNt V-4 ’.0 4) 0% Sn 1 2r 4o 04 11) IN S -44 a1. w 0 1- 4 0 0 ~ 4 44 0 H 01 4e Pll~ gas chromatography

  5. Phytochemical and antiinflammatory studies on Terminalia catappa.

    PubMed

    Fan, Y M; Xu, L Z; Gao, J; Wang, Y; Tang, X H; Zhao, X N; Zhang, Z X

    2004-06-01

    The antiinflammatory activity of Terminalia catappa leaves ethanolic extract was studied using 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in acute and chronic models. A bioassay-oriented fractionation procedure showed that the activity concentrates in the chloroform fraction. Ursolic acid (1) and 2alpha,3beta,23-trihydroxyurs-12-en-28-oic acid (2), isolated from the chloroform fraction, exhibited strong antiinflammatory activities. The results suggest that the triterpenic acids 1 and 2 are responsible for the antiinflammatory activity of T. catappa leaves.

  6. Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice

    SciTech Connect

    Solanki, V.; Slaga, T.J.

    1981-04-01

    The binding of (20-/sup 3/H)phorbol 12,13-dibutyrate ((/sup 3/H)PDB) to intact living epidermal cells in monolayer culture was characterized. At 37/sup 0/C, the maximum specific (/sup 3/H)PDB binding (binding displaceable by 30 ..mu..M unlabeled PDB) was attained in 15 to 20 min and was followed by a rapid decrease (down regulation) of radioactivity bound to the cells. The activity lost by the cells during this decrease was found in the incubation medium. Prior exposure of cells to phorbol 12-myristate 13-acetate (PMA; 12-O-tetradecanoylphorbol 13-acetate) but not to phorbol for 2 h at 37/sup 0/C caused approx. 55% reduction in the number of measurable binding sites for (/sup 3/H)PDB. The down regulation was temperature sensitive; there was no loss of radioactivity after 1 h at 4/sup 0/C. The specific binding of (/sup 3/H)PDB at 4/sup 0/C reached equilibrium in 15 to 20 min and was saturable and freely reversible. At equilibrium, epidermal cells contained 1.2 x 10/sup 5/ binding sites per cell, and binding sites had a K/sub D/ of 10 nM. Specificity of binding was shown by the observation that the biologically active phorbol esters PMA and 12-deoxyphorbol 13-decanoate inhibited the binding, whereas the inactive parent compound phorbol and the nonphorbol tumor promoter anthralin did not have any effect. The abilities of these compounds to inhibit (/sup 3/H)PDB binding directly correlates with their tumor promoting activities. Epidermal cells exposed to retinoic acid or fluocinolone acetonide for 24 h had similar (/sup 3/H)PDB binding characteristics as untreated cells suggesting that inhibition of tumor promotion induced by these compounds is not mediated through alterations in the phorbol ester binding sites.

  7. Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice.

    PubMed Central

    Solanki, V; Slaga, T J

    1981-01-01

    The binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDB) to intact living epidermal cells in monolayer culture was characterized. At 37 degrees C, the maximum specific [3H]PDB binding (binding displaceable by 30 microM unlabeled PDB) was attained in 15--20 min and was followed by a rapid decrease (down regulation) of radioactivity bound to the cells. The activity lost by the cells during this decrease was found in the incubation medium. Prior exposure of cells to phorbol 12-myristate 13-acetate (PMA; 12-O-tetradecanoylphorbol 13-acetate) but not to phorbol for 2 hr at 37 degrees C caused approximately 55% reduction in the number of measurable binding sites for [3H]PDB. The down regulation was temperature sensitive; there was no loss of radioactivity after 1 hr at 4 degrees C. The specific binding of [3H]PDB at 4 degrees C reached equilibrium in 15--20 min and was saturable and freely reversible. At equilibrium, epidermal cells contained 1.2 x 10(5) binding sites per cell, and binding sites had a KD of 10 nM. Specificity of binding was shown by the observation that the biologically active phorbol esters PMA and 12-deoxyphorbol 13-decanoate inhibited the binding, whereas the inactive parent compound phorbol and the nonphorbol tumor promoter anthralin did not have any effect. The abilities of these compounds to inhibit [3H]PDB binding directly correlates with their tumor promoting activities. Epidermal cells exposed to retinoic acid or fluocinolone acetonide for 24 hr had similar [3H]PDB binding characteristics as untreated cells suggesting that inhibition of tumor promotion induced by these compounds is not mediated through alterations in the phorbol ester binding sites. PMID:6941309

  8. Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

    SciTech Connect

    Onali, P.; Olianas, M.C.

    1987-03-23

    In rat striatal synaptosomes, 4..beta..-phorbol 12-myristate 13-acetate (PMA) and 4 ..beta..-phorbol 12,13-dibutyrate (PDBu), two activators of Ca/sup 2 +/-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C) tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 ..mu..M PMA and 1 ..mu..M PDBu. 4 ..beta..-Phorbol and 4 ..beta..-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 ..mu..M. PMA did not change the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C)DOPA. Addition of 1 mM EGTA to a Ca/sup 2 +/-free incubation medium failed to affect PMA stimulation. KCl (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KCl addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis. 37 references, 3 figures, 3 tables.

  9. Inhibition of carcinogen induced c-Ha-ras and c-fos proto-oncogenes expression by dietary curcumin

    PubMed Central

    Limtrakul, Porn-ngarm; Anuchapreeda, Songyot; Lipigorngoson, Suwiwek; Dunn, Floyd W

    2001-01-01

    Background We investigated the chemopreventive action of dietary curcumin on 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12,0-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor formation in Swiss albino mice. Curcumin, a yellow coloring matter isolated from roots of Curcuma longa Linn, is a phenolic compound possessing antioxidant, free radical scavenger, and antiinflammatory properties. It has been shown by previously reported work that TPA-induced skin tumors were inhibited by topical application of curcumin, and curcumin has been shown to inhibit a variety of biological activities of TPA. Topical application of curcumin was reported to inhibit TPA-induced c-fos, c-jun and c-myc gene expression in mouse skin. This paper reports the effects of orally administered curcumin, which was consumed as a dietary component at concentrations of 0.2 % or 1 %, in ad libitum feeding. Results Animals in which tumors had been initiated with DMBA and promoted with TPA experienced significantly fewer tumors and less tumor volume if they ingested either 0.2% or 1% curcumin diets. Also, the dietary consumption of curcumin resulted in a significantly decreased expression of ras and fos proto-oncogenes in the tumorous skin, as measured by enhanced chemiluminesence Western blotting detection system (Amersham). Conclusions Whereas earlier work demonstrated that topical application of curcumin to mouse skin inhibited TPA-induced expression of c-fos, c-jun and c-myc oncogenes, our results are the first to show that orally consumed curcumin significantly inhibited DMBA- and TPA-induced ras and fos gene expression in mouse skin. PMID:11231886

  10. PKCα activation down-regulates ATM and radio-sensitizes androgen-sensitive human prostate cancer cells in vitro and in vivo

    PubMed Central

    Truman, Jean-Philip; Rotenberg, Susan A.; Kang, Ji-Hye; Lerman, Gabriel; Fuks, Zvi; Kolesnick, Richard; Marquez, Victor E.; Haimovitz-Friedman, Adriana

    2009-01-01

    We previously demonstrated that treatment of human androgen-responsive prostate cancer cell lines LNCaP and CWR22-Rv1 with 12-O-tetradecanoylphorbol 13-acetate (TPA), a known protein kinase C (PKC) activator, decreases ATM protein levels, thus de-repressing the enzyme ceramide synthase (CS) and promoting apoptosis as well as radio-sensitizing these cells.1 Here we show that PKCα mediates the TPA effect on ATM expression, since ATM suppression and apoptosis induced by either TPA or diacylglycerol-lactone (DAG-lactone), both inducing PKCα activation,2 are abrogated in LNCaP cells following transfection of a kinase-dead PKCα mutant (KD-PKCα). Similarly, KD-PKCα blocks the apoptotic response elicited by combination of TPA and radiation, whereas expression of constitutively active PKCα is sufficient to sensitize cells to radiation alone, without a need to pre-treat the cells with TPA. These findings identify CS activation as a downstream event of PKCα activity in LNCaP cells. Similar results were obtained in CWR22-Rv1 cells with DAG-lactone treatment. Using the LNCaP orthotopic prostate model it is shown that treatment with TPA or DAG-lactone induces significant reduction in tumor ATM levels coupled with tumor growth delay. Furthermore, while fractionated radiation alone produces significant tumor growth delay, pretreatment with TPA or DAG-lactone significantly potentiates tumor cure. These findings support a model in which activation of PKCα downregulates ATM, thus relieving CS repression by ATM and enhancing apoptosis via ceramide generation. This model may provide a basis for the design of new therapies in prostate cancer. PMID:19029835

  11. Inflammation, oxidative DNA damage, and carcinogenesis.

    PubMed Central

    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 thought 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. Many studies support the multistep nature of carcinogenesis, and a significant amount of evidence indicates that more than one genetic event is necessary for neoplastic transformation. Selective growth stimulation of initiated cells by TPA does not explain how further genetic events may occur by chronic exposure to this nongenotoxic agent. We 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 H2O2 and oxidized lipid products. Furthermore, macrophage populations that release both H2O2 and metabolites of arachidonic acid (AA) are more efficient at inducing oxidative DNA damage in surrounding cells than populations which only release H2O2 or metabolites of AA. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin than C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H2O2 and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice.(ABSTRACT TRUNCATED AT 250 WORDS) Images FIGURE 8. A FIGURE 8. B PMID:3129286

  12. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    PubMed Central

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Background Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. Methods PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Results Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. Conclusion PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration. PMID:19077250

  13. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  14. Effects of combined phytochemicals on skin tumorigenesis in SENCAR mice

    PubMed Central

    KOWALCZYK, MAGDALENA C.; JUNCO, JACOB J.; KOWALCZYK, PIOTR; TOLSTYKH, OLGA; HANAUSEK, MARGARET; SLAGA, THOMAS J.; WALASZEK, ZBIGNIEW

    2013-01-01

    The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans. PMID:23835587

  15. Effects of combined phytochemicals on skin tumorigenesis in SENCAR mice.

    PubMed

    Kowalczyk, Magdalena C; Junco, Jacob J; Kowalczyk, Piotr; Tolstykh, Olga; Hanausek, Margaret; Slaga, Thomas J; Walaszek, Zbigniew

    2013-09-01

    The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans.

  16. Protein kinase C and regulation of the local competence of Xenopus ectoderm

    SciTech Connect

    Otte, A.P.; Kramer, I.M.; Durston, A.J. )

    1991-02-01

    The limited competence of embryonic tissue to respond to an inductive signal has an essential, regulatory function in embryonic induction. The molecular basis for the competence of Xenopus ectoderm to differentiate into neural tissue was investigated. Dorsal mesoderm or 12-O-tetradecanoyl phorbol-13-acetate (TPA) caused in vivo activation of protein kinase C (PKC) and neural differentiation mainly in dorsal ectoderm and to a lesser extent in ventral ectoderm. These data correlate with the observations that PKC preparations from dorsal and ventral ectoderm differ, the dorsal PKC preparation being more susceptible to activation by TPA and diolein than is the ventral PKC preparation. Monoclonal antibodies against the bovine PKC {alpha} plus {beta} or {gamma} isozymes immunostained dorsal and ventral ectoderm, respectively, which suggests different localizations of PKC isozymes. These results suggest that PKC participates in the establishment of embryonic competence.

  17. Anti-carcinogenic activity of Taraxacum plant. I.

    PubMed

    Takasaki, M; Konoshima, T; Tokuda, H; Masuda, K; Arai, Y; Shiojima, K; Ageta, H

    1999-06-01

    An extract of the roots of Taraxacum japonicum (Compositae) exhibited strong anti-tumor-promoting activities on the two-stage carcinogenesis of mouse skin tumor induced by dimethylbenz[a] anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter, as well as on that induced by DMBA and fumonisin B1. Further, the extract exhibited anti-tumor-initiating activity on the two-stage carcinogenesis of mouse skin tumor induced by (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexen amide (NOR-1) as an initiator and TPA as a promoter. These results suggested that an extract of the roots of the Taraxacum plant could be a valuable chemopreventive agent against chemical carcinogenesis.

  18. Anti-carcinogenic activity of Taraxacum plant. II.

    PubMed

    Takasaki, M; Konoshima, T; Tokuda, H; Masuda, K; Arai, Y; Shiojima, K; Ageta, H

    1999-06-01

    Eleven triterpenoids (1-11) from the roots of Taraxacum japonicum (Compositae) were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetrade-canoylphorbol-13-acetate (TPA), in Raji cells as a primary screening test for anti-tumor-promoters (cancer chemopreventive agents). Of these triterpenoids, taraxasterol (1) and taraxerol (7) exhibited significant inhibitory effects on EBV-EA induction, but the inhibitory effects of their acetates 2 and 8 were weaker than those of 1 and 7. Furthermore, 1 and 7 exhibited potent anti-tumor-promoting activity in the two-stage carcinogenesis tests of mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter, and 1 showed a remarkable inhibitory effect on mouse spontaneous mammary tumors using C3H/OuJ mouse. These results strongly suggested that taraxasterol (1) could be a valuable chemopreventive agent.

  19. Modulation of the estrogen receptor structure, evidence of a heterogeneity

    SciTech Connect

    Toulas, C.; Guilbaud, N.; Delassus, F.; Bayard, F.; Faye, J.C. )

    1990-01-01

    In order to analyse the molecular weight polymorphism of the estrogen receptor (ER) in MCF-7 cells, we have developed a procedure which allowed in situ linkage of ER by (3H) tamoxifen aziridine and provided labelled proteins in conditions which minimized protease activities. After labelling, cell lysis was performed in SDS buffer containing various concentrations of mercaptoethanol. Proteins extracted with phenolic solution and precipitated by cold acetone were analysed by SDS PAGE. It appears that beside the form of 67 kDa already described, binding entities of tamoxifen aziridine were also present at a molecular mass of 110 kDa and 45 kDa. On the other hand, investigations on the effect of 12-0-Tetradecanoyl Phorbol 13-Acetate (TPA) showed that TPA induces a decrease of the 67 kDa entity.

  20. Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation.

    PubMed Central

    Filvaroff, E; Stern, D F; Dotto, G P

    1990-01-01

    Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation. Images PMID:1689456

  1. Influence of activating hormones on human platelet membrane Ca/sup 2 +/-ATPase activity

    SciTech Connect

    Resink, T.J.; Dimitrov, D.; Stucki, S.; Buehler, F.R.

    1986-07-16

    Intact platelets were pretreated with hormones and thereafter membranes were prepared and Ca/sup 2 +/-ATPase activity determined. Thrombin decreased the V/sub max/ of Ca/sup 2 +/-ATPase after pretreatment of intact platelets. Platelet activating factor, vasopressin and ADP also decreased Ca/sup 2 +/-ATPase activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) or A23187 or ionomycin alone had no effect, while the simultaneous pretreatment with TPA and Ca/sup 2 +/-ionophore decreased Ca/sup 2 +/-ATPase activity. cAMP elevating agents prostaglandin E/sub 1/ (PGE/sub 1/) and forskolin had no influence per se on Ca/sup 2 +/-ATPase, but antagonized the inhibitory effect of thrombin. The data suggest a close connection between phosphoinositide metabolism and the Ca/sup 2 +/-ATPase system.

  2. Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

    SciTech Connect

    Xun, C.Q.; Ensor, C.M.; Tai, H.H. )

    1991-06-28

    Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

  3. Two overlapping sequence motifs within the polyomavirus enhancer are independently the targets of stimulation by both the tumor promoter 12-O-tetradecanoylphorbol-3-acetate and the Ha-ras oncogene

    SciTech Connect

    Yamaguchi, Yyko; Satake, Masanobu; Ito, Yoshiaki

    1989-03-01

    A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly stimulates the activity of polyomavirus enhancer in a human erythroleukemia cell line, K562. The target of stimulation was the previously defined A element (from nucleotides 5107 to 5130) of the enhancer. The authors found that within the A element, two partly overlapping sequence motifs (one from nucleotides 5107 to 5117, the other from nucleotides 5113 to 5121) were independently the targets of TPA stimulation. The former is homologous to the enhancer core sequence of the adenovirus type 5 E1A gene, and the latter shares the consensus AP-1-binding site. In addition, transiently expressed Ha-ras oncogene also stimulated these two subelements in K562 cells, as they reported for NIH 3T3 cells previously.

  4. Topical anti-inflammatory activity of 2alpha-hydroxy pentacyclic triterpene acids from the leaves of Ugni molinae.

    PubMed

    Aguirre, María C; Delporte, Carla; Backhouse, Nadine; Erazo, Silvia; Letelier, María Eugenia; Cassels, Bruce K; Silva, Ximena; Alegría, Sergio; Negrete, Rosa

    2006-08-15

    Leaf extracts of Ugni molinae Turcz. are used in the Chilean cosmetic industry on the assumption that they have decongestant, regenerative, and anti-aging properties. A bioassay-guided fractionation of this plant material showed that some extracts have potent anti-inflammatory activities. Further fractionation led to the isolation and identification of betulinic acid, a mixture of ursolic and oleanolic acids, and the 2alpha-hydroxy derivatives alphitolic, asiatic, and corosolic acids. The latter three were evaluated in vivo in the mouse ear assay for their topical anti-inflammatory activity, inducing inflammation with either arachidonic acid (AA) or 12-O-tetradecanoylphorbol-13 acetate (TPA). Only corosolic acid was active in the AA assay, with similar potency to nimesulide, but all three triterpene acids inhibited TPA-induced inflammation with potencies comparable to that of indomethacin.

  5. Epidermal Expression of Intercellular Adhesion Molecule 1 is Not a Primary Inducer of Cutaneous Inflammation in Transgenic Mice

    NASA Astrophysics Data System (ADS)

    Williams, Ifor R.; Kupper, Thomas S.

    1994-10-01

    Keratinocytes at sites of cutaneous inflammation have increased expression of intercellular adhesion molecule 1 (ICAM-1), a cytokine-inducible adhesion molecule which binds the leukocyte integrins LFA-1 and Mac-1. Transgenic mice were prepared in which the expression of mouse ICAM-1 was targeted to basal keratinocytes by using the human K14 keratin promoter. The level of constitutive expression attained in the transgenic mice exceeded the peak level of ICAM-1 expression induced on nontransgenic mouse keratinocytes in vitro by optimal combinations of interferon γ and tumor necrosis factor α or in vivo by proinflammatory stimuli such as phorbol 12-myristate 13-acetate. In vitro adhesion assays demonstrated that cultured transgenic keratinocytes were superior to normal keratinocytes as a substrate for the LFA-1-dependent binding of mouse T cells, confirming that the transgene-encoded ICAM-1 was expressed in a functional form. However, the high level of constitutive ICAM-1 expression achieved on keratinocytes in vivo in these transgenic mice did not result in additional recruitment of CD45^+ leukocytes into transgenic epidermis, nor did it elicit dermal inflammation. Keratinocyte ICAM-1 expression also did not potentiate contact-hypersensitivity reactions to epicutaneous application of haptens. The absence of a spontaneous phenotype in these transgenic mice was not the result of increased levels of soluble ICAM-1, since serum levels of soluble ICAM-1 were equal in transgenic mice and controls. We conclude that elevated ICAM-1 expression on keratinocytes cannot act independently to influence leukocyte trafficking and elicit cutaneous inflammation.

  6. Bioassay-guided isolation and identification of anti-platelet-active compounds from the root of Ashitaba (Angelica keiskei Koidz.).

    PubMed

    Son, Dong Ju; Park, Ye Oak; Yu, Chengguang; Lee, Sung Eun; Park, Young Hyun

    2014-01-01

    Platelet aggregation is fundamental to a wide range of physiological and pathological processes, including the induction of thrombosis and arteriosclerosis. Anti-platelet activity of a crude methanol extract and solvent fractions of Ashitaba roots (Angelica keiskei Koidz.) was evaluated using a turbidimetric method using washed rabbit platelets. We identified the anti-platelet activities of two chalcones, 4-hydroxyderricin and xanthoangelol, isolated from the ethyl acetate-soluble fraction of Ashitaba roots by using a bioassay-guided isolation method. 4-Hydroxyderricin and xanthoangelol effectively inhibited platelet aggregation induced by collagen (IC50 of 41.9 and 35.9 μM, respectively), platelet-activating factor (IC50 of 46.1 and 42.3 μM, respectively) and phorbol 12-myristate 13-acetate (IC50 of 16.5 and 45.9 μM, respectively). These compounds did not inhibit thrombin-induced platelet aggregation (IC50 of>80 μM). The results suggest that the chalcones 4-hydroxyderricin and xanthoangelol may be potent anti-thrombotic components of A. keiskei Koidz.

  7. Persistent induction of c-fos and c-jun expression by asbestos

    SciTech Connect

    Heintz, N.H.; Mossman, B.T. ); Janssen, Y.M. Univ. of Limburg, Maastricht )

    1993-04-15

    To investigate the mechanisms of asbestos-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in rat pleural mesothelial cells and hamster tracheal epithelial cells after exposure to crocidolite or chrysotile asbestos. In contrast to phorbol 12-myristate 13-acetate, which induces rapid and transient increases in c-fos and c-jun mRNA, asbestos causes 2- to 5-fold increases in c-fos and c-jun mRNA that persist for at least 24 hr in mesothelial cells. The induction of c-fos and c-jun mRNA by asbestos in mesothelial cells is dose-dependent and is most pronounced with crocidolite, the type of asbestos most pathogenic in the causation of pleural mesothelioma. Induction of c-jun gene expression by asbestos occurs in tracheal epithelial cells but is not accompanied by a corresponding induction of c-fos gene expression. In both cell types, asbestos induces increases in protein factors that bind specifically to the DNA sites that mediate gene expression by the AP-1 family of transcription factors. The persistent induction of AP-1 transcription factors by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of the early response gene pathway that includes c-jun and/or c-fos. 30 refs., 5 figs.

  8. Fc gamma receptor type III (CD16) is included in the zeta NK receptor complex expressed by human natural killer cells.

    PubMed Central

    Anderson, P; Caligiuri, M; O'Brien, C; Manley, T; Ritz, J; Schlossman, S F

    1990-01-01

    We recently reported that CD3- natural killer (NK) cells express the zeta chain of the T-cell receptor complex (zeta NK) in association with higher molecular weight structures whose expression differs between individual NK cell clones. Because NK cell cytolytic activity is known to be triggered by perturbation of the type III Fc gamma receptor (CD16), we sought to determine whether this activating molecule is included in the zeta NK molecular complex. Biochemical evidence for a physical association between CD16 and zeta NK was obtained by comparing immunoprecipitates formed using monoclonal antibodies reactive with each of these molecules by SDS/polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping. In both clonal and polyclonal populations of CD3- NK cells, CD16 and zeta NK specifically associated with one another. Functional evidence for a specific association between CD16 and zeta NK in intact cells was obtained by demonstrating a coordinate down-modulation of both of these molecules induced by either phorbol 12-myristate 13-acetate or monoclonal antibodies reactive with CD16. Our results suggest that Fc gamma receptor type III (CD16) is included in the zeta NK complex and that this complex is likely to play an important role in NK cell activation. Images PMID:2138330

  9. Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

    PubMed Central

    Reiser, H; Freeman, G J; Razi-Wolf, Z; Gimmi, C D; Benacerraf, B; Nadler, L M

    1992-01-01

    We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex. Images PMID:1370349

  10. In vitro effects of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Dobrowolska-Zachwieja, Agnieszka; Grajek, Włodzimierz

    2009-01-01

    Oxidative stress and inflammation are involved in the development of obesity. Beetroot (Beta vulgaris var. rubra) is a food ingredient containing betalain pigments that show antioxidant activity. The in vitro effect of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals has been investigated. Fifteen obese women (aged 45 +/- 9 years, BMI >30 kg/m2) and nine healthy controls (women, aged 29 +/- 11 years, BMI = 22.2 +/- 1.6 kg/m2) were examined. The investigated products were used as concentrates and after transport and digestion in an artificial gastrointestinal tract. Neutrophil oxidant production, in response to phorbol 12-myristate 13-acetate, was characterized by luminol-dependent chemiluminescence and a flow cytometric dichlorofluorescin oxidation assay. Caspase-3 activity, a marker of apoptosis, was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Neutrophils from obese individuals had a significantly higher ROS production compared with the controls (p < 0.05). Beetroot products inhibited neutrophil oxidative metabolism in a concentration-dependent manner. Also observed were the pro-apoptotic effects of beetroot at a concentration range of 0.1-10% in 24 h culture of stimulated neutrophils. These natural products (in both the liquid and solid state) have antioxidant and antiinflammatory capacity, and could be an important adjunct in the treatment of obesity.

  11. The beetroot component betanin modulates ROS production, DNA damage and apoptosis in human polymorphonuclear neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Kostrzewa, Artur; Łuczak, Michał; Jagodziński, Paweł P; Baer-Dubowska, Wanda

    2012-06-01

    The aim of this study was to evaluate the effect of betanin, one of the beetroot major components, on ROS production, DNA damage and apoptosis in human resting and stimulated with phorbol 12-myristate13-acetate polymorphonuclear neutrophils, one of the key elements of the inflammatory response. Incubation of neutrophils with betanin in the concentration range 2-500 µM resulted in significant inhibition of ROS production (by 15-46%, depending on the ROS detection assay). The antioxidant capacity of betanin was most prominently expressed in the chemiluminescence measurements. This compound decreased also the percentage of DNA in comet tails in stimulated neutrophils, but only at the 24 h time point. In resting neutrophils an increased level of DNA in comet tails was observed. Betanin did not affect the activity of caspase-3, in resting neutrophils, but significantly enhanced the enzyme activity in stimulated neutrophils. The western blot analysis showed, however, an increased level of caspase-3 cleavage products as a result of betanin treatment both in resting and stimulated neutrophils. The results indicate that betanin may be responsible for the effect of beetroot products on neutrophil oxidative metabolism and its consequences, DNA damage and apoptosis. The dose and time dependent effects on these processes require further studies.

  12. Anti-allergic effect of the naturally-occurring conjugated linolenic acid isomer, jacaric acid, on the activated human mast cell line-1

    PubMed Central

    LIU, WAI NAM; LEUNG, KWOK NAM

    2015-01-01

    The present study aimed to investigate the immunomodulatory effect of jacaric acid, a naturally-occurring conjugated linolenic acid isomer that can be found in jacaranda seed oil, on the activated human mast cell line-1 (HMC-1). Our previous studies have demonstrated that jacaric acid only exerted minimal, if any, cytotoxicity on normal murine cells. In the present study, jacaric acid at concentrations ≤100 µM did not exhibit direct cytotoxicity on human peripheral blood mononuclear cells after 72 h of incubation, as determined by the MTT reduction assay. By contrast, jacaric acid could alleviate the calcium ionophore A23187 and phorbol 12-myristate 13-acetate-triggered allergic response in the HMC-1 cells at concentrations that were non-cytotoxic to the HMC-1 cells. Following pre-treatment with jacaric acid, the secretion of two inflammatory mediators, β-N-acetylglucosaminidase and tryptase, as well as the T helper 2 cytokines [interleukin (IL)-4 and IL-13] was significantly reduced in HMC-1 cells. The alleviation of allergic response was accompanied by downregulation of the matrix metalloproteinase-2 and −9 proteins and upregulation of the tissue inhibitor of metalloproteinase-1 protein. Collectively, the results indicated that the naturally-occurring jacaric acid exhibits a suppressive effect on the allergic response in activated human mast cells in vitro, and this could not be attributed to the direct cytotoxicity of jacaric acid on the treated cells. PMID:26623027

  13. Anti-allergic effect of the naturally-occurring conjugated linolenic acid isomer, jacaric acid, on the activated human mast cell line-1.

    PubMed

    Liu, Wai Nam; Leung, Kwok Nam

    2015-11-01

    The present study aimed to investigate the immunomodulatory effect of jacaric acid, a naturally-occurring conjugated linolenic acid isomer that can be found in jacaranda seed oil, on the activated human mast cell line-1 (HMC-1). Our previous studies have demonstrated that jacaric acid only exerted minimal, if any, cytotoxicity on normal murine cells. In the present study, jacaric acid at concentrations ≤100 µM did not exhibit direct cytotoxicity on human peripheral blood mononuclear cells after 72 h of incubation, as determined by the MTT reduction assay. By contrast, jacaric acid could alleviate the calcium ionophore A23187 and phorbol 12-myristate 13-acetate-triggered allergic response in the HMC-1 cells at concentrations that were non-cytotoxic to the HMC-1 cells. Following pre-treatment with jacaric acid, the secretion of two inflammatory mediators, β-N-acetylglucosaminidase and tryptase, as well as the T helper 2 cytokines [interleukin (IL)-4 and IL-13] was significantly reduced in HMC-1 cells. The alleviation of allergic response was accompanied by downregulation of the matrix metalloproteinase-2 and -9 proteins and upregulation of the tissue inhibitor of metalloproteinase-1 protein. Collectively, the results indicated that the naturally-occurring jacaric acid exhibits a suppressive effect on the allergic response in activated human mast cells in vitro, and this could not be attributed to the direct cytotoxicity of jacaric acid on the treated cells.

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

    PubMed Central

    Rupasinghe, H. P. Vasantha; Boehm, Mannfred M. A.; Sekhon-Loodu, Satvir; Parmar, Indu; Bors, Bob; Jamieson, Andrew R.

    2015-01-01

    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. PMID:26043379

  15. Reactive oxygen species mediate phorbol ester-stimulated cAMP response in human eosinophils.

    PubMed

    Ezeamuzie, Charles I; Taslim, Najla

    2006-08-14

    Recently, we showed that phorbol 12-myristate 13-acetate (PMA) can cause a direct, PKC-dependent, stimulation of intracellular cAMP in human eosinophils. Since PMA also stimulates the release of reactive oxygen species in these cells, we have investigated whether reactive oxygen species are involved in the cAMP response. Provided eosinophils were incubated for <20 min at 37 degrees C before stimulation, PMA potently stimulated cAMP generation that surpassed that of histamine. Pre-treatment of the cells with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI) and apocynin, strongly inhibited the cAMP production induced by PMA, but not that induced by histamine. This treatment also strongly inhibited the release of superoxide anions (O(2)(-)). The cAMP response was also inhibited by pre-treatment with the specific peroxide scavenger, ebselen, but not superoxide dismutase, or NG-nitro-l-arginine methyl ester (L-NAME), thus, suggesting the possible involvement of a peroxide rather than O(2)(-) or nitric oxide (NO). These results reveal a novel involvement of intracellular reactive oxygen species in protein kinase C (PKC)-dependent stimulation of cAMP production in human eosinophils.

  16. Neutrophil Extracellular Traps of Cynoglossus semilaevis: Production Characteristics and Antibacterial Effect

    PubMed Central

    Zhao, Ming-li; Chi, Heng; Sun, Li

    2017-01-01

    Neutrophil extracellular traps (NETs) are structures released by neutrophils as a cellular immune defense against microbial invasion. The process of NETs generation, netosis (NETosis), can take place via either a suicidal mechanism, during which the NETs-releasing cells became dead, or a “live” mechanism, during which the NETs-releasing cells remain vital. NETosis has been studied intensively in mammals in recent years, but very little is known about the NETosis in fish. In this study, we examined NETosis in tongue sole (Cynoglossus semilaevis), a species of teleost with important economic values. We found that following stimulation with phorbol 12-myristate 13-acetate (PMA) and three common fish bacterial pathogens, abundant NETs structures were released by neutrophils that were most likely in a live state. The released NETs captured, but did not kill, the bacterial pathogens; however, the replication of extracellular, but not intracellular, pathogens was inhibited by NETs to significant extents. Reactive oxygen species (ROS), nitric oxide (NO), and myeloperoxidase (MPO) production were observed to be enhanced in NETosing neutrophils, and blocking the production of these factors by inhibitors significantly decreased NETs production induced by PMA and all three bacteria. Taken together, these results indicate for the first time that in teleost there exists a non-cell death pathway of NETosis that produces NETs with antibacterial effects in a ROS-, NO-, and MPO-dependent manner. PMID:28382034

  17. 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.

    PubMed

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-06-16

    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.

  18. 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

    PubMed Central

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-01-01

    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. PMID:27322248

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

    PubMed

    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.

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

    PubMed

    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.

  1. Simultaneous Real-Time Monitoring of Oxygen Consumption and Hydrogen Peroxide Production in Cells Using Our Newly Developed Chip-Type Biosensor Device

    PubMed Central

    Prasad, Ankush; Kikuchi, Hiroyuki; Inoue, Kumi Y.; Suzuki, Makoto; Sugiura, Yamato; Sugai, Tomoya; Tomonori, Amano; Tada, Mika; Kobayashi, Masaki; Matsue, Tomokazu; Kasai, Shigenobu

    2016-01-01

    All living organisms bear its defense mechanism. Immune cells during invasion by foreign body undergoes phagocytosis during which monocyte and neutrophil produces reactive oxygen species (ROS). The ROS generated in animal cells are known to be involved in several diseases and ailments, when generated in excess. Therefore, if the ROS generated in cells can be measured and analyzed precisely, it can be employed in immune function evaluation and disease detection. The aim of the current study is to introduce our newly developed chip-type biosensor device with high specificity and sensitivity. It comprises of counter electrode and working electrodes I and II. The counter electrode is a platinum plate while the working electrodes I and II are platinum microelectrode and osmium-horseradish peroxidase modified gold electrode, respectively which acts as oxygen and hydrogen peroxide (H2O2) detection sensors. Simultaneous measurement of oxygen consumption and H2O2 generation were measured in animal cells under the effect of exogenous addition of differentiation inducer, phorbol 12-myristate 13-acetate. The results obtained showed considerable changes in reduction currents in the absence and presence of inducer. Our newly developed chip-type biosensor device is claimed to be a useful tool for real-time monitoring of the respiratory activity and precise detection of H2O2 in cells. It can thus be widely applied in biomedical research and in clinical trials being an advancement over other H2O2 detection techniques. PMID:27065878

  2. HTLV-1 Tax impairs K63-linked ubiquitination of STING to evade host innate immunity.

    PubMed

    Wang, Jie; Yang, Shuai; Liu, Lu; Wang, Hui; Yang, Bo

    2017-03-15

    The cellular antiviral innate immune system is essential for host defense and viruses have evolved a variety of strategies to evade the innate immunity. Human T lymphotropic virus type 1 (HTLV-1) belongs to the deltaretrovirus family and it can establish persistent infection in human beings for many years. However, how this virus evades the host innate immune responses remains unclear. Here we report a new strategy used by HTLV-1 to block innate immune responses. We observed that stimulator of interferon genes (STING) limited HTLV-1 protein expression and was critical to HTLV-1 reverse transcription intermediate (RTI) ssDNA90 triggered interferon (IFN)-β production in phorbol12-myristate13-acetate (PMA)-differentiated THP1 (PMA-THP1) cells. The HTLV-1 protein Tax inhibited STING overexpression induced transcriptional activation of IFN-β. Tax also impaired poly(dA:dT), interferon stimulatory DNA (ISD) or cyclic GMP-AMP (cGAMP) -stimulated IFN-β production, which was dependent on STING activation. Coimmunoprecipitation assays and confocal microscopy indicated that Tax was associated with STING in the same complex. Mechanistic studies suggested that Tax decreased the K63-linked ubiquitination of STING and disrupted the interactions between STING and TANK-binding kinase 1 (TBK1). These findings may shed more light on the molecular mechanisms underlying HTLV-1 infection.

  3. Adhesion of human leukocytes to biomaterials: an in vitro study using alkanethiolate monolayers with different chemically functionalized surfaces.

    PubMed

    Barbosa, Judite N; Barbosa, Mário A; Aguas, Artur P

    2003-06-15

    The adhesion of human leukocytes to self-assembled monolayers of well-defined surface chemistry was investigated in vitro. Polymorphonuclear (PMN) and mononuclear leukocytes were isolated from human blood by centrifugation techniques. The effect on adhesion of cell activation produced by pre-incubation of leukocytes with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) was also studied. Gold substrates were modified by treatment with alkanethiols with three different terminal chemical groups: COOH, OH, and CH(3). After incubation with the two subpopulations of leukocytes, the monolayers were washed, treated with fixative, stained with a Giemsa method, and observed by light microscopy to quantify the number of attached leukocytes. Comparative quantification of the density of leukocyte adhesion to the three types of self-assembled monolayers was determined. The hydrophobic surface expressing CH(3) was found to be the one that induced the highest adhesion density of leukocytes, both of PMN and mononuclear cells. In vitro activation of both mononuclear and PMN leukocytes further increased cell adhesion to the chemically defined monolayers that were used. This enhancement was higher for PHA-activated than for PMA-stimulated mononuclear cells, whereas PMA treatment of neutrophils resulted in a higher rate of adhesion of these cells than PHA stimulation.

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

    SciTech Connect

    Jung, Se-Hui; Park, Jin-Young; Joo, Jung-Hoon; Kim, Young-Myeong; Ha, Kwon-Soo

    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.

  5. Inhibitory effect of arctigenin on lymphocyte activation stimulated with PMA/ionomycin.

    PubMed

    Sun, Cheng-Hong; Lai, Xin-Qiang; Zhang, Li; Yao, Jing-Chun; Guan, Yong-Xia; Pan, Li-Hong; Yan, Ying

    2014-04-01

    This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.

  6. Silver nanoparticles impede phorbol myristate acetate-induced monocyte-macrophage differentiation and autophagy

    NASA Astrophysics Data System (ADS)

    Xu, Yingying; Wang, Liming; Bai, Ru; Zhang, Tianlu; Chen, Chunying

    2015-09-01

    Monocytes/macrophages are important constituents of the innate immune system. Monocyte-macrophage differentiation is not only crucial for innate immune responses, but is also related to some cardiovascular diseases. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials because of their broad-spectrum antimicrobial properties. However, the effect of AgNPs on the functions of blood monocytes is scarcely reported. Here, we report the impedance effect of AgNPs on THP-1 monocyte differentiation, and that this effect was mediated by autophagy blockade and lysosomal impairment. Firstly, AgNPs inhibit phorbol 12-myristate 13-acetate (PMA)-induced monocyte differentiation by down-regulating both expression of surface marker CD11b and response to lipopolysaccharide (LPS) stimulation. Secondly, autophagy is activated during PMA-induced THP-1 monocyte differentiation, and the autophagy inhibitor chloroquine (CQ) can inhibit this process. Thirdly, AgNPs block the degradation of the autophagy substrate p62 and induce autophagosome accumulation, which demonstrates the blockade of autophagic flux. Fourthly, lysosomal impairments including alkalization and decrease of lysosomal membrane stability were observed in AgNP-treated THP-1 cells. In conclusion, we demonstrate that the impedance of monocyte-macrophage differentiation by AgNPs is mediated by autophagy blockade and lysosomal dysfunction. Our results suggest that crosstalk exists in different biological effects induced by AgNPs.

  7. Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-11-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.

  8. Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum†

    PubMed Central

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-01-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes. PMID:16227584

  9. Possible Involvement of the Inhibition of NF-κB Factor in Anti-Inflammatory Actions That Melatonin Exerts on Mast Cells.

    PubMed

    Maldonado, M D; García-Moreno, H; González-Yanes, C; Calvo, J R

    2016-08-01

    Melatonin is a molecule endogenously produced in a wide variety of immune cells, including mast cells (RBL-2H3). It exhibits immunomodulatory, anti-inflammatory and anti-apoptotic properties. The physiologic mechanisms underlying these activities of melatonin have not been clarified in mast cells. This work is designed to determine the anti-inflammatory effect and mechanism of action of melatonin on activated mast cells. RBL-2H3 were pre-treated with exogenous melatonin (MELx) at physiological (100nM) and pharmacological (1 mM) doses for 30 min, washed and activated with PMACI (phorbol 12-myristate 13-acetate plus calcium ionophore A23187) for 2 h and 12 h. The data shows that pre-treatment of MELx in stimulated mast cells, significantly reduced the levels of endogenous melatonin production (MELn), TNF-α and IL-6. These effects are directly related with the MELx concentration used. MELx also inhibited IKK/NF-κB signal transduction pathway in stimulated mast cells. These results indicate a molecular basis for the ability of melatonin to prevent inflammation and for the treatment of allergic inflammatory diseases through the down-regulation of mast cell activation. J. Cell. Biochem. 117: 1926-1933, 2016. © 2016 Wiley Periodicals, Inc.

  10. Functionality and opposite roles of two interleukin 4 haplotypes in immune cells

    PubMed Central

    Anovazzi, G; Medeiros, M C; Pigossi, S C; Finoti, L S; Souza Moreira, T M; Mayer, M P A; Zanelli, C F; Valentini, S R; Rossa-Junior, C; Scarel-Caminaga, R M

    2017-01-01

    Cytokines expression can be influenced by polymorphisms in their respective coding genes. We associated the CTI/TTD haplotype (Hap-1) and TCI/CCI haplotype (Hap-2) in the IL4 gene formed by the −590, +33 and variable number of tandem repeat polymorphisms with the severity of chronic periodontitis in humans. The functionality of these IL4 haplotypes in the response of immune cells to phorbol 12-myristate 13-acetate (PMA) with Ionomycin and IL-1β (as inflammatory stimuli) was evaluated. Gene expression (quantitative real-time PCR), profile of secreted cytokines (multiplex) and phenotypic polarization of T cells (flow cytometry) were the outcomes assessed. Green fluorescent protein reporter plasmid constructs containing specific IL4 haplotype were transiently transfected into JM cells to assess the influence of the individual haplotypes on promoter activity. In response to inflammatory stimuli the immune cells from Hap-1 haplotype had increased expression of anti-inflammatory IL4; conversely, the Hap-2 haplotype showed higher levels of pro-inflammatory cytokines. The haplotype CTI proved to be the most important for the regulation of IL4 promoter, regardless of the nature of the inflammatory stimulation; whereas the polymorphism in the promoter region had the least functional effect. In conclusion, IL4 haplotypes studied are functional and trigger opposite immune responses: anti-inflammatory (Hap-1) and pro-inflammatory (Hap-2). In addition, we identified the CTI haplotype as the main responsible for the regulation of IL4 transcriptional activity. PMID:28053321

  11. Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes.

    PubMed

    Baj-Krzyworzeka, Monika; Szatanek, Rafał; Weglarczyk, Kazimierz; Baran, Jarosław; Urbanowicz, Barbara; Brański, Piotr; Ratajczak, Mariusz Z; Zembala, Marek

    2006-07-01

    This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.

  12. Photon Counts Statistics in Leukocyte Cell Dynamics

    NASA Astrophysics Data System (ADS)

    van Wijk, Eduard; van der Greef, Jan; van Wijk, Roeland

    2011-12-01

    In the present experiment ultra-weak photon emission/ chemiluminescence from isolated neutrophils was recorded. It is associated with the production of reactive oxygen species (ROS) in the "respiratory burst" process which can be activated by PMA (Phorbol 12-Myristate 13-Acetate). Commonly, the reaction is demonstrated utilizing the enhancer luminol. However, with the use of highly sensitive photomultiplier equipment it is also recorded without enhancer. In that case, it can be hypothesized that photon count statistics may assist in understanding the underlying metabolic activity and cooperation of these cells. To study this hypothesis leukocytes were stimulated with PMA and increased photon signals were recorded in the quasi stable period utilizing Fano factor analysis at different window sizes. The Fano factor is defined by the variance over the mean of the number of photon within the observation time. The analysis demonstrated that the Fano factor of true signal and not of the surrogate signals obtained by random shuffling increases when the window size increased. It is concluded that photon count statistics, in particular Fano factor analysis, provides information regarding leukocyte interactions. It opens the perspective to utilize this analytical procedure in (in vivo) inflammation research. However, this needs further validation.

  13. Short-term and long-term effects of protein kinase C on the trafficking and stability of human organic anion transporter 3

    PubMed Central

    Zhang, Qiang; Suh, Wonmo; Pan, Zui; You, Guofeng

    2012-01-01

    Human organic anion transporter 3 (hOAT3) belongs to a family of organic anion transporters that play critical roles in the body disposition of numerous clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. In the current study, we investigated the short-term and long-term regulation of hOAT3 by protein kinase C (PKC). We showed that short-term activation of PKC by phobol 12-Myristate 13-Acetate (PMA) inhibited hOAT3 activity through accelerating its internalization from cell surface to intracellular recycling endosomes. The colocalization of hOAT3 with EEA1-positive recycling endosomes was demonstrated by immunolocalization with confocal microscopy. Furthermore, we showed that long-term activation of PKC resulted in the enhanced degradation of cell surface hOAT3. The pathways for hOAT3 degradation were further examined using proteasomal and lysosomal inhibitors. Our results showed that both proteasomal inhibitors and the lysosomal inhibitors significantly blocked hOAT3 degradation. These results demonstrate that PKC plays critical roles in the trafficking and the stability of hOAT3. PMID:22773962

  14. The anti-malarial artemisinin inhibits pro-inflammatory cytokines via the NF-κB canonical signaling pathway in PMA-induced THP-1 monocytes.

    PubMed

    Wang, Yue; Huang, Zhouqing; Wang, Liansheng; Meng, Shu; Fan, Yuqi; Chen, Ting; Cao, Jiatian; Jiang, Rujia; Wang, Changqian

    2011-02-01

    Several kinds of sesquiterpene lactones have been proven to inhibit NF-κB and to retard atherosclerosis by reducing lesion size and changing plaque composition. The anti-malarial artemisinin (Art) is a pure sesquiterpene lactone extracted from the Chinese herb Artemisia annua (qinghao, sweet wormwood). In the present study, we demonstrate that artemisinin inhibits the secretion and the mRNA levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in a dose-dependent manner in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 human monocytes. We also found that the NF-κB specific inhibitor, Bay 11-7082, inhibited the expression of these pro-inflammatory cytokines, suggesting that the NF-κB pathway may be involved in the decreased cytokine release. At all time-points (1-6 h), artemisinin impeded the phosphorylation of IKKα/ß, the phosphorylation and degradation of IκBα and the nuclear translocation of the NF-κB p65 subunit. Additionally, artemisinin inhibited the translocation of the NF-κB p65 subunit as demonstrated by confocal laser scanning microscopic analysis and by NF-κB binding assays. Our data indicate that artemisinin exerts an anti-inflammatory effect on PMA-induced THP-1 monocytes, suggesting the potential role of artemisinin in preventing the inflammatory progression of atherosclerosis.

  15. Luminol-, isoluminol- and lucigenin-enhanced chemiluminescence of rat blood phagocytes stimulated with different activators.

    PubMed

    Pavelkova, Martina; Kubala, Lukas

    2004-01-01

    Luminol-, isoluminol- or lucigenin-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen species by rat blood leukocytes. Opsonized zymosan (OZ), phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 (Ca-I) or N-formyl-Met-Leu-Phe (fMLP) were used as activators. The CL signal of isolated blood leukocytes decreased in rank order of luminol > isoluminol > lucigenin. The kinetic profiles of luminol- and isoluminol-enhanced CL were similar upon stimulation by each activator tested. The remarkably higher luminol and isoluminol CL responses were obtained after OZ stimulation when compared with other activators. However, when lucigenin was used, the PMA- and OZ-stimulated CL were comparable. The presence of plasma increased OZ-activated CL because of the enhanced phagocytosis of OZ. This was demonstrated by determining the phagocytosis of the fluorescent OZ using a flow cytometer. In contrast, the presence of plasma decreased PMA-activated CL, due to the antioxidant properties of plasma as determined by the CL method. As far as whole blood is concerned, only OZ activated luminol-enhanced CL was reliable. Blood volumes over 5 microL decreased CL activity due to the scavenging ability of erythrocytes. The results suggest that 0.5 microL whole blood is sufficient for routine luminol-enhanced CL analysis of whole blood oxidative burst in rats.

  16. [Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation].

    PubMed

    Roshchupkin, D I; Belakina, N S; Murina, M A

    2006-01-01

    The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.

  17. Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

    PubMed

    Jing, Tengyang; Lai, Zhangxing; Wu, Lidan; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2016-12-06

    Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

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

    PubMed

    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.

  19. Stimulation of fibroblast proliferation by neokyotorphin requires Ca influx and activation of PKA, CaMK II and MAPK/ERK.

    PubMed

    Sazonova, Olga V; Blishchenko, Elena Yu; Tolmazova, Anna G; Khachin, Dmitry P; Leontiev, Konstantin V; Karelin, Andrey A; Ivanov, Vadim T

    2007-01-01

    Neokyotorphin [TSKYR, hemoglobin alpha-chain fragment (137-141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4beta-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+ L-type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin.

  20. Differential role of protein kinase C in desensitization of muscarinic receptor induced by phorbol esters and receptor agonists

    SciTech Connect

    Lai, Wi Sheung.

    1989-01-01

    PKC, a phorbol ester receptor, copurified with specific binding sites of ({sup 3}H)phorbol-12,13,-dibutyrate (({sup 3}H)PDBu). The specific binding of ({sup 3}H)PDBu to intact cells was saturable to a single class of binding sites. The PKC and phorbol ester receptors in N1E-115 cells can be down regulated by prolonged phorbol ester incubation. Phorbol 12-myristate 13-acetate (PMA) suppressed muscarinic receptor-mediated cyclic GMP response in a time-dependent and a concentration-dependent fashion and the suppressive effect of PMA could be attenuated by a protein kinase inhibitor, H-7, as well as by down-regulation of the PKC through long-term incubation with PDBu. Exposure of the cells to the muscarinic agonist carbamylcholine also desensitized subsequent CBC-mediated cyclic GMP response. However, pretreatment with carbamylcholine did not desensitize histamine-induced cyclic GMP formation while treatment with PMA suppressed this histamine-mediated response. Preincubation of the cells with CBC, but not with phorbol ester, resulted in down-regulation of muscarinic receptors. The loss of muscarinic receptors induced by agonist even occurred when the phosphoinositide hydrolysis response was suppressed.

  1. Regulation of muscarinic acetylcholine receptors in the 1321N1 human astrocytoma cell line

    SciTech Connect

    Hoover, R.K.

    1989-01-01

    The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of 1321N1 human astrocytoma cells was studied. Binding was studied in both intact cells and cell lysates. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand ({sup 3}H)QNB or the hydrophilic radioligand ({sup 3}H)NMS. In contrast, full agonists exhibited markedly lower apparent affinities in intact cells with ({sup 3}H)QNB than with ({sup 3}H)NMS. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs ({sup 3}H)QNB were markedly higher. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. In other studies, the effects of the protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) on muscarinic receptor downregulation and internalization in 1321N1 cells were determined. PMA alone did not induce muscarinic receptor downregulation but instead decreased both the rate and final extent of downregulation induced by the agonist carbachol. The specificity of other protein kinase C activators for inhibiting carbachol-induced downregulation indicated involvement of protein kinase C. Furthermore, the protein kinase C inhibitor staurosporine prevented the inhibitory effect of PMA on downregulation. However, staurosporine did not inhibit agonist-induced downregulation.

  2. Arjunolic acid ameliorates reactive oxygen species via inhibition of p47phox-serine phosphorylation and mitochondrial dysfunction

    PubMed Central

    Miriyala, Sumitra; Chandra, Mini; Maxey, Benjamin; Day, Alicia; St. Clair, Daret K.; Panchatcharam, Manikandan

    2015-01-01

    Impaired cardiovascular function during acute myocardial infarction (MI) is partly associated with recruitment of activated polymorphonuclear neutrophils. The protective role of arjunolic acid (AA; 2:3:23-Trihydroxy olean-12-en-28-oic acid) is studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation. Neutrophils were isolated from normal and acute MI mice to find out the efficacy of AA in reducing oxidative stress. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) resulted in an oxidative burst of superoxide anion (O2•−) and enhanced release of lysosomal enzymes. The treatment of neutrophils with PMA induced phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase. Furthermore, we observed activated ERK induced phosphorylation of Ser345 in MI neutrophils. Treatment with AA significantly inhibited the phosphorylation of P47phox and ERK in the stimulated controls and MI neutrophils. Oxidative phosphorylation activities in MI cells were lower than in control, while the glycolysis rates were elevated in MI cells compared to the control. In addition, we observed AA decreased intracellular oxidative stress and reduced the levels of O2•− in neutrophils. This study therefore identifies targets for AA in activated neutrophils mediated by the MAPK pathway on p47phox involved in ROS generation. PMID:26319153

  3. Long non-coding RNA Malat1 promotes neurite outgrowth through activation of ERK/MAPK signalling pathway in N2a cells.

    PubMed

    Chen, Lei; Feng, Peimin; Zhu, Xi; He, Shixu; Duan, Jialan; Zhou, Dong

    2016-11-01

    Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are playing critical roles in neurogenesis, yet the underlying molecular mechanisms remain largely elusive. Neurite outgrowth is an early step in neuronal differentiation and regeneration. Using in vitro differentiation of neuroblastoma-derived Neuro-2a (N2a) cell as a model, we performed expression profiling to identify lncRNAs putatively relevant for neurite outgrowth. We identified that Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was one of the most significantly up-regulated lncRNAs during N2a cell differentiation. Malat1 knockdown resulted in defects in neurite outgrowth as well as enhanced cell death. To pinpoint signalling pathways perturbed by Malat1 depletion, we then performed a reporter-based screening to examine the activities of 50 signalling pathways in Malat1 knockdown cells. We found that Malat1 knockdown resulted in conspicuous inhibition of Mitogen-Activated Protein Kinase (MAPK) signaling pathway as well as abnormal activation of Peroxisome proliferator-activated receptor (PPAR) and P53 signalling pathway. Inhibition of ERK/MAPK pathway with PD98059 potently blocked N2a cell neurite outgrowth, whereas phorbol 12-myristate 13-acetate-induced ERK activation rescued defects in neurite outgrowth and cell death induced by Malat1 depletion. Together, our results established a critical role of Malat1 in the early step of neuronal differentiation through activating ERK/MAPK signalling pathway.

  4. Comparison of the hypertrophic effect of phorbol ester, norepinephrine, angiotensin II and contraction on cultured cardiomyocytes

    SciTech Connect

    Allo, S.N.; Carl, L.L.; Morgan, H.E. )

    1991-03-15

    Phorbol 12-myristate 13-acetate (PMA), norepinephrine (NE), angiotensin II (AII) and contraction stimulate cardiomyocyte growth. Differences exist in the time course and extent of protein and RNA accumulation. Cells plated at 4 {times} 10{sup 6} cells/60mm dish and arrested with 50 mM KCl demonstrated no significant growth. Treatment with PMA stimulated growth to a maximum of 17% at 48 h. In contrast, maximal stimulation of growth was 36% at 48 h and 31% at 72 h for contracting and NE treated cells, respectively. Maximal stimulation of the capacity for protein synthesis was 32% for PMA treated cells at 24 h as compared to 59% and 77% for NE treated and contracting cells respectively at 72 h. In support of a primary role for altered capacity in the regulation of protein synthesis, there was a significant correlation between RNA and protein content independent of the stimulus used. AII increased RNA content by 28% at 48h, but had no effect on growth up to 72h. Treatment with staurosporine blocked the stimulation of growth, suggestive of a role for protein kinase C (PKC). However, the inhibition of contraction-induced growth was due in part to a reduction in the rate of contraction. It was concluded that: significant differences existed in the time course of growth stimulation and RNA accumulation, depending on the stimulus; and growth inhibition by staurosporine is suggestive of an important role of PKC in hypertrophic growth induced by these stimuli.

  5. Tetradecanol reduces EL-4 T cell growth by the down regulation of NF-κB mediated IL-2 secretion.

    PubMed

    Park, Jung Up; Kang, Bok Yun; Lee, Hwa-Jeong; Kim, Sunoh; Bae, Donghyuck; Park, Jong-Hwan; Kim, Young Ran

    2017-03-15

    Tetradecanol is a straight-chain saturated fatty alcohol purified from Dendropanax morbifera leaves. We found that tetradecanol (30μM) reduced specifically the growth of T cells such as EL-4 T cell and isolated murine CD4(+) T cells. In this study, we investigated the effects of tetradecanol on the regulation of interlukin-2 (IL-2), a potent T cell growth factor. Tetradecanol significantly inhibited IL-2 secretion in EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) and also in isolated murine CD4(+) T cells activated with anti-CD3 and anti-CD28 antibodies. Next, we examined the effect of tetradecanol on the transcriptional activity related to IL-2 production in T cells. Tetradecanol decreased PMA/Io-induced promoter activity of NF-κB in EL-4 T cells, but did not show any significant effects on the promoters of activator protein 1 (AP-1) and nuclear factor of activated T cells (NF-AT). Tetradecanol inhibited IκBα degradation and nuclear translocation of NF-κB subunit, p65 in PMA/Io-activated EL-4 T cells. These results suggest that tetradecanol might have immunosuppressive effects on T cell mediated disorders. Using a chronic allergic contact dermatitis model induced by repeated application of oxazolone, we showed that tetradecanol reduced ear thickness induced by oxazolone.

  6. Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

    PubMed

    Lee, Hee Doo; Koo, Bon-Hun; Kim, Yeon Hyang; Jeon, Ok-Hee; Kim, Doo-Sik

    2012-07-01

    A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

  7. Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kB-like transcription factor

    SciTech Connect

    Edbrooke, M.R.; Cheshire, J.K.; Woo, P.; Burt, B.W.

    1989-05-01

    The authors have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). They found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, they placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by others as the binding site for the nuclear factor NF/kappa/B. In a cotransfection competition experiment, they could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF/kappa/B-binding sequence. The same long-terminal-repeat DNA containing mutant NF/kappa/B-binding sequences did not affect CAT expression, which suggested that binding by an NF/kappa/B-like factor is required for increased SAA transcription.

  8. PMA-induced dissociation of Ku86 from the promoter causes transcriptional up-regulation of histamine H1 receptor

    PubMed Central

    Mizuguchi, Hiroyuki; Miyagi, Kohei; Terao, Takuma; Sakamoto, Noriko; Yamawaki, Yosuke; Adachi, Tsubasa; Ono, Shohei; Sasaki, Yohei; Yoshimura, Yoshiyuki; Kitamura, Yoshiaki; Takeda, Noriaki; Fukui, Hiroyuki

    2012-01-01

    Histamine H1 receptor (H1R) gene is up-regulated in patients with allergic rhinitis, and its expression level strongly correlates with the severity of symptoms. However, the mechanism underlying this remains unknown. Here we report the mechanism of H1R gene up-regulation. The luciferase assay revealed the existence of two promoter regions, A and B1. Two AP-1 and one Ets-1 bound to region A, while Ku86, Ku70, and PARP-1 bound to region B1. Ku86 was responsible for DNA binding and poly(ADP-ribosyl)ated in response to phorbol-12-myristate-13-acetate stimulation, inducing its dissociation from region B1 that is crucial for promoter activity. Knockdown of Ku86 gene enhanced up-regulation of H1R gene expression. Experiments using inhibitors for MEK and PARP-1 indicate that regions A and B1 are downstream regulatory elements of the PKCδ/ERK/PARP-1 signaling pathway. Data suggest a novel mechanism for the up-regulation of H1R gene expression. PMID:23209876

  9. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    PubMed

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  10. Inhibitory effects of norlignans isolated from Anemarrhena asphodeloides on degranulation of rat basophilic leukemia- 2H3Cells.

    PubMed

    Bak, Jong Phil; Cho, Young Mi; Kim, Inhye; Park, Dae Won; Kwon, Jung Eun; Jeong, Yong Joon; Kwak, Jong Hwan; Kang, Se Chan

    2016-12-01

    Anemarrhena asphodeloides is known to suppress inflammation and lower various fevers. To determine the active component of A. asphodeloides, ethanol (EtOH) extract of A. asphodeloides rhizomes was fractionized. The compounds isolated from the dichloromethane (CH2Cl2) soluble fraction were identified as 4'-O-methylnyasol (1), nyasol (2), 3″-methoxynyasol (3), 3″-hydroxy-4″-methoxy-4″-dehydroxynyasol (4), 4-hydroxybenzaldehyde (5), and 4-hydroxyacetophenone (6). The four norlignans (1-4) potently inhibited the release of β-hexosaminidase from immunoglobulin E (IgE)/dinitrophenol-conjugated bovine serum albumin (DNP-BSA)-treated rat basophilic leukemia (RBL)-2H3 and A23187 plus phorbol 12-myristate 13-acetate co-treated isolated rat primary mast cells, as markers of degranulation and histamine release. The intraperitoneal treatment with the EtOH extract significantly suppressed the fetal reaction, and serum histamine release induced by compound 48/80 in mice. These results suggest that the four active norlignan compounds and the EtOH extract of A. asphodeloides may have potential to be developed as medicines for the treatment of allergies by inhibiting the activation of mast cells.

  11. Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells.

    PubMed

    Miyajima, Chiharu; Itoh, Yuka; Inoue, Yasumichi; Hayashi, Hidetoshi

    2015-01-01

    Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.

  12. Thrombomodulin regulates monocye differentiation via PKCδ and ERK1/2 pathway in vitro and in atherosclerotic artery

    PubMed Central

    Tsai, Chien-Sung; Lin, Yi-Wen; Huang, Chun-Yao; Shih, Chun-Min; Tsai, Yi-Ting; Tsao, Nai-Wen; Lin, Chin-Sheng; Shih, Chun-Che; Jeng, Hellen; Lin, Feng-Yen

    2016-01-01

    Thrombomodulin (TM) modulates the activation of protein C and coagulation. Additionally, TM regulates monocyte migration and inflammation. However, its role on monocyte differentiation is still unknown. We investigated the effects of TM on monocyte differentiation. First, we found that TM was increased when THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA). Overexpression of TM enhanced the macrophage markers, CD14 and CD68 expression in PMA-induced THP-1. TM siRNA depressed the PMA-induced increase of p21Cip1/WAF1 via ERK1/2-NF-kB p65 signaling. TM regulated cytoskeletal reorganization via its interaction with paxillin, cofilin, LIMK1, and PYK2. In addition, PMA-induced p21Cip1/WAF1 expression, CD14-positive cell labeling intensity and ERK1/2 phosphorylation were markedly inhibited when protein kinase C-δ (PKCδ) was knocked down. We identified that TM directly interacts with PKCδ. PKCδ was highly expressed in human atherosclerotic arteries and colocalized with TM in CD68-positive infiltrated macrophages of plaques, indicating that the coordination between TM and PKCδ in macrophages participated in atherogenesis. TM may act as a scaffold for PKCδ docking, which keeps PKCδ in the region close to the monocyte membrane to promote the activation of ERK1/2. Taken together, our findings suggest that TM-PKCδ interaction may contribute to cardiovascular disorders by affecting monocye differentiation, which may develop future therapeutic applications. PMID:27910925

  13. Theanine is a candidate amino acid for pharmacological stabilization of mast cells.

    PubMed

    Kim, N H; Jeong, H J; Kim, H M

    2012-05-01

    The increasing occurrences of allergic disorders may be attributed to exposure to environmental factors that contribute to the pathogenesis of allergy. The health benefits of green tea have been widely reported but are largely unsubstantiated. Theanine is the major amino acid present in green tea. In this study, we investigated the role of theanine in both IgE- and non- IgE-induced allergic response. Theanine inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling responses. IgE-mediated passive cutaneous anaphylaxis was inhibited by the oral administration or pharmaceutical acupuncture of theanine. Histamine release from mast cells was decreased with the treatment of theanine. Theanine also repressed phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced TNF-α, IL-1β, IL-6, and IL-8 secretion by suppressing NF-κB activation. Furthermore, theanine suppressed the activation of caspase-1 and the expression of receptor interacting protein-2. The current study demonstrates for the first time that theanine might possess mast cell-stabilizing capabilities.

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

    PubMed

    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.

  15. Inhibition of neutrophil priming and tyrosyl phosphorylation by cepharanthine, a nonsteroidal antiinflammatory drug.

    PubMed

    Kobuchi, H; Li, M J; Matsuno, T; Yasuda, T; Utsumi, K

    1992-12-01

    Receptor-mediated superoxide (O2.-)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O2.- generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O2.- generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methionyl-leucyl- phenylalanine (FMLP)-mediated O2.- generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O2.- generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.

  16. Sinomenine influences capacity for invasion and migration in activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, and CD147

    PubMed Central

    Ou, Yang-qiong; Chen, Li-hua; Li, Xue-jun; Lin, Zhi-bin; Li, Wei-dong

    2009-01-01

    Aim: The aim of this study was to investigate the mechanism of the effects of Sinomenine (SIN) on the invasion and migration ability of activated human monocytic THP-1 cells (A-THP-1). Sinomenine is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum. Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Cells were treated with different concentrations of SIN. The invasion and migration ability of cells was tested by in vitro transwell assays. The levels of CD147 and MMPs were evaluated by flow cytometric analysis and zymographic analysis, respectively. The mRNA expression of CD147, MMP-2, and MMP-9 was measured by RT-PCR. Results: The invasion and migration ability of A-THP-1 cells was significantly inhibited by SIN in a concentration-dependent fashion; at the same time, the levels of CD147, MMP-2, and MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 mmol/L and 1.00 mmol/L (P<0.01). Conclusion: A possible mechanism of the inhibitory effect of SIN on cell invasion and migration ability is repression of the expression of MMP-2 and MMP-9, which strongly correlates with the inhibition of CD147 activity. PMID:19305422

  17. TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

    PubMed

    Pype, S; Declercq, W; Ibrahimi, A; Michiels, C; Van Rietschoten, J G; Dewulf, N; de Boer, M; Vandenabeele, P; Huylebroeck, D; Remacle, J E

    2000-06-16

    CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

  18. Loss-of-function of the protein kinase C δ (PKCδ) causes a B-cell lymphoproliferative syndrome in humans.

    PubMed

    Kuehn, Hye Sun; Niemela, Julie E; Rangel-Santos, Andreia; Zhang, Mingchang; Pittaluga, Stefania; Stoddard, Jennifer L; Hussey, Ashleigh A; Evbuomwan, Moses O; Priel, Debra A Long; Kuhns, Douglas B; Park, C Lucy; Fleisher, Thomas A; Uzel, Gulbu; Oliveira, João B

    2013-04-18

    Defective lymphocyte apoptosis results in chronic lymphadenopathy and/or splenomegaly associated with autoimmune phenomena. The prototype for human apoptosis disorders is the autoimmune lymphoproliferative syndrome (ALPS), which is caused by mutations in the FAS apoptotic pathway. Recently, patients with an ALPS-like disease called RAS-associated autoimmune leukoproliferative disorder, in which somatic mutations in NRAS or KRAS are found, also were described. Despite this progress, many patients with ALPS-like disease remain undefined genetically. We identified a homozygous, loss-of-function mutation in PRKCD (PKCδ) in a patient who presented with chronic lymphadenopathy, splenomegaly, autoantibodies, elevated immunoglobulins and natural killer dysfunction associated with chronic, low-grade Epstein-Barr virus infection. This mutation markedly decreased protein expression and resulted in ex vivo B-cell hyperproliferation, a phenotype similar to that of the PKCδ knockout mouse. Lymph nodes showed intense follicular hyperplasia, also mirroring the mouse model. Immunophenotyping of circulating lymphocytes demonstrated expansion of CD5+CD20+ B cells. Knockdown of PKCδ in normal mononuclear cells recapitulated the B-cell hyperproliferative phenotype in vitro. Reconstitution of PKCδ in patient-derived EBV-transformed B-cell lines partially restored phorbol-12-myristate-13-acetate-induced cell death. In summary, homozygous PRKCD mutation results in B-cell hyperproliferation and defective apoptosis with consequent lymphocyte accumulation and autoantibody production in humans, and disrupts natural killer cell function.

  19. Activity of phospholipase C and release of prostaglandin F2 alpha by endometrial tissue from ovariectomized ewes receiving progesterone and estradiol.

    PubMed

    Raw, R E; Silvia, W J

    1991-03-01

    Progesterone and estradiol interact to regulate secretion of prostaglandin (PG) F2 alpha from the ovine endometrium in response to oxytocin. Two experiments were conducted to determine if these effects were due to changes in activity of phospholipase C or in the second messenger responsive pathways that regulate production of PGF2 alpha. In both experiments, ovariectomized ewes were assigned to one of four treatment groups (control, estradiol, progesterone, progesterone and estradiol). Steroids were administered, in vivo, to mimic the changes that occur during the estrous cycle. On Day 16 of steroid treatment, endometrial tissue was collected and incubated, in vitro, to measure activity of phospholipase C and release of PGF2 alpha. Treatment with progesterone, in vivo, enhanced basal and oxytocin-induced activity of phospholipase C and release of PGF2 alpha, in vitro. Estradiol suppressed oxytocin-induced activity of phospholipase C, both in the presence and absence of progesterone. In contrast to its effects on phospholipase C, estradiol inhibited basal and oxytocin-induced release of PGF2 alpha when administered alone, but not when administered with progesterone. Steroids had similar effects on the release of PGF2 alpha induced by phorbol 12-myristate 13-acetate and A23187. It was concluded that progesterone and estradiol regulate endometrial release of PGF2 alpha by affecting both the activity of phospholipase C and its associated second messenger responsive pathways that may regulate production of PGF2 alpha.

  20. Identification and characterization of an enhancer in the coding region of the genome of human immunodeficiency virus type 1.

    PubMed Central

    Verdin, E; Becker, N; Bex, F; Droogmans, L; Burny, A

    1990-01-01

    Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by cis-acting DNA elements located in the viral long terminal repeats, by viral transregulatory proteins, and by cellular transcription factors acting in concert to modulate the degree of viral expression. We demonstrate that a DNA fragment corresponding to the central portion of the HIV-1 genome exhibits enhancer activity when cloned upstream of the thymidine kinase promoter of herpes simplex virus. This enhancer is inducible by phorbol 12-myristate 13-acetate in HeLa cells and is independent of its position and orientation with respect to the promoter. We have mapped the activity of the enhancer to two independent domains encompassing nucleotides 4079-4342 (end of the pol gene) and nucleotides 4781-6026 (vif gene and first coding exon of tat). This intragenic enhancer and its subdomains demonstrate cellular specificity because they are only active in specific cell lines. The presence of similar intragenic enhancer elements in other retroviruses suggests that they might be a conserved feature of this family of viruses. Images PMID:2352955

  1. Differential usage of signal transduction pathways defines two types of serum response factor target gene.

    PubMed

    Gineitis, D; Treisman, R

    2001-07-06

    Activation of the transcription factor serum response factor (SRF) is dependent on Rho-controlled changes in actin dynamics. We used pathway-specific inhibitors to compare the roles of actin dynamics, extracellular signal-regulated kinase (ERK) signaling, and phosphatidylinositol 3-kinase in signaling either to SRF itself or to four cellular SRF target genes. Serum, lysophosphatidic acid, platelet-derived growth factor, and phorbol 12-myristate 13-acetate (PMA) each activated transcription of a stably integrated SRF reporter gene dependent on functional RhoA GTPase. Inhibition of mitogen-activated protein kinase-ERK kinase (MEK) signalling reduced activation of the SRF reporter by all stimuli by about 50%, except for PMA, which was effectively blocked. Inhibition of phosphatidylinositol 3-kinase slightly reduced reporter activation by serum and lysophosphatidic acid but substantially inhibited activation by platelet-derived growth factor and PMA. Reporter induction by all stimuli was absolutely dependent on actin dynamics. Regulation of the SRF (srf) and vinculin (vcl) genes was similar to that of the SRF reporter gene; activation by all stimuli was Rho-dependent and required actin dynamics but was largely independent of MEK activity. In contrast, activation of fos and egr1 occurred independently of RhoA and actin polymerization but was almost completely dependent on MEK activation. These results show that at least two classes of SRF target genes can be distinguished on the basis of their relative sensitivity to RhoA-actin and MEK-ERK signaling pathways.

  2. Regulation of hepatic EAAT-2 glutamate transporter expression in human liver cholestasis

    PubMed Central

    Najimi, Mustapha; Stéphenne, Xavier; Sempoux, Christine; Sokal, Etienne

    2014-01-01

    AIM: To investigate the activity and expression of EAAT2 glutamate transporter in both in vitro and in vivo models of cholestasis. METHODS: This study was conducted on human hepatoblastoma HepG2 cell cultures, the liver of bile duct ligated rats and human specimens from cholestatic patients. EAAT2 glutamate transporter activity and expression were analyzed using a substrate uptake assay, immunofluorescence, reverse transcription-polymerase chain reaction, and immunohistochemistry, respectively. RESULTS: In HepG2 cells, cholestasis was mimicked by treating cells with the protein kinase C activator, phorbol 12-myristate 13-acetate. Under such conditions, EAAT2 transporter activity was decreased both at the level of substrate affinity and maximal transport velocity. The decreased uptake was correlated with intracellular translocation of EAAT2 molecules as demonstrated using immunofluorescence. In the liver of bile duct ligated rats, an increase in EAAT2 transporter protein expression in hepatocytes was demonstrated using immunohistochemistry. The same findings were observed in human liver specimens of cholestasis in which high levels of γ-glutamyl transpeptidase were documented in patients with biliary atresia and progressive familial intrahepatic cholestasis type 3. CONCLUSION: This study demonstrates the alteration in glutamate handling by hepatocytes in liver cholestasis and suggests a potential cross-talk between glutamatergic and bile systems. PMID:24587631

  3. A human T-cell line with inducible production of interleukins 5 and 4. A model for studies of gene expression.

    PubMed

    Mordvinov, V A; Peroni, S E; De Boer, M L; Kees, U R; Sanderson, C J

    1999-08-31

    The production of interleukin-5 (IL5) and interleukin-4 (IL4) by activated T-cells is important in the pathogenesis of helminth infections and allergy. Human Jurkat cells express IL4 but one of the main factors restricting studies of human IL5 expression has been the lack of human T-cell lines which express significant levels of IL5 in an inducible fashion. We report that the human T-cell leukemia cell line (PER-117), previously shown to produce IL2, also produces IL5 and IL4, and is a useful model for the study of the regulation of IL5 and IL4 gene expression. We show that expression of IL5 and IL4 mRNAs in PER-117 cells is stimulation dependent. IL5 and IL4 reporter constructs are also transiently expressed in these cells in an inducible fashion. IL5 production in the PER-117 cell line can be activated by phorbol 12-myristate 13-acetate alone and further enhanced by calcium ionophore A23187, cyclic adenosine 3', 5'-monophosphate or anti-CD28 antibodies. The conditions used to stimulate the PER-117 cells determined whether IL5 production was inhibited by cyclosporin A or dexamethasone. These data indicate that the PER-117 cell line is a model to study signal transduction and transcriptional activation of the human IL5 gene in human T-cells.

  4. Mosla dianthera inhibits mast cell-mediated allergic reactions through the inhibition of histamine release and inflammatory cytokine production

    SciTech Connect

    Lee, Dong-Hee; Kim, Sang-Hyun . E-mail: shkim72@knu.ac.kr; Eun, Jae-Soon; Shin, Tae-Yong . E-mail: tyshin@woosuk.ac.kr

    2006-11-01

    In this study, we investigated the effect of the aqueous extract of Mosla dianthera (Maxim.) (AEMD) on the mast cell-mediated allergy model and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases such as asthma, sinusitis and rheumatoid arthritis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. AEMD inhibited compound 48/80-induced systemic reactions in mice. AEMD decreased immunoglobulin E-mediated local allergic reactions, passive cutaneous anaphylaxis. AEMD attenuated intracellular calcium level and release of histamine from rat peritoneal mast cells activated by compound 48/80. Furthermore, AEMD attenuated the phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated TNF-{alpha}, IL-8 and IL-6 secretion in human mast cells. The inhibitory effect of AEMD on the pro-inflammatory cytokines was nuclear factor-{kappa}B (NF-{kappa}B) dependent. AEMD decreased PMA and A23187-induced degradation of I{kappa}B{alpha} and nuclear translocation of NF-{kappa}B. Our findings provide evidence that AEMD inhibits mast cell-derived immediate-type allergic reactions and involvement of pro-inflammatory cytokines and NF-{kappa}B in these effects.

  5. P2X receptors in cochlear Deiters' cells.

    PubMed

    Chen, C; Bobbin, R P

    1998-05-01

    1. The ionotropic purinoceptors in isolated Deiters' cells of guinea-pig cochlea were characterized by use of the whole-cell variant of the patch-clamp technique. 2. Extracellular application of adenosine 5'-triphosphate (ATP) induced a dose-dependent inward current when the cells were voltage-clamped at -80 mV. The ATP-induced current showed desensitization and had a reversal potential around -4 mV. 3. Increasing intracellular free Ca2+ by decreasing the concentration of EGTA in the pipette solution reduced the amplitude of the ATP-gated current. 4. The order of agonist potency was: 2-methylthioATP (2-meSATP)>ATP>benzoylbenzoyl-ATP (BzATP)>alpha,beta-methyleneATP (alpha,beta,meATP>adenosine 5'-diphosphate (ADP)>uridine 5'-triphosphate (UTP)>adenosine 5'-monophosphate (AMP)=adenosine (Ad). 5. Pretreatment with forskolin (10 microM), 8-bromoadenosine-3',5'-cyclophosphate (8-Br-cyclic AMP, 1 mM), 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or phorbol-12-myristate-13-acetate (PMA, 1 microM) reversibly reduced the ATP-induced peak current. 6. The results are consistent with molecular biological data which indicate that P2X2 purinoceptors are present in Deiters' cells. In addition, the reduction of the ATP-gated current by activators of protein kinase A and protein kinase C indicates that these P2X2 purinoceptors can be functionally modulated by receptor phosphorylation.

  6. Expression of the cytochrome P450 epoxygenase CYP2J2 in human monocytic leukocytes.

    PubMed

    Nakayama, Kaeko; Nitto, Takeaki; Inoue, Teruo; Node, Koichi

    2008-08-29

    CYP2J2 is one of the cytochrome P450 epoxygenases involved in the metabolism of arachidonic acid. CYP2J2 has been identified in several tissues, especially cardiovascular tissues. CYP2J2 has cardiovascular effects, as epoxyeicosatrienoic acid, one of its metabolites, has anti-inflammatory and vasodilative activities. We investigated the expression of CYP2J2 in human leukocytes using reverse transcription-polymerase chain reaction, immunoblotting and immunostaining. Human monocytic cells, but not human neutrophils, exhibited constitutive expression of CYP2J2. Furthermore, the expression of CYP2J2 mRNA increased when the human monocytic cell line THP-1 cells and human monocytes were stimulated with phorbol 12-myristate 13-acetate and macrophage-colony stimulating factor in combination with granulocyte/macrophage-colony stimulating factor, respectively. These results suggest that expression of CYP2J2 was up-regulated when human monocytes differentiated into macrophages and that human monocytic cells and macrophages have a pathway to metabolize arachidonic acid using CYP epoxygenases.

  7. Induction of chondroitin sulfate proteoglycan synthesis and secretion in lymphocytes and monocytes

    PubMed Central

    1983-01-01

    The ability of mononuclear leukocytes to synthesize and secrete proteoglycans was evaluated. Using radiolabeling with H2 35SO4, it is shown that peripheral blood mononuclear cells (PBMC) and their major subpopulations (B cells, T cells, and monocytes), as well as mouse spleen cells, all secreted easily detectable proteoglycan. After 24-h labeling periods, 90% of macromolecular 35S could be detected in culture media. This material was primarily (greater than 95%) chondroitin-4-sulfate proteoglycan (CSPG). Production and secretion of CSPG could be stimulated more than 200% in PBMC and 300% in T cell populations by high concentrations of concanavalin A and phorbol 12- myristate-13-acetate; lipopolysaccharide induced a small (twofold) but reproducible increase in CSPG secretion by adherent mononuclear leukocytes. The CSPG secreted by PBMC was relatively small in size compared to chondrocyte CSPG (130,000 daltons vs. 2-4 million daltons) but possessed similar sizes of glycosaminoglycan chains and greater solubility in low ionic strength solutions. This sulfated polyanion, which was produced endogenously by leukocytes and was actively secreted, might function as a co-mediator or "second messenger" in certain immune responses. PMID:6604059

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

    SciTech Connect

    Kim, Chi-Kyeong; Hirose, Yuko; Sakudo, Akikazu; Takeyama, Natsumi; Kang, Chung-Boo; Taniuchi, Yojiro; Matsumoto, Yoshitsugu; Itohara, Shigeyoshi; Sakaguchi, Suehiro; Onodera, Takashi . E-mail: aonoder@mail.ecc.u-tokyo.ac.jp

    2007-06-29

    Splenocytes of wild-type (Prnp {sup +/+}) and prion protein gene-deficient (Prnp {sup -/-}) 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{sup C}) expression was enhanced following ConA stimulation, but not PMA + Io or LPS in Prnp {sup +/+} splenocytes. Rikn Prnp {sup -/-} splenocytes elicited lower cell proliferations than Prnp {sup +/+} or Zrch I Prnp {sup -/-} 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{sup C} and PrPLP/Doppel.

  9. Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

    SciTech Connect

    Kasai, Kikuo; Hiraiwa, Masaki; Emoto, Tatsushi; Hattori, Yoshiyuki; Shimoda, Shin-Ichi ); Ohmori, Takeshi; Koizumi, Narumi; Hosoya, Toichiro )

    1989-01-01

    The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03 - 0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor and phorbol 12-myristate 13-acetate completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.

  10. Effects of inorganic iodide, epidermal growth factor and phorbol ester on hormone synthesis by porcine thyroid follicles cultured in suspension

    SciTech Connect

    Kasai, Kikuo; Ichimura, Kenichi; Banba, Nobuyuki; Emoto, Tatsushi; Hiraiwa, Masaki; Hishinuma, Akira; Hattori, Yoshiyuki; Shimoda, Shinichi ); Yamaguchi, Fumihiko; Hosoya, Toichiro )

    1992-01-01

    Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining in the contents of thyroxine (T{sub 4}) and triiodothyronine (T{sub 3}) in the media and by paperchromatographic analysis of {sup 125}I-labeled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI. The maximal response was obtained at 1 {mu}M. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred {mu}M and NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor and phorbol 12-myristate 13-acetate inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.

  11. Stimulation of prostaglandin E/sub 2/ production by phorbol esters and epidermal growth factor in porcine thyroid cells

    SciTech Connect

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

    1987-07-13

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

  12. Quantifying transient psychological stress using a novel technique: changes to PMA-induced leukocyte production of ROS in vitro.

    PubMed

    Shelton-Rayner, Graham K; Mian, Rubina; Chandler, Simon; Robertson, Duncan; Macdonald, David W

    2011-01-01

    Although much work has been conducted to quantify the long-term physiological effects of psychological stress, measures of short-term, low-level stress have been more elusive. This study assessed the effect of exposure of volunteers to a mild, brief, psychologically stressful event, on the functional ability of leukocytes in whole blood to respond to phorbol 12-myristate 13-acetate (PMA) in vitro. Volunteers operated a car electric window and adjusted it to 4 pre-determined positions. Between each operation the mechanism's polarity was covertly altered (group B) or remained unaltered (group A). For each treatment group 10 different subjects provided capillary blood samples pre- and post-stressor. Using a chemiluminescent technique termed leukocyte coping capacity, the ability of leukocytes to produce reactive oxygen species (ROS) in vitro was assessed. ROS release differed significantly at 10 min post-stressor between treatment groups, suggesting exposure to acute psychological stress leads to a reduced ability to respond to bacterial challenge.

  13. Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM.

    PubMed Central

    Fuleihan, R; Ramesh, N; Loh, R; Jabara, H; Rosen, R S; Chatila, T; Fu, S M; Stamenkovic, I; Geha, R S

    1993-01-01

    B lymphocytes from patients with X chromosome-linked immunoglobulin deficiency with normal or elevated serum IgM are unable to switch from the synthesis of IgM/IgD to that of other immunoglobulin isotypes. Isotype switch recombination was evaluated in three affected males by examining interleukin 4-driven IgE synthesis. T-cell-dependent IgE synthesis was completely absent in the B lymphocytes of the patients. In contrast, CD40 mAb plus interleukin 4 induced the patients' B cells to synthesize IgE and to undergo deletional switch recombination. Because interaction between CD40 and its ligand on activated T cells is critical for T-cell-driven isotype switching, we examined CD40 ligand expression. In contrast to normal T cells, lymphocytes from the patients expressed no detectable CD40 ligand on their surface after stimulation with phorbol 12-myristate 13-acetate and ionomycin, although the mRNA of the ligand was expressed normally. These results suggest that defective expression of the CD40 ligand underlies the failure of isotype switching in this disease. Images Fig. 1 Fig. 3 PMID:7681587

  14. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1997-03-14

    The authors have examined the role of integrins and extracellular matrix (ECM) proteins in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M-CSF). Increased {beta}{sub 1} integrin and fibronectin (FN) gene expression was observed in PMA-treated HL-60 cells and PMA- or M-CSF-treated monocytes, even at a time preceding the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} (PKC{beta}) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Restoration of PKC{beta} resulted in PMA-induced FN gene expression and macrophage differentiation. The macrophage phenotype induced in HL-60 cells or monocytes was attenuated by anti-{beta}{sub 1} integrin or anti-FN MAbs. The authors suggest that macrophage differentiation involves activation of PKC and expression of specific integrins and ECM proteins. The stimulated cells, through their integrins, attach and spread on these substrates by binding to the deposited ECM proteins. This attachment and spreading in turn, through integrin signaling, leads to the macrophage phenotype.

  15. Regulation of 90-kilodalton ribosomal S6 kinase phosphorylation in the rat pineal gland.

    PubMed

    Ho, A K; Mackova, M; Cho, C; Chik, C L

    2003-08-01

    In this study we investigated diurnal changes in the activation state of the 90-kDa ribosomal S6 kinase (p90RSK) in the rat pineal gland. In animals housed under a lighting regimen with 12 h of light, we found an increase in phosphorylated p90RSK during the dark phase, and this increase was abolished by treatment with propranolol or continuous exposure to light. To determine the intracellular mechanism involved, rat pinealocytes were treated with norepinephrine. Norepinephrine caused a parallel increase in phosphorylated p42/44 MAPK (p42/44(MAPK)) and p90RSK that was reduced by prazosin or propranolol, indicating involvement of both alpha(1)- and beta-adrenergic receptors. Treatment with dibutyryl cGMP, 4beta-phorbol 12-myristate 13-acetate, or ionomycin mimicked norepinephrine-stimulated p90RSK phosphorylation, whereas dibutyryl cAMP caused a decrease in p90RSK phosphorylation. Inhibition of p42/44(MAPK) activation by UO126 was effective in reducing norepinephrine-stimulated p90RSK phosphorylation. Moreover, UO126 had an inhibitory effect on norepinephrine-stimulated arylalkyl-N-acetyltransferase activity. These results indicate that the adrenergically regulated nocturnal increase in p90RSK phosphorylation is mainly mediated through a cGMP-->p42/44(MAPK)-dependent mechanism.

  16. Induction of megakaryocytic colony-stimulating activity in mouse skin by inflammatory agents and tumor promoters

    SciTech Connect

    Clark, D.A.; Dessypris, E.N.; Koury, M.J.

    1987-03-01

    The production of megakaryocytic colony-stimulating activity (MEG-CSA) was assayed in acetic acid extracts of skin from mice topically treated with inflammatory and tumor-promoting agents. A rapid induction of MEG-CSA was found in skin treated both with phorbol 12-myristate 13-acetate (PMA), a strong tumor promoter, and with mezerein, a weak tumor promoter, but no induction was found in untreated skin. The time course of induction of MEG-CSA following treatment of skin with PMA or mezerein was very similar to that previously demonstrated for the induction of granulocyte-macrophage colony-stimulating activity in mouse skin by these agents. The induced MEG-CSA was found in both the epidermis and the dermis. Pretreatment of the skin with US -methasone abrogated the MEG-CSA induction. The cell number response curve suggests that the MEG-CSA acts directly on the progenitor cells of the megakaryocyte colonies. That topical administration of diterpene esters results in the rapid, local induction of MEG-CSA which can be blocked by US -methasone pretreatment suggests a mechanism for the thrombocytosis associated with some inflammatory states. The indirect action in which diterpene esters induce in certain cells the production or release of growth regulatory factors for other cell types may also aid in understanding their carcinogenic properties.

  17. Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation

    SciTech Connect

    Bloom, T. )

    1991-03-01

    Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development. In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with ({sup 32}P)orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four-cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four-cell embryos. Inhibition of protein synthesis at the eight-cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight-cell but not in four-cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight-cell embryos incubated in phorbol 12-myristate 13-acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphoprotein spots associated with the eight-cell but not with the four-cell stage. Embryos incubated in Ca2(+)-free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.

  18. Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes.

    PubMed Central

    Burn, P; Kupfer, A; Singer, S J

    1988-01-01

    Members of the family of transmembrane integral membrane proteins called integrins have been implicated in forming attachments to actin microfilaments of the cytoskeleton. These attachments are thought to involve one or more intervening peripheral membrane proteins linked to integrin. To detect such possible linkages in vivo, the integrin molecules on the surfaces of intact chicken peripheral blood lymphocytes were collected into caps by cross-linking with specific antibodies, and the capped cells were examined by double immunofluorescence to determine whether particular cytoskeletal proteins were co-collected with the integrin. With resting lymphocytes, the capping of integrin did not result in any detectable redistribution of either talin, vinculin, or alpha-actinin inside the cells. However, if the capping was carried out upon the addition of phorbol 12-myristate 13-acetate (PMA) to the cells, then talin, but not vinculin or alpha-actinin, was found associated with the integrin caps. PMA is known to activate protein kinase C. These results suggest that after, but not before, PMA stimulation of intact cells, talin becomes linked either directly or indirectly with integrin, reflecting the formation of a membrane-cytoskeletal association that is metabolically regulated. Images PMID:3124107

  19. Abrogation of TNF-mediated cytotoxicity by space flight involves protein kinase C

    NASA Technical Reports Server (NTRS)

    Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Experiments conducted on STS-50 indicated that space flight significantly inhibited tumor necrosis factor (TNF)-mediated killing of LM929 cells compared to ground controls. In ground-based studies, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also inhibited TNF-mediated killing of LM929 cells. Therefore, we used PKC inhibitors to determine if the inhibitory effects of spaceflight on TNF-mediated cytotoxicity involved the activation of PKC. In experiments conducted onboard space shuttle mission STS-54, we saw that in the presence of the protein kinase C inhibitors H7 and H8, TNF-mediated cytotoxicity was restored to levels of those observed in the ground controls. Subsequent experiments done during the STS-57 mission tested the dose response of two protein kinase inhibitors, H7 and HA1004. We again saw that killing was restored in a dose-dependent manner, with inhibitor concentrations known to inhibit PKC being most effective. These data suggest that space flight ameliorates the action of TNF by affecting PKC in target cells.

  20. Effect of magnetic resonance imaging on human respiratory burst of neutrophils.

    PubMed

    Heine, J; Scheinichen, D; Jaeger, K; Herzog, T; Sümpelmann, R; Leuwer, M

    1999-03-05

    It is known that low intensity magnetic fields increase superoxide anion production during the respiratory burst of rat peritoneal neutrophils in vitro. We investigated whether the high intensity magnetic fields (1.5 T) during magnetic resonance imaging can influence the human neutrophil function under in vivo conditions. Blood samples were obtained from 12 patients immediately before and after magnetic resonance imaging (mean time 27.6(+/-11.4 min)). The induced respiratory burst was investigated by the intracellular oxidative transformation of dihydrorhodamine 123 to the fluorescent dye rhodamine 123 via flow cytometry. The respiratory burst was induced either with phorbol 12-myristate 13-acetate, Escherichia coli, N-formyl-methionyl-leucylphenylalanine or priming with tumor necrosis factor followed by FMLP stimulation. There was no significant difference between the respiratory burst before and after magnetic resonance imaging, irrespective of the stimulating agent. Short time exposure to a high intensity magnetic field during magnetic resonance imaging seems not to influence the production of radical species in living neutrophils.

  1. 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

    PubMed Central

    Daga, Martina; Cetrangolo, Giovanni Paolo; Ciamporcero, Eric Stefano; Petrella, Claudia; Graf, Maria; Uchida, Koji; Mamone, Gianfranco; Ferranti, Pasquale; Ames, Paul R. J.

    2015-01-01

    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. PMID:26078803

  2. Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields

    SciTech Connect

    Holian, O.; Reyes, H.M.; Attar, B.M.; Walter, R.J.; Astumian, R.D.; Lee, R.C.

    1996-12-31

    The authors examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5--1,000 mV/cm p-p) were applied for 1 h to cells (10{sup 7}/ml) in Teflon chambers at 37 C in the presence or absence of 2 {micro}M phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100--1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA, EFs (33--330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents.

  3. Effects of Lupenone, Lupeol, and Taraxerol Derived from Adenophora triphylla on the Gene Expression and Production of Airway MUC5AC Mucin

    PubMed Central

    Yoon, Yong Pill; Lee, Hyun Jae; Lee, Dong-Ung; Lee, Sang Kook; Hong, Jang-Hee

    2015-01-01

    Background Adenophora triphylla var. japonica is empirically used for controlling airway inflammatory diseases in folk medicine. We evaluated the gene expression and production of mucin from airway epithelial cells in response to lupenone, lupeol and taraxerol derived from Adenophora triphylla var. japonica. Methods Confluent NCI-H292 cells were pretreated with lupenone, lupeol or taraxerol for 30 minutes and then stimulated with tumor necrosis factor α (TNF-α) for 24 hours. The MUC5AC mucin gene expression and production were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Additionally, we examined whether lupenone, lupeol or taraxerol affects MUC5AC mucin production induced by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), the other 2 stimulators of airway mucin production. Results Lupenone, lupeol, and taraxerol inhibited the gene expression and production of MUC5AC mucin induced by TNF-α from NCI-H292 cells, respectively. The 3 compounds inhibited the EGF or PMA-induced production of MUC5AC mucin in NCI-H292 cells. Conclusion These results indicated that lupenone, lupeol and taraxerol derived from Adenophora triphylla var. japonica regulates the production and gene expression of mucin, by directly acting on airway epithelial cells. In addition, the results partly explain the mechanism of of Adenophora triphylla var. japonica as a traditional remedy for diverse inflammatory pulmonary diseases. PMID:26175774

  4. Riluzole attenuates excitatory amino acid transporter type 3 activity in Xenopus oocytes via protein kinase C inhibition.

    PubMed

    Choi, Jung-Seok; Ryu, Jung-Hee; Zuo, Zhiyi; Yang, Seong-Mi; Chang, Hye-Won; Do, Sang-Hwan

    2013-08-05

    This study aimed to evaluate the effect of riluzole on the activity of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, and to investigate the role of protein kinase C (PKC) in this effect. EAAT3 expression was induced in Xenopus oocytes by injecting EAAT3 mRNA. Using the two-electrode voltage clamping method, membrane currents were recorded before, during, and after applying l-glutamate (30 μM) in the absence and presence of prior incubation with riluzole (0.3-100 μM). To study the effect of PKC on the riluzole-induced change in EAAT3 activity, oocytes were preincubated with 100 μM phorbol-12-myristate-13-acetate (PMA), a PKC activator, or PKC inhibitors (2 µM staurosporine and 100 µM chelerythrine) before the recording. Responses were quantified by integrating current traces and are reported in microCoulombs (μC). Riluzole reduced EAAT3 activity in a concentration-dependent manner (0.3-100 μM). Treatment of oocytes with PMA significantly increased the baseline and riluzole-reduced EAAT activity (P<0.05). In addition, treatment of oocytes with PKC inhibitors reduced basal transporter currents, but did not show a further significant decrease in the riluzole-reduced EAAT3 activity. These results suggest that riluzole reduces EAAT3 activity through PKC inhibition.

  5. Propofol reverses oxidative stress-attenuated glutamate transporter EAAT3 activity: evidence of protein kinase C involvement.

    PubMed

    Yun, Jung-Yeon; Park, Kum-Suk; Kim, Jin-Hee; Do, Sang-Hwan; Zuo, Zhiyi

    2007-06-22

    The authors investigated the effects of propofol on EAAT3 (excitatory amino acid transporter 3) activity under oxidative stress induced by tert-butyl hydroperoxide (t-BHP), and the mediation of these effects by protein kinase C (PKC). Rat EAAT3 was expressed in Xenopus oocytes and L-glutamate (30 microM)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to t-BHP (1-20 mM) for 10 min dose-dependently decreased EAAT3 activity, and t-BHP (5 mM) significantly decreased the Vmax, but not the Km of EAAT3 for glutamate, and propofol (1-100 microM) dose-dependently reversed this t-BHP-attenuated EAAT3 activity. Phorbol-12-myristate-13-acetate (a PKC activator), also abolished this t-BHP-induced reduction in EAAT3 activity, whereas staurosporine (a PKC inhibitor), significantly decreased EAAT3 activity. However, as compared with staurosporine or t-BHP alone, t-BHP and staurosporine in combination did not further reduce EAAT3 activity. A similar pattern was observed for chelerythrine (also a PKC inhibitor). In oocytes pretreated with combinations of t-BHP and PMA (or staurosporine), propofol failed to change EAAT3 activity. Our results suggest that propofol restores oxidative stress-reduced EAAT3 activity and that these effects of propofol may be PKC-mediated.

  6. Topical azithromycin and clarithromycin inhibit acute and chronic skin inflammation in sensitized mice, with apparent selectivity for Th2-mediated processes in delayed-type hypersensitivity.

    PubMed

    Ivetić Tkalčević, Vanesa; Cužić, Snježana; Kramarić, Miroslava Dominis; Parnham, Michael J; Eraković Haber, Vesna

    2012-02-01

    Macrolide antibiotics inhibit the secretion of Th1 cytokines while their effects on the release of Th2 cytokines are variable. We investigated molecular and cellular markers of Th1- and Th2-mediated inflammatory mechanisms and the anti-inflammatory activity of azithromycin and clarithromycin in phorbol 12-myristate 13-acetate (PMA) and oxazolone (OXA)-induced skin inflammation. Dexamethasone (50 μg/ear), azithromycin, and clarithromycin (500 μg/ear) reduced TNF-α and interleukin (IL)-1β concentration in ear tissue by inhibiting inflammatory cell accumulation in PMA-induced inflammation. In OXA-induced early delayed-type hypersensitivity (DTH), the macrolides (2 mg/ear) and dexamethasone (25 μg/ear) reduced ear tissue inflammatory cell infiltration and secretion of IL-4 while clarithromycin also decreased IFN-γ concentration. Macrolides showed better activity when administered after the challenge. In OXA-induced chronic DTH, azithromycin (1 mg/ear) reduced the number of ear tissue mast cells and decreased the concentration of IL-4 in ear tissue and of immunoglobulin (Ig)E in serum. Clarithromycin (1 mg/ear) reduced serum IgE concentration, possibly by a mechanism independent of IL-4, while both macrolides attenuated mast cell degranulation. In conclusion, azithromycin and clarithromycin attenuate pro-inflammatory cytokine production and leukocyte infiltration during innate immune reactions, while selectively affecting Th2 rather than Th1 immunity in DTH reactions.

  7. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  8. New lanostanes and naphthoquinones isolated from Antrodia salmonea and their antioxidative burst activity in human leukocytes.

    PubMed

    Shen, Chien-Chang; Shen, Yuh-Chiang; Wang, Yea-Hwey; Lin, Lie-Chwen; Don, Ming-Jaw; Liou, Kuo-Tong; Wang, Wen-Yen; Hou, Yu-Chang; Chang, Tun-Tschu

    2006-02-01

    Four new compounds were isolated from the basidiomata of the fungus Antrodia salmonea, a newly identified species of Antrodia (Aphyllophorales) in Taiwan. These new compounds are named as lanosta-8,24-diene-3beta,15alpha,21-triol (1), 24-methylenelanost-8-ene-3beta,15alpha,21-triol (2), 2,3-dimethoxy-5-(2',5'-dimethoxy-3',4'-methylenedioxyphenyl)-7-methyl-[1,4]-naphthoquinone (3), and 2,3-dimethoxy-6-(2',5'-dimethoxy-3',4'-methylenedioxyphenyl)-7-methyl-[1,4]-naphthoquinone (4), respectively. Their structures were elucidated by spectroscopic methods. An in vitro cellular functional assay was performed to evaluate their anti-oxidative burst activity in human leukocytes. They showed inhibitory effects against phorbol 12-myristate-13-acetate (PMA), a direct protein kinase C activator, induced oxidative burst in neutrophils (PMN) and mononuclear cells (MNC) with 50 % inhibitory concentration (IC(50)) ranging from 3.5 to 25.8 microM. The potency order of these compounds in PMA-activated leukocytes was as 1 > 3 > 4 > 2. They were relatively less effective in formyl-Met-Leu-Phe (fMLP), a G-protein coupled receptor agonist, induced oxidative burst, except for compounds 3 and 4 in fMLP-activated PMN. These results indicated that three (1, 3, and 4) of these four newly identified compounds displayed anti-oxidative effect in human leukocytes with different potency and might confer anti-inflammatory activity to these drugs.

  9. An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking.

    PubMed Central

    Miller, C L; Lee, J H; Kieff, E; Longnecker, R

    1994-01-01

    The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-type control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases. Images Fig. 2 Fig. 3 Fig. 4 PMID:8290598

  10. Interrelationship between growth factor-induced pH changes and intracellular Ca/sup 2 +/

    SciTech Connect

    Ives, H.E.; Daniel, T.O.

    1987-04-01

    Many mitogens cause rapid changes in intracellular pH and Ca/sup 2 +/. The authors studied the patterns of pH and Ca/sup 2 +/ changes after exposure of murine fibroblasts to platelet-derived growth factor (PDGF), bombesin, phorbol 12-myristate 13-acetate (PMA), and the vasoactive peptide bradykinin. Intracellular pH and Ca/sup 2 +/ were measured by using the fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2. Three distinct patterns of intracellular pH change were observed. (i) PDGF and bombesin caused a rapid cytoplasmic acidification of 0.03 pH unit followed by a slower alkalinization of approx. = 0.11 pH unit above the resting pH of 6.88. (ii) PMA caused alkalinization without causing the early acidification. (iii) Bradykinin caused rapid acidification without the slower net alkalinization. All acidification responses were amiloride resistant. Patterns of intracellular Ca/sup 2 +/ response were also determined for each agent. In Ca/sup 2 +/-buffered cells, PDGF, bombesin, bradykinin, and ionomycin failed to induce cellular acidification, but alkalinization responses to PDGF, bombesin, and PMA persisted. They propose that the transient acidification seen with PDGF, bombesin, and other agents is the result of increased intracellular Ca/sup 2 +/. However, growth factor-induced alkalinization via the Na/sup +//H/sup +/ exchanger is independent of changes in Ca/sup 2 +/.

  11. Control of EGF receptor function by protein kinase C

    SciTech Connect

    Whiteley, B.J.

    1986-01-01

    Treatment of human epidermoid carcinoma A431 cells with nanomolar concentrations of the potent tumor promotor, phorbol 12-myristate 13-acetate (PMA), is shown to attentuate the ability of epidermal growth factor (EGF) or serum to activate Na/sup +//H/sup +/ exchange, which is measured as an amiloride-inhibitable pH/sub i/ increase or /sup 22/Na/sup +/ uptake. The ability of PMA to directly activate Na/sup +//H/sup +/ exchange is also reported, but PMA-induced pH/sub i/ increases are modest with respect to those of EGF or serum and require relatively high concentrations of PMA. The effects of PMA on mitogen receptor-stimulated Na/sup +//H/sup +/ exchange were examined in the mouse fibroblast NR6 cell line using platelet-derived growth factor (PDGF). The results were similar to those in A431 cells, except that PMA in NR6 cells causes pH/sub i/ increases at lower concentrations. Phorbol diester action is mediated by the activity of the enzyme protein kinase C. The results summarized above support the hypothesis that PMA-induced protein kinase C activity opposes mitogenic stimulation. The presumed endogenous PMA analog is diacylglycerol, which is generated by phosphoinositide hydrolysis and has been reported to be produced in response to the mitogens, EGF and PDGF.

  12. Gamma irradiation enhances biological activities of mulberry leaf extract

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

    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, PGE2, 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.

  13. Inhibitory effects of Chikusetsusaponin IVa on lipopolysaccharide-induced pro-inflammatory responses in THP-1 cells.

    PubMed

    Wang, H; Qi, J; Li, L; Wu, T; Wang, Y; Wang, X; Ning, Q

    2015-09-01

    This study investigated anti-inflammatory effects and possible mechanisms of Chikusetsusaponin IVa (Chi IVa), one of the main bioactive components in saponins from Panacis japonica (SPJ), which is used in traditional Tujia and Hmong Chinese medicine. To this end, changes in the inflammatory profiles of lipopolysacchride (LPS)-stimulated phrobol 12-myristate 13-acetate(PMA)-differented THP-1 macrophages were evaluated following Chi IVa treatment. The results showed that Chi IVa markedly decreased the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) at both the mRNA and protein level, which proved to be dose-dependent. Further studies revealed that Chi IVa strongly suppressed NF-κB activation and downregulated the phosphorylation of ERK, p38, and JNK. Our present study demonstrates that Chi IVa suppresses the production of iNOS, COX-2, IL-1β, IL-6, and TNF-α in LPS-stimulated THP-1 cells likely by inhibiting NF-κB activation and ERK, JNK, and p38 signal pathway phosphorylation.

  14. Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca2+ clearance from MCF-7 breast cancer cells.

    PubMed

    Varga, Karolina; Pászty, Katalin; Padányi, Rita; Hegedűs, Luca; Brouland, Jean-Philippe; Papp, Béla; Enyedi, Agnes

    2014-02-01

    The expression of the plasma membrane Ca2+ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca2+ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression - either by treatment or overexpression - led to enhanced Ca2+ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca2+ homeostasis. These results suggest that modulation of Ca2+ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.

  15. Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells.

    PubMed Central

    Goldgaber, D; Harris, H W; Hla, T; Maciag, T; Donnelly, R J; Jacobsen, J S; Vitek, M P; Gajdusek, D C

    1989-01-01

    We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter. Images PMID:2508093

  16. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.

    PubMed

    Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth J; Siller, Richard; Srsen, Vlastimil; Morgan, Katie; Bryans, Anna; Kozlowska, Ada; Koulovasilopoulos, Andreas; Underwood, Ian; Smith, Stewart; Del-Pozo, Jorge; Moss, Sharon; Thompson, Alexandra Inés; Henderson, Neil C; Hayes, Peter C; Plevris, John N; Bagnaninchi, Pierre-Olivier; Nelson, Leonard J

    2017-01-30

    Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

  17. Human immunodeficiency virus type 1 RNA detection in peripheral blood mononuclear cells by polymerase chain reaction: enhanced sensitivity after mitogenic stimulation.

    PubMed

    Tetali, S K; Oyaizu, N; Paul, M; Pahwa, S

    1993-01-01

    The aim of this study was to investigate whether stimulus-induced up-regulation of human immunodeficiency virus type 1 (HIV-1) expression in peripheral blood mononuclear cells (PBMC) could enhance the diagnostic sensitivity of the polymerase chain reaction (PCR). PBMC derived from 11 HIV-1-infected asymptomatic adults were cultured with a stimulus of phytohemagglutinin (PHA) plus phorbol 12-myristate 13-acetate (PMA) for 36 h prior to lysing the cells for PCR. In all 11 patients studied, the intensity of PCR-assisted HIV RNA amplification (RNA-PCR) performed on stimulated cells was significantly (p < 0.001) higher than that obtained on unstimulated cells. A comparison of conventional PCR-assisted DNA amplification (DNA-PCR) with that of RNA-PCR was made on seven patients. The sensitivity of DNA-PCR was also increased by prior stimulation of cells, although not to the same extent as was observed for RNA-PCR. The results of our study indicate that the sensitivity of PCR can be significantly enhanced by prior activation of cells with PHA and PMA.

  18. ADAM-10-Mediated N-cadherin Cleavage is Protein Kinase C-α–Dependent and Promotes Glioblastoma Cell Migration

    PubMed Central

    Kohutek, Zachary A.; diPierro, Charles G.; Redpath, Gerard T.; Hussaini, Isa M.

    2009-01-01

    Matrix metalloproteinases (MMPs) and the related ‘a disintegrin and metalloproteinases’ (ADAMs) promote tumorigenesis by cleaving extracellular matrix and protein substrates, including N-cadherin. While N-cadherin is thought to regulate cell adhesion, migration and invasion, its role has not been characterized in glioblastomas (GBMs). In this study, we investigated the expression and function of post-translational N-cadherin cleavage in GBM cells as well as its regulation by protein kinase C (PKC). N-cadherin cleavage occurred at a higher level in glioblastoma cells than in non-neoplastic astrocytes. Treatment with the PKC-activator phorbol 12-myristate 13-acetate (PMA) increased N-cadherin cleavage, which was reduced by pharmacological inhibitors and siRNA specific for ADAM-10 or PKC-α. Furthermore, treatment of GBM cells with PMA induced the translocation of ADAM-10 to the cell membrane, the site where N-cadherin was cleaved, and this translocation was significantly reduced by the PKC-α inhibitor Gö6976 or PKC-α shRNA. In functional studies, N-cadherin cleavage was required for GBM cell migration, as depletion of N-cadherin cleavage by N-cadherin siRNA, ADAM-10 siRNA, or a cleavage-site mutant N-cadherin, decreased GBM cell migration. Taken together, these results suggest that N-cadherin cleavage is regulated by a PKC-α-ADAM-10 cascade in GBM cells and may be involved in mediating GBM cell migration. PMID:19357285

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

    PubMed

    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.

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

    SciTech Connect

    Moors, Michaela . E-mail: moors@uni-duesseldorf.de; Cline, Jason E. . E-mail: jason.cline@uni-duesseldorf.de; Abel, Josef . E-mail: josef.abel@uni-duesseldorf.de; Fritsche, Ellen . E-mail: ellen.fritsche@uni-duesseldorf.de

    2007-05-15

    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.

  1. Effect of cinnamon water extract on monocyte-to-macrophage differentiation and scavenger receptor activity

    PubMed Central

    2014-01-01

    Background Water soluble cinnamon extract has been shown to increase insulin sensitivity and modulate macrophage activation, a desirable trait for the management of obesity or atherosclerosis. Our present study investigated whether cinnamon water extract (CWE) may influence the differentiation of monocytes into macrophages and the activity of macrophage scavenger receptors, commonly observed in atherosclerotic lesions. Methods We investigated the effect of CWE on the expression of various surface markers and the uptake of acetylated low density lipoprotein (LDL) in phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cells. The protein levels of PMA or macrophage-colony stimulating factor (M-CSF)-stimulated type 1 macrophage scavenger receptor (SRA) were analyzed. Finally, the role of extracellar signal-related kinase (ERK) 1/2 in SRA synthesis and the effect of CWE on PMA-stimulated ERK1/2 were determined. Results CWE inhibited the differentiation of monocyte by decreasing the expression of CD11b, CD36 and SRA and the uptake of acetyl LDL. CWE suppressed the upregulation of SRA by M-CSF and modulated ERK1/2 activity, which was required for PMA-induced SRA synthesis. Conclusions Our results demonstrate that CWE was able to interfere with monocyte differentiation and macrophage scavenger activity, indicating its potential in preventing the development of atherosclerotic lesions. PMID:24602512

  2. Mechanism for uptake of silica particles by monocytic U937 cells.

    PubMed

    Hetland, G; Namork, E; Schwarze, P E; Aase, A

    2000-07-01

    We examined the mechanism for uptake by monocytic cells of particles found in the atmosphere of some industrial work places. As a model system, irregular crystalline silica particles (SPs), sphere-like cryptocrystalline microsilica particles (MPs) and carbon particles (CPs) were exposed to pro-monocytic U937 cells. Plasma-treated SP and MP, but not CP, activated the alternative complement pathway, but bound little C3b. However, all particles adsorbed serum IgG, IgA and IgM unspecifically. Phenotyping of U937 cells for complement receptors (CRs) and Fcgamma receptors (FcgammaRs) showed that interferon gamma (INFgamma) increased expression of FcgammaRI, CR3 (CD11b/CD18) and CR4 (CD11c/CD18) and that phorbol-12-myristate-13-acetate (PMA) increased expression of CR4. Scanning electron microscopy (SEM) demonstrated higher phagocytosis of plasma-treated SP than native SP by both PMA- and INFgamma-stimulated, but not unstimulated, cells. MP and CP could not be distinguished from cellular structures. Inhibition experiments in SEM revealed uptake of heparin-plasma-treated SP via FcgammaRI on INFgamma-stimulated U937 cells, but could not exclude possible participation of CR3. The results indicate that plasma-treated SPs bind Ig and are internalized by differentiated monocytic cells via FcgammaRI, which is known to trigger cellular production of toxic oxygen species that may induce pulmonary inflammation in vivo.

  3. Multiple receptors mobilize calcium through a pertussis toxin (PT) sensitive GTP-binding protein in human neutrophils (PMN's)

    SciTech Connect

    Lad, P.M.; Olson, C.V.; Grewal, I.S.; Frolich, M.; Scott, S.J.

    1986-03-05

    Treatment of PMN's with PT causes an abolition of chemotaxis, enzyme release, superoxide generation and aggregation caused by f-met-leu-phe (FMLP),C5a and platelet activating factor (PAF). Lectin (Con-A) induced capping and receptor induced shape change are abolished, but phagocytosis is unaltered. In whole cells, calcium mobilization induced by FMLP, PAF and Con-A inhibited by PT although the FMLP-mediated effect is more susceptible to PT's effects. Treatment of PMN's with phorbol 12-myristate 13 acetate (PMA) causes an abolition of calcium mobilization by all agents in a range which also inhibits cap formation. Investigation of calcium uptake reveals PT sensitive and insensitive components. Reciprocal interactions between Ns and Ni proteins are also observed since pretreatment with FMLP and PAF causes a stimulation of Ns-mediated cyclic AMP enhancement while pretreatment with Ns linked receptors (PGE/sub 1/ and beta receptor agonists) inhibits calcium mobilization. Comparative peptide mapping studies indicate substantial similarity between Ni proteins in PMN's, platelets and human erythrocytes. The authors results suggest that the Ni linked calcium mobilization sensitive to PMA is important to the regulation of the human neutrophil.

  4. Epidermal growth factor (EGF) stimulated Ca/sup 2 +/ mobilization in hepatocytes is abolished by phorbol esters, pertussis toxin and partial hepatectomy

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1986-05-01

    EGF has been demonstrated to increase free intracellular Ca/sup 2 +/ levels in isolated hepatocytes putatively by generation of the second messenger inositol trisphosphate (IP/sub 3/). Pretreatment of cells with phorbol 12-myristate 13-acetate (PMA) inhibited the EGF (66 nM) stimulated Ca/sup 2 +/ response as measured by quin2. Inhibition by PMA was maximal within 3 min and was concentration dependent (IC/sub 50/ = 13.5 nM). Four other active phorbol ester analogues blocked the Ca/sup 2 +/ response while inactive analogues did not. EGF was unable to increase intracellular Ca/sup 2 +/ levels in hepatocytes isolated from rats treated with pertussis toxin for 72 hrs. Neither PMA nor toxin pretreatment was able to inhibit the Ca/sup 2 +/ response to angiotensin II (Ang II). In hepatocytes isolated 24 hrs after partial hepatectomy, the Ca/sup 2 +/ response to EGF (as measured by phosphorylase activity, EC/sub 50/ = 5 nM) was completely abolished and remained attenuated for 7 days post-hepatectomy. The Ca/sup 2 +/ response to Ang II in this model system was also blunted but required 3 days for development of the full effect and within 7 days full activity is nearly restored. The results suggest that fundamental differences exist in the transduction mechanisms used by these two Ca/sup 2 +/-linked hormones to mobilize intracellular Ca/sup 2 +/ (and putatively increase IP/sub 3/ formation).

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

    SciTech Connect

    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 ({sup 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. ({sup 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 ({sup 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 ({sup 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.

  6. Intracellular calcium rise is not a necessary step for the stimulated actin polymerization

    SciTech Connect

    Yassin, R.

    1986-03-01

    Stimulation of rabbit peritoneal neutrophils by many chemotactic (formyl Methionyl-Leucyl-Phenylalanine (fMLP), Leukotriene B/sub 4/ (LTB/sub 4/)) and non-chemotactic (phorbol 12-myristate, 13-acetate (PMA), platelet activating factor (PAF), and the calcium ionophore A23187) factors produces rapid and dose dependent increases in the amount of actin associated with the cytoskeleton. The stimulated increase in cytoskeletal actin does not appear to require a rise in the intracellular concentration of free calcium. The increase in cytoskeletal actin produced by A23187 is transient and does not depend on the presence of calcium in the suspending medium. In the presence of extracellular calcium, the effect of the ionophore is biphasic with respect to concentration. The increases in actin association with cytoskeletal produced by fMLP, LTB/sub 4/, and A23187 but not by PMA, are inhibited by hyperosmolarity and pertussis toxin pretreatment. On the other hand, the addition of hyperosmolarity or pertussis toxin has small effect on the rise in the intracellular calcium produced by A23187. The results presented here suggest that an increase in the intracellular concentration of free calcium is not necessary for the stimulated increases in cytoskeletal actin.

  7. Nonrandom duplication of the chromosome bearing a mutated Ha-ras-1 allele in mouse skin tumors.

    PubMed Central

    Bianchi, A B; Aldaz, C M; Conti, C J

    1990-01-01

    We analyzed the normal/mutated allelic ratio of the Ha-ras-1 gene in mouse skin squamous cell carcinomas induced by initiation with dimethylbenz[a]anthracene and promotion with phorbol 12-myristate 13-acetate. DNA for these studies was obtained from short-term tumor cultures (24-72 hr) to eliminate the contribution of stromal and inflammatory cells to the sample. The allelotypic analysis was performed in 25 squamous cell carcinomas by quantitative radio-analysis of the Xba I restriction fragment length polymorphism as detected by BS9, a v-Ha-ras probe, and rehybridization of the Southern blots with probes for chromosomes 7 and 8. Approximately 85% of the tumors presented overrepresentation of the mutated allele in the form of 1 normal/2 mutated (12 tumors), 0 normal/3 mutated (4 tumors), 0 normal/2 mutated (3 tumors), and gene amplification (3 tumors). No tumor was found with a 2 normal/1 mutated allelic ratio. These results support our previous cytogenetic studies, indicating that trisomy of chromosome 7 is present in the majority of these tumors and show that nonrandom duplication of the chromosome carrying the mutated Ha-ras-1 allele appears to be a major mechanism by which the mutated gene is overrepresented. Images PMID:1697691

  8. Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes.

    PubMed

    Kalgraff, Cathrine A K; Wergeland, Heidrun I; Pettersen, Eirin Fausa

    2011-09-01

    The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.

  9. Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout, Oncorhynchus mykiss (Walbaum).

    PubMed

    Chettri, J K; Holten-Andersen, L; Buchmann, K

    2010-07-01

    Abstract Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and beta-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15-20 degrees C and a reduced activity at temperature extremes (1, 4 and 30 degrees C). Highest activity was found at a cell concentration of 1 x 10(7) cells mL(-1). Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.

  10. Caffeic acid phenethyl ester inhibits T-cell activation by targeting both nuclear factor of activated T-cells and NF-kappaB transcription factors.

    PubMed

    Márquez, Nieves; Sancho, Rocío; Macho, Antonio; Calzado, Marco A; Fiebich, Bernd L; Muñoz, Eduardo

    2004-03-01

    Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been shown to reveal anti-inflammatory properties. Since T-cells play a key role in the onset of several inflammatory diseases, we have evaluated the immunosuppressive activity of CAPE in human T-cells, discovering that this phenolic compound is a potent inhibitor of early and late events in T-cell receptor-mediated T-cell activation. Moreover, we found that CAPE specifically inhibited both interleukin (IL)-2 gene transcription and IL-2 synthesis in stimulated T-cells. To further characterize the inhibitory mechanisms of CAPE at the transcriptional level, we examined the DNA binding and transcriptional activities of nuclear factor (NF)-kappaB, nuclear factor of activated cells (NFAT), and activator protein-1 (AP-1) transcription factors in Jurkat cells. We found that CAPE inhibited NF-kappaB-dependent transcriptional activity without affecting the degradation of the cytoplasmic NF-kappaB inhibitory protein, IkappaBalpha. However, both NF-kappaB binding to DNA and transcriptional activity of a Gal4-p65 hybrid protein were clearly prevented in CAPE-treated Jurkat cells. Moreover, CAPE inhibited both the DNA-binding and transcriptional activity of NFAT, a result that correlated with its ability to inhibit phorbol 12-myristate 13-acetate plus ionomycin-induced NFAT1 dephosphorylation. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory activities of this natural compound.

  11. Protein kinase C involvement in homologous desensitization of delta-opioid receptor coupled to Gi1-phospholipase C activation in Xenopus oocytes.

    PubMed

    Ueda, H; Miyamae, T; Hayashi, C; Watanabe, S; Fukushima, N; Sasaki, Y; Iwamura, T; Misu, Y

    1995-11-01

    We have developed the coexpression system of both delta-opioid receptor (DOR1) and M2-muscarinic receptor (M2) which mediate agonist-evoked currents due to common post-receptor mechanisms including Gi1 and phospholipase C (PLC) activation in Xenopus oocytes reconstituted with Gi1 alpha. The DOR1-currents by 100 nM D-Ser2-leu-enkephalin-Thr6 (DSLET) were selectively desensitized by 10 nM phorbol 12-myristate 13-acetate (PMA). The PMA-desensitization of DSLET-currents was abolished in the presence of calphostin C, a protein kinase C inhibitor, or reversed by an intracellular injection of calcineurin, a protein phosphatase 2B. When a higher concentration (3 microM) of DSLET was used, DSLET-currents were rapidly desensitized by repeated challenges of DSLET itself. However, repeated challenges of 10 microM ACh caused no influence on such DSLET- or M2-currents. The desensitization of DSLET-currents was selectively reversed by protein kinase C inhibitors. Similar results were also obtained with various delta-opioid agonists. These results suggest that protein kinase C is involved in the homologous desensitization of delta-opioid receptors.

  12. Ca2+/Calmodulin-Dependent Kinase Kinase α Is Expressed by Monocytic Cells and Regulates the Activation Profile

    PubMed Central

    Guest, Christopher B.; Deszo, Eric L.; Hartman, Matthew E.; York, Jason M.; Kelley, Keith W.; Freund, Gregory G.

    2008-01-01

    Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca2+/calmodulin-dependent kinase kinase α (CaMKKα) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKα by mass spectrometry and Western analysis. The function of CaMKKα in monocyte activation was examined using the CaMKKα inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKα, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKα to the nucleus. Finally, to further examine monocyte activation profiles, TNFα and IL-10 secretion were studied. CaMKKα inhibition attenuated PMA-dependent IL-10 production and enhanced TNFα production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKα in the differentiation of monocytic cells. PMID:18270593

  13. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    PubMed

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.

  14. The natural compound magnolol inhibits invasion and exhibits potential in human breast cancer therapy

    PubMed Central

    Liu, Ying; Cao, Wei; Zhang, Bo; Liu, Yong-qiang; Wang, Zhong-yuan; Wu, Yan-ping; Yu, Xian-jun; Zhang, Xu-dong; Ming, Ping-hong; Zhou, Guang-biao; Huang, Laiqiang

    2013-01-01

    Invasion and metastasis are the main causes of treatment failure and death in breast cancer. Thus, novel invasion-based therapies such as those involving natural agents are urgently required. In this study, we examined the effects of magnolol (Mag), a compound extracted from medicinal herbs, on breast cancer cells in vitro and in vivo. Highly invasive cancer cells were found to be highly sensitive to treatment. Mag markedly inhibited the activity of highly invasive MDA-MB-231 cells. Furthermore, Mag significantly downregulated matrix metalloproteinase-9 (MMP-9) expression, an enzyme critical to tumor invasion. Mag also inhibited nuclear factor-κB (NF-κB) transcriptional activity and the DNA binding of NF-κB to MMP-9 promoter. These results indicate that Mag suppresses tumor invasion by inhibiting MMP-9 through the NF-κB pathway. Moreover, Mag overcame the promoting effects of phorbol 12-myristate 13-acetate (PMA) on the invasion of MDA-MB-231 cells. Our findings reveal the therapeutic potential and mechanism of Mag against cancer. PMID:24226295

  15. Ec sub. gamma. receptor type III (CD16) is included in the. zeta. NK receptor complex expressed by human natural killer cells

    SciTech Connect

    Anderson, P.; Caligiuri, M.; O'Brien, C.; Manley, T.; Ritz, J.; Schlossman, S.F. )

    1990-03-01

    The authors recently reported that CD3{sup {minus}} natural killer (NK) cells express the {zeta} chain of the T-cell receptor complex ({zeta} NK) in association with higher molecular weight structures whose expression differs between individual NK cell clones. Because NK cell cytolytic activity is known to be triggered by perturbation of the type III Fc{sub {gamma}} receptor (CD16), they sought to determine whether this activating molecule is included in the {zeta}NK molecular complex. Biochemical evidence for a physical association between CD16 and {zeta}NK was obtained by comparing immunoprecipitates formed using monoclonal antibodies reactive with each of these molecules by SDS/polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping. In both clonal and polyclonal populations of CD3{sup {minus}}NK cells, CD16 and {zeta}NK specifically associated with one another. Functional evidence for a specific association between CD16 and {zeta}NK in intact cells was obtained by demonstrating a coordinate down-modulation of both of these molecules induced by either phorbol 12-myristate 13-acetate or monoclonal antibodies reactive with CD16. The results suggest that Fc{sub {gamma}} receptor type III (CD16) is included in the {zeta}NK complex and that this complex is likely to play an important role in NK cell activation.

  16. Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein.

    PubMed

    Xu, Long; Ting-Lou; Lv, Nonghua; Zhu, Xuan; Chen, Youxiang; Yang, Jing

    2009-08-01

    Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway.

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

    PubMed

    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.

  18. Aspalathin and nothofagin from rooibos (Aspalathus linearis) inhibit endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Kwak, Soyoung; Han, Min-Su; Bae, Jong-Sup

    2015-01-01

    Aspalathin (Asp) and nothofagin (Not) are two major active dihydrochalcones found in green rooibos, which have been reported for their anti-oxidant activity. Increasing evidence has demonstrated that beyond its role in the activation of protein C, endothelial cell protein C receptor (EPCR) is also involved in vascular inflammation. EPCR activity is markedly changed by ectodomain cleavage and its release as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of Asp and Not on EPCR shedding. Our results demonstrated that Asp and Not induced potent inhibition of phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α-, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-induced EPCR shedding. Asp and Not also inhibited the expression and activity of PMA-induced TACE in endothelial cells. Asp and Not also suppressed CLP-induced protein C decrease in mice and thrombin generation in HUVECs. In addition, treatment with Asp and Not resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of Asp and Not as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

  19. Priming by grepafloxacin on respiratory burst of human neutrophils: its possible mechanism.

    PubMed

    Niwa, Masayuki; Kanamori, Yutaka; Hotta, Koichi; Matsuno, Hiroyuki; Kozawa, Osamu; Fujimoto, Sadaki; Uematsu, Toshihiko

    2002-10-01

    Grepafloxacin is a broad-spectrum fluoroquinolone derivative that has good tissue penetration. We demonstrated that grepafloxacin showed a priming effect on neutrophil respiratory burst, triggered by either a chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4), but not by the phorbol ester phorbol 12-myristate 13-acetate (PMA). The priming effect of grepafloxacin on fMLP-stimulated superoxide generation by human neutrophils correlated with the penetration of grepafloxacin into cells. Removal of extracellular grepafloxacin did not inhibit the priming effect on fMLP-stimulated superoxide generation. Furthermore, grepafloxacin induced the translocation of p47-phox and p67-phox to the membrane fraction of neutrophils, whereas tyrosine phosphorylation was hardly observed in neutrophils exposed to grepafloxacin. The priming effect of grepafloxacin on superoxide generation from neutrophils was not inhibited by treatment with pertussis toxin, a protein-tyrosine kinase inhibitor (ST-638) or a protein kinase C inhibitor (calphostin C), or chelation of extracellular calcium. Grepafloxacin did not change the fMLP receptor-binding properties. Taken together, these findings suggest that grepafloxacin evokes a priming effect on neutrophil superoxide generation intracellularly through the translocation of p47-phox and even p67-phox protein to the membrane fractions. GTP binding protein, protein-tyrosine phosphorylation and protein kinase C activation are not involved in the priming effect.

  20. Suplatast tosilate alleviates nasal symptoms through the suppression of nuclear factor of activated T-cells-mediated IL-9 gene expression in toluene-2,4-diisocyanate-sensitized rats.

    PubMed

    Mizuguchi, Hiroyuki; Orimoto, Naoki; Kadota, Takuya; Kominami, Takahiro; Das, Asish K; Sawada, Akiho; Tamada, Misaki; Miyagi, Kohei; Adachi, Tsubasa; Matsumoto, Mayumi; Kosaka, Tomoya; Kitamura, Yoshiaki; Takeda, Noriaki; Fukui, Hiroyuki

    2016-03-01

    Histamine H1 receptor (H1R) gene is upregulated in patients with pollinosis; its expression level is highly correlated with the nasal symptom severity. Antihistamines are widely used as allergy treatments because they inhibit histamine signaling by blocking H1R or suppressing H1R signaling as inverse agonists. However, long-term treatment with antihistamines does not completely resolve toluene-2,4-diisocyanate (TDI)-induced nasal symptoms, although it can decrease H1R gene expression to the basal level, suggesting additional signaling is responsible for the pathogenesis of the allergic symptoms. Here, we show that treatment with suplatast tosilate in combination with antihistamines markedly alleviates nasal symptoms in TDI-sensitized rats. Suplatast suppressed TDI-induced upregulation of IL-9 gene expression. Suplatast also suppressed ionomycin/phorbol-12-myristate-13-acetate-induced upregulation of IL-2 gene expression in Jurkat cells, in which calcineurin (CN)/nuclear factor of activated T-cells (NFAT) signaling is known to be involved. Immunoblot analysis demonstrated that suplatast inhibited binding of NFAT to DNA. Furthermore, suplatast suppressed ionomycin-induced IL-9 mRNA upregulation in RBL-2H3 cells, in which CN/NFAT signaling is also involved. These data suggest that suplatast suppressed NFAT-mediated IL-9 gene expression in TDI-sensitized rats and this might be the underlying mechanism of the therapeutic effects of combined therapy of suplatast with antihistamine.

  1. Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

    2016-12-01

    Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos.

  2. CD107a as a marker of activation in chicken cytotoxic T cells.

    PubMed

    Wattrang, Eva; Dalgaard, Tina S; Norup, Liselotte R; Kjærup, Rikke B; Lundén, Anna; Juul-Madsen, Helle R

    2015-04-01

    The study aimed to evaluate cell surface mobilisation of CD107a as a general activation marker on chicken cytotoxic T cells (CTL). Experiments comprised establishment of an in vitro model for activation-induced CD107a mobilisation and design of a marker panel for the detection of CD107a mobilisation on chicken CTL isolated from different tissues. Moreover, CD107a mobilisation was analysed on CTL isolated from airways of infectious bronchitis virus (IBV)-infected birds direct ex vivo and upon in vitro stimulation. Results showed that phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin was a consistent inducer of CD107a cell surface mobilisation on chicken CTL in a 4h cell culture model. In chickens experimentally infected with IBV, higher frequencies of CTL isolated from respiratory tissues were positive for CD107a on the cell surface compared to those from uninfected control chickens indicating in vivo activation. Moreover, upon in vitro PMA+ ionomycin stimulation, higher proportions of CTL isolated from the airways of IBV-infected chickens showed CD107a mobilisation compared to those from uninfected control chickens. Monitoring of CD107a cell surface mobilisation may thus be a useful tool for studies of chicken CTL cytolytic potential both in vivo and in vitro.

  3. Insulin and phorbol ester stimulate conductive Na/sup +/ transport through a common pathway

    SciTech Connect

    Civan, M.M.; Peterson-Yantorno, K.; O'Brien, T.G.

    1988-02-01

    Insulin stimulates Na/sup +/ transport across frog skin, toad urinary bladder, and the distal renal nephron. This stimulation reflects an increase in apical membrane Na/sup +/ permeability and a stimulation of the basolateral membrane Na,K-exchange pump. Considerable indirect evidence has suggested that the apical natriferic effect of insulin is mediated by activation of protein kinase C. However, no direct information has been available documenting that insulin and protein kinase C indeed share a common pathway in stimulating Na/sup +/ transport across frog skin. In the present work, the authors have studied the interaction of insulin and phorbol 12-myristate 13-acetate (PMA), a documented activator of protein kinase C. Preincubation of skins with 1,2-dioctanoylglycerol, another activator of protein kinase C, increases baseline Na/sup +/ transport and reduces the subsequent natriferic response to PMA. Preincubation with PMA markedly reduces the subsequent natriferic action of insulin. This effect does not appear to primarily reflect PMA-induced internalization of insulin receptors. The insulin receptors are localized on the basolateral surface of frog skin, but the application of PMA to this surface is much less effective than mucosal treatment in reducing the response to insulin. The current results provide documentation that insulin and protein kinase C share a common pathway in stimulating Na/sup +/ transport across frog skin. The data are consistent with the concept that the natriferic effect of insulin on frog skin is, at least in part, mediated by activation of protein kinase C.

  4. Protective role of intracellular zinc in myocardial ischemia/reperfusion is associated with preservation of protein kinase C isoforms.

    PubMed

    Karagulova, Gulnura; Yue, Yuankun; Moreyra, Abel; Boutjdir, Mohamed; Korichneva, Irina

    2007-05-01

    The recent discovery of zinc signals and their essential role in the redox signaling network implies that zinc homeostasis and the function of zinc-containing proteins are probably altered as a result of oxidative stress, suggesting new targets for pharmacological intervention. We hypothesized that the level of intracellular labile zinc is changed in hearts subjected to ischemia/reperfusion (I/R) and investigated whether the maintenance of myocardial zinc status protected heart functions. Using fluorescent imaging, we demonstrated decreased levels of labile zinc in the I/R hearts. Phorbol 12-myristate 13-acetate, a known trigger of zinc release, liberated zinc ions in control hearts but failed to produce any increase in zinc levels in the I/R rat hearts. Adding the zinc ionophore pyrithione at reperfusion improved myocardial recovery up to 100% and reduced the incidence of arrhythmias more than 2-fold. This effect was dose-dependent, and high concentrations of zinc were toxic. Adding membrane-impermeable zinc chloride was ineffective. Hearts from rats receiving zinc pyrithione supplements in their diet fully recovered from I/R. The recovery was associated with the prevention of degradation of the two protein kinase C isoforms, delta and epsilon, during I/R. In conclusion, our results suggest a protective role of intracellular zinc in myocardial recovery from oxidative stress imposed by I/R. The data support the potential clinical use of zinc ionophores in the settings of acute redox stress in the heart.

  5. Anti-inflammatory drugs and tumor necrosis factor-alpha production from monocytes: role of transcription factor NF-kappa B and implication for rheumatoid arthritis therapy.

    PubMed

    Lavagno, Luisa; Gunella, Gabriele; Bardelli, Claudio; Spina, Simona; Fresu, Luigia Grazia; Viano, Ilario; Brunelleschi, Sandra

    2004-10-06

    Inhibition of tumor necrosis factor-alpha (TNF-alpha) represents a relevant target in rheumatoid arthritis therapy. Besides inhibiting cyclooxygenase, anti-inflammatory drugs can affect the activation of transcription factors. We investigated the ability of dexamethasone, indomethacin, and rofecoxib to modulate nuclear factor-kappaB (NF-kappaB) activation and TNF-alpha release from human monocytes challenged with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Both stimuli induced NF-kappaB nuclear translocation and TNF-alpha secretion. Dexamethasone potently inhibited TNF-alpha release, indomethacin inhibited only PMA-evoked release, while rofecoxib had no effect. In the electrophoretic mobility shift assay, dexamethasone and rofecoxib dose-dependently inhibited the DNA binding activity of NF-kappaB in stimulated monocytes, whereas indomethacin failed to inhibit the LPS-evoked one. These results were further confirmed by evaluating the drugs' ability to reduce nuclear NF-kappaB subunits, as well as the amount of phosphorylated IkappaBalpha in cytosolic fractions. In conclusion, these results indicate that anti-inflammatory drugs differ largely in their ability to inhibit NF-kappaB activity and/or TNF-alpha release from human monocytes. These effects can be relevant to rheumatoid arthritis therapy.

  6. Interaction between phosphoinositide turnover system and cyclic AMP pathway for the secretion of pancreastatin and somatostatin from QGP-1N cells.

    PubMed

    Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y

    1992-06-30

    It is found that secretion of pancreastatin and somatostatin from QGP-1N cells is regulated through muscarinic receptor-mediated activation of phosphatidylinositide hydrolysis system. In this report, whether the cAMP pathway interacts with the phosphoinositide turnover system for the secretion of pancreastatin and somatostatin from QGP-1N cells through muscarinic receptors was studied. Stimulation of QGP-1N cells with carbachol increased intracellular cAMP levels. The carbachol-induced increase in cAMP levels was inhibited by atropine. Calcium ionophore (A23187) and phorbol 12-myristate 13-acetate increased cAMP synthesis. Dibutyryl cAMP, forskolin and theophylline stimulated secretion of pancreastatin and somatostatin. When either dibutyryl cAMP, forskolin or theophylline was added in culture medium with A23187, phorbol ester or carbachol, a synergistic effect was found on pancreastatin and somatostatin secretion. These results suggest that interaction between the phosphoinositide turnover system and the cAMP pathway occurs in QGP-1N cells through muscarinic receptor stimulation for the secretion of pancreastatin and somatostatin.

  7. Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression inJurkat Cells

    PubMed Central

    Pan, Yao; Wei, Xuetao; Hao, Weidong

    2015-01-01

    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. PMID:26343699

  8. Modification of intracellular free calcium in cultured A10 vascular smooth muscle cells by exogenous phosphatidic acid.

    PubMed

    Bhugra, Praveen; Xu, Yan-Jun; Rathi, Satyajeet; Dhalla, Naranjan S

    2003-06-15

    Exogenous phosphatidic acid (PA) was observed to produce a concentration-dependent increase in [Ca(2+)](i) in cultured A10 vascular smooth muscle cells. Preincubation of cells with sarcoplasmic reticulum Ca(2+)-ATPase inhibitors (cyclopiazonic acid and thapsigargin), a phospholipase C inhibitor (2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate), inositol 1,4,5-trisphosphate receptor antagonists (2-aminoethoxydiphenyl borate and xestospongin), and an activator of protein kinase C (PKC) (phorbol 12-myristate 13-acetate) depressed the PA-evoked increase in [Ca(2+)](i). Although EGTA, an extracellular Ca(2+) chelator, decreased the PA-induced increase in [Ca(2+)](i), sarcolemmal Ca(2+)-channel blockers (verapamil or diltiazem) did not alter the action of PA. On the other hand, inhibitors of PKC (bisindolylmaleimide I) and G(i)-protein (pertussis toxin) potentiated the increase in [Ca(2+)](i) evoked by PA significantly. These results suggest that the PA-induced increase in [Ca(2+)](i) in vascular smooth muscle cells may occur upon the activation of phospholipase C and the subsequent release of Ca(2+) from the inositol 1,4,5-trisphosphate-sensitive Ca(2+) pool in the sarcoplasmic reticulum. This action of PA may be mediated through the involvement of PKC.

  9. Thymus cell antigen 1 (Thy1, CD90) is expressed by lymphatic vessels and mediates cell adhesion to lymphatic endothelium.

    PubMed

    Jurisic, Giorgia; Iolyeva, Maria; Proulx, Steven T; Halin, Cornelia; Detmar, Michael

    2010-10-15

    The lymphatic vascular system plays an important role in inflammation and cancer progression, although the molecular mechanisms involved are poorly understood. As determined by comparative transcriptional profiling studies of ex vivo isolated mouse intestinal lymphatic endothelial cells versus blood vascular endothelial cells, thymus cell antigen 1 (Thy1, CD90) was expressed at much higher levels in lymphatic endothelial cells than in blood vascular endothelial cells. These findings were confirmed by quantitative PCR, and at the protein level by FACS and immunofluorescence analyses. Thy1 was also strongly expressed by tumor-associated lymphatic vessels, as evaluated in a B16 melanoma footpad model in mice. Blockade of Thy1 inhibited tumor cell adhesion to cultured mouse lymphatic endothelial cells. Importantly, treatment of human dermal microvascular endothelial cells with tumor necrosis factor or phorbol 12-myristate 13-acetate resulted in Thy1 upregulation in podoplanin-expressing lymphatic endothelial cells, but not in podoplanin-negative blood vascular endothelial cells. Moreover, adhesion of human polymorphonuclear and mononuclear leukocytes to human lymphatic endothelial cells was Thy1-dependent. Together, these results identify Thy1 as a novel lymphatic vessel expressed gene and suggest its potential role in the cell adhesion processes required for tumor progression and inflammation.

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

    PubMed Central

    Xu, Yan-shan; Chen, Gang; Guo, Yan; Wang, Dan-yi; Meng, Gui-bin

    2017-01-01

    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. PMID:28182096

  11. The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Zhang, Tao

    2011-05-01

    Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl-1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA, activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1, 4, 5-trisphosphate (IP3) but not PKC.

  12. Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine.

    PubMed

    Barber, Latorya A; Palascak, Mary B; Qi, Xiaoyang; Joiner, Clinton H; Franco, Robert S

    2015-11-01

    Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol-12-myristate-13-acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA-activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA-dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.

  13. Discrete control of TRPV4 channel function in the distal nephron by protein kinases A and C.

    PubMed

    Mamenko, Mykola; Zaika, Oleg L; Boukelmoune, Nabila; Berrout, Jonathan; O'Neil, Roger G; Pochynyuk, Oleh

    2013-07-12

    We have recently documented that the Ca(2+)-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca(2+) responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca(2+)]i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca(2+)]i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca(2+)]i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca(2+)]i responses and greatly increased basal [Ca(2+)]i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.

  14. Relationship between interleukin-5 production and variations in eosinophil counts during HIV infection in West Africa: influence of Mycobacterium tuberculosis infection.

    PubMed

    Diagbouga, S; Aldebert, D; Fumoux, F; Capron, M; Ledru, E

    1999-02-01

    Eosinophils are important effectors of the non-specific immune response and we studied whether perturbations in the production of the type 2 cytokine, interleukin-5 (IL-5), could account for the variations in eosinophil counts observed in human immunodeficiency virus (HIV) infection. HIV-infected patients without helminthiasis were investigated in a cross-sectional study in West Africa. Eosinophil counts were significantly higher in CDC-B patients than in controls, but were dramatically decreased at the CDC-C stage. Phorbol 12-myristate 13-acetate (PMA)+ ionomycin-induced IL-5 production by peripheral blood mononuclear cells (PBMC) was decreased from the A stage of the disease, and significant correlations were observed between IL-5 production and eosinophil counts in tuberculosis (TB)-negative HIV-1-positive, TB-positive HIV-1-positive and TB-positive HIV-negative patient groups. Nevertheless, the production of IL-5 was not decreased in HIV-positive patients with TB, in contrast to HIV-positive patients without TB presenting with the same ranges of CD4+ counts. Our data suggest that, during HIV infection, the impairment in IL-5 production is one of the factors associated with the 'paradoxal' eosinopenia observed in tropical areas, but that IL-5 production during active TB is compensated by cellular subsets, yet to be identified.

  15. Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine dephosphorylation and inhibition of mitogen-activated protein kinase in human mononuclear phagocytes. Role of the Src homology 2 containing tyrosine phosphatase 1.

    PubMed

    Knutson, K L; Hmama, Z; Herrera-Velit, P; Rochford, R; Reiner, N E

    1998-01-02

    Lipoarabinomannan (LAM) is a putative virulence factor of Mycobacterium tuberculosis that inhibits monocyte functions, and this may involve antagonism of cell signaling pathways. The effects of LAM on protein tyrosine phosphorylation in cells of the human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein kinase. To examine whether these effects of LAM could be related to activation of a phosphatase, fractions from LAM-treated cells were analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells, using phosphorylated mitogen-activated protein kinase as substrate, indicated that LAM promoted increased activity of SHP-1 in vivo. LAM also activated SHP-1 directly in vitro. Exposure of cells to LAM also attenuated the expression of tumor necrosis factor-alpha, interleukin-12, and major histocompatibility class II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.

  16. The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release.

    PubMed

    Bystrzycka, Weronika; Moskalik, Aneta; Sieczkowska, Sandra; Manda-Handzlik, Aneta; Demkow, Urszula; Ciepiela, Olga

    2016-01-01

    Neutrophil extracellular traps (NETs) are threads of nuclear DNA complexed with antimicrobial proteins released by neutrophils to extracellular matrix to bind, immobilise, and kill different pathogens. NET formation is triggered by different physiological and non-physiological stimulants. It is also suggested that antibiotics could be non-physiological compounds that influence NET release. The aim of the study was to investigate the effect of clindamycin and amoxicillin on NET release and the phagocyte function of neutrophils. Neutrophils isolated from healthy donors by density centrifugation method were incubated with amoxicillin or clindamycin for two hours, and then NET release was stimulated with phorbol 12-myristate 13-acetate (PMA). After three hours of incubation with PMA NETs were quantified as amount of extracellular DNA by fluorometry and visualised by immunofluorescent microscopy. The percent of phagocyting cells was measured by flow cytometry. We showed that amoxicillin induces NET formation (increase of extracellular DNA fluorescence, p = 0.03), while clindamycin had no influence on NET release (p > 0.05), as confirmed by quantitative measurement and fluorescent microscopy. Regarding phagocyte function, both antibiotics increased bacterial uptake (43.3% and 61.6% median increase for amoxicillin and clindamycin, respectively). We concluded that the ability of antibiotics to modulate NET release depends on the antibiotic used and is not associated with their ability to influence phagocytosis.

  17. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    SciTech Connect

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  18. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    SciTech Connect

    Menschikowski, Mario; Hagelgans, Albert; Eisenhofer, Graeme; Siegert, Gabriele

    2009-09-10

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), but not interferon-{gamma} and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1{beta} and TNF-{alpha} correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1{beta} and TNF-{alpha}, and downstream by MAP kinase signaling pathways and metalloproteinases.

  19. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

    PubMed Central

    Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth J.; Siller, Richard; Srsen, Vlastimil; Morgan, Katie; Bryans, Anna; Kozlowska, Ada; Koulovasilopoulos, Andreas; Underwood, Ian; Smith, Stewart; del-Pozo, Jorge; Moss, Sharon; Thompson, Alexandra Inés; Henderson, Neil C.; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre-Olivier; Nelson, Leonard J.

    2017-01-01

    Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization. PMID:28134251

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

    USGS Publications Warehouse

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

    2014-01-01

    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.

  1. Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling

    SciTech Connect

    Kilfeather, S.A.; Stein, M.; O'Malley, K. )

    1991-01-01

    Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate for 10 minutes enhanced cyclic AMP accumulation in human neutrophils under basal conditions and in response to the beta-adrenergic receptor agonist isoproterenol (ISO, 1{mu}M) and the adenylate cyclase activator forskolin (FSK, 10mM). Potentiation of responses to ISO by PMA was dose-dependent between 0.1 and 100nM PMA. The diacylglycerol analogue, 1-oleoyl-2-actylgylcerol (OAG) (50 {mu}M) also elevated beta-receptor responses, but 4beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 {mu}M) also elevated neutrophil cyclic AMP accumulation. All potentiating effects of PMA on cyclic AMP production were inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H{sub 7}). PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalized compartments, or the capacity of ISO to induce beta-receptor internalization. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation int he presence of PMA were not elevated by PMA.

  2. Activation of the human. beta. sub 2 -interferon/hepatocyte-stimulating factor/interleukin 6 promoter by cytokines, viruses, and second messenger agonists

    SciTech Connect

    Ray, A.; Tatter, S.B.; May, L.T.; Sehgal, P.B. )

    1988-09-01

    The hallmark of {beta}{sub 2}-interferon (IFN-{beta}{sub 2})/hepatocyte-stimulating factor/interleukin 6 gene expression is its inducibility in different types of human cells (fibroblasts, monocytes, epithelial cells, and endothelial cells) by different stimuli, which include cytokines such as tumor necrosis factor, interleukin 1 (IL-1) and platelet-derived growth factor, different viruses, and bacterial products such as endotoxin. The activation by cytokines, viruses, and second messenger agonists of the IFN-{beta}{sub 2} promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was studied after transfection into HeLa cells. A chimeric gene containing IFN-{beta}{sub 2} DNA from -1180 to +13 linked to the CAT gene was inducible {approx}10-fold by phorbol 12-myristate 13-acetate (PMA), followed, in decreasing order, by pseudorabies and Sendai viruses; serum; the cytokines tumor necrosis factor, IL-1, and epidermal growth factor; the cAMP agonists BrcAMP and forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine; poly(I){center dot}poly(C); 1,2-diacylglycerol and the calcium ionophore A23187. The region between -225 and -113 in IFN-{beta}{sub 2}, which contains DNA motifs similar to the regulatory elements in the human c-fos gene, appears to contain the major cis-acting regulatory elements responsible for the activation of the IFN-{beta}{sub 2} promoter by several different cytokines, viruses, and second messenger agonists.

  3. Enhanced cAMP accumulation by a phorbol ester in cerebral cortical cells

    SciTech Connect

    Beeler, J.F.; Davis, C.W.

    1987-05-01

    Phorbol 12-myristate-13-acetate (PMA) was found to be selective in its ability to alter cAMP accumulations in cultured rat cerebral cortical cells. Basal levels of cAMP in cultured neuronal and nonneuronal cells preincubated in the absence or presence of PMA were 14 pmol/mg protein and 16 pmol/mg protein, respectively. Adenosine increased cAMP levels in a dose-dependent manner. cAMP accumulation in response to low concentrations of adenosine was not significantly altered by pretreatment with PMA but marked potentiation of adenosine elicited accumulations was observed at 10 and 100 ..mu..M adenosine. Longer preincubation with PMA resulted in a decreased ability of PMA to enhance adenosine elicited accumulations of cAMP. PMA did not significantly alter cAMP accumulation by forskolin (FOR) and enhanced norepinephrine stimulated cAMP by only 2-fold. For similarly potentiated adenosine/sub 2/ (A/sub 2/)- receptor elicited accumulation of cAMP which could be further enhanced by PMA. These results suggest that the effects of the phorbol ester are more specific for potentiating adenosine stimulated cAMP accumulation and may occur as a result of a more efficient coupling between the A/sub 2/-receptor, N-protein and adenylate cyclase.

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

    SciTech Connect

    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 ({sup 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.

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

    SciTech Connect

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

    1986-05-01

    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 (/sup 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.

  6. Moisture damage in home associates with systemic inflammation in children.

    PubMed

    Mustonen, K; Karvonen, A M; Kirjavainen, P; Roponen, M; Schaub, B; Hyvärinen, A; Frey, U; Renz, H; Pfefferle, P I; Genuneit, J; Vaarala, O; Pekkanen, J

    2016-06-01

    This study investigated the association between confirmed moisture damage in homes and systemic subclinical inflammation in children. Home inspections were performed in homes of 291 children at the age of 6 years. Subclinical inflammation at the age of 6 years was assessed by measuring the circulating levels of C-reactive protein (CRP) and leukocytes in peripheral blood and fractional exhaled nitric oxide (FeNO). Proinflammatory cytokines interleukin (IL)-1β and IL-6 and tumor necrosis factor (TNF)-α were measured in unstimulated, and in phorbol 12-myristate 13-acetate and ionomycin (PI), lipopolysaccharide (LPS), or peptidoglycan (PPG)-stimulated whole blood. Major moisture damage in the child's main living areas (living room, kitchen, or child's bedroom) and moisture damage with mold in the bathroom were associated with increased levels of CRP and stimulated production of several proinflammatory cytokines. There were no significant associations between moisture damage/visible mold and leukocyte or FeNO values. The results suggest that moisture damage or mold in home may be associated with increased systemic subclinical inflammation and proinflammatory cytokine responsiveness.

  7. Copper egress is induced by PMA in human THP-1 monocytic cell line.

    PubMed

    Afton, Scott E; Caruso, Joseph A; Britigan, Bradley E; Qin, Zhenyu

    2009-06-01

    Copper egress is an essential regulator of the kinetics of cellular copper and is primarily regulated by ATP7A, a copper-transporting P-type ATPase. However, little is known under which physiological condition copper egress is induced and its molecular consequence. In current manuscript, using THP-1 cells, a human monocytic cell line, we found that ATP7A expression was increased in cells exposed to phorbol-12-myristate-13-acetate (PMA), a potent inducer of neovascularization and cancer. Inductively coupled plasma mass spectrometry revealed that PMA also induced copper egress. Inhibition of ATP7A expression using small interfering RNA abrogated PMA induced copper egress. PMA treatment in THP-1 cells resulted in increased expression of matrix metalloproteinase (MMP) 9 and vascular endothelial growth factor receptor 1 (VEGFR1), whereas inhibition of ATP7A resulted in suppression of PMA-induced expression of VEGFR1, but not MMP9. Finally, addition of exogenous copper into the conditioned medium did not change VEGFR1 expression in THP-1 cells. Collectively, we demonstrate that PMA induces copper egress in THP-1 cells, which is regulated by ATP7A, and ATP7A regulates VEGFR1 expression. Considering the involvement of copper in neovascularization, our current finding provides the potential evidence to interpret the molecular mechanism.

  8. Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages.

    PubMed

    Hong, Yi-Fan; Kim, Hangeun; Kim, Hye Sun; Park, Woo Jung; Kim, Joo-Yun; Chung, Dae Kyun

    2016-01-01

    Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent.

  9. Thioredoxin Ameliorates Cutaneous Inflammation by Regulating the Epithelial Production and Release of Pro-Inflammatory Cytokines

    PubMed Central

    Tian, Hai; Matsuo, Yoshiyuki; Fukunaga, Atsushi; Ono, Ryusuke; Nishigori, Chikako; Yodoi, Junji

    2013-01-01

    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. PMID:24058364

  10. A novel benzofuran, 4-methoxybenzofuran-5-carboxamide, from Tephrosia purpurea suppressed histamine H1 receptor gene expression through a protein kinase C-δ-dependent signaling pathway.

    PubMed

    Shill, Manik Chandra; Mizuguchi, Hiroyuki; Karmakar, Sanmoy; Kadota, Takuya; Mukherjee, Pulok K; Kitamura, Yoshiaki; Kashiwada, Yoshiki; Nemoto, Hisao; Takeda, Noriaki; Fukui, Hiroyuki

    2016-01-01

    Histamine H1 receptor (H1R) gene is upregulated in patients with allergic rhinitis (AR), and its expression level is strongly correlated with the severity of allergic symptoms. We previously reported isolation of the putative anti-allergic compound, 4-methoxybenzofuran-5-carboxamide (MBCA) from Tephrosia purpurea and its chemical synthesis (Shill et al., Bioorg Med Chem 2015;23:6869-6874). However, the mechanism underlying its anti-allergic activity remains to be elucidated. Here, we report the mechanism of MBCA on phorbol 12-myristate-13-acetate (PMA)- or histamine-induced upregulation of H1R gene expression in HeLa cells, and in vivo effects of MBCA were also determined in toluene-2,4-diisocyanate (TDI)-sensitized rats. MBCA suppressed PMA- and histamine-induced upregulation of H1R expression at both mRNA and protein levels and inhibited PMA-induced phosphorylation of PKCδ at Tyr(311) and subsequent translocation to the Golgi. Furthermore, MBCA ameliorated allergic symptoms and suppressed the elevation of H1R and helper T cell type 2 (Th2) cytokine mRNAs in TDI-sensitized rats. Data suggest that MBCA alleviates nasal symptoms in TDI-sensitized rats through the inhibition of H1R and Th2 cytokine gene expression. The mechanism of its H1R gene suppression underlies the inhibition of PKCδ activation.

  11. Dexamethasone Inhibits S. aureus-Induced Neutrophil Extracellular Pathogen-Killing Mechanism, Possibly through Toll-Like Receptor Regulation

    PubMed Central

    Wan, Ting; Zhao, Yingying; Fan, Fangli; Hu, Renjian; Jin, Xiuming

    2017-01-01

    Neutrophils release neutrophil extracellular traps (NETs) in a pathogen-killing process called NETosis. Excessive NETs formation, however, is implicated in disease pathogenesis. Therefore, to understand how NETosis is regulated, we examined the effect of dexamethasone (DXM), an anti-inflammatory drug, on this process and the role of toll-like receptors (TLRs). We stimulated human neutrophils with phorbol 12-myristate 13-acetate (PMA) or Staphylococcus aureus (S. aureus) and quantified NETs formation. We also examined the effect of DXM on the bactericidal effect of NETs and the role of reactive oxygen species (ROS) and nuclear factor (NF)-κB in DXM-regulated NETosis. DXM significantly inhibited S. aureus-induced NETosis and extracellular bacterial killing. ROS production and NF-κB activation were not involved in DXM-regulated NETosis. TLR2 and TLR4, but not TLR5 or TLR6, modified S. aureus-induced NETs formation. Neither DXM nor TLRs were involved in PMA-induced NETosis. Furthermore, TLR2 and TLR4 agonists rescued DXM-inhibited NETosis, and neither TLR2 nor TLR4 antagonists could further inhibit NETosis reduction induced by DXM, indicating that DXM may inhibit NETosis by regulating TLR2 and TLR4. In conclusion, the mechanisms of S. aureus- and PMA-induced NETosis are different. DXM decreases NETs formation independently of oxidant production and NF-κB phosphorylation and possibly via a TLR-dependent mechanism. PMID:28232829

  12. Acrylonitrile-induced extracellular signal-regulated kinase (ERK) activation via protein