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Sample records for 12-myristate 13-acetate tpa

  1. Phorbol 12-myristate 13-acetate promotes nuclear translocation of hepatic steroid response element binding protein-2.

    PubMed

    Wong, Tsz Yan; Tan, Yan Qin; Lin, Shu-Mei; Leung, Lai K

    2016-06-01

    Sterol regulatory element-binding protein (SREBP)-2 is a pivotal transcriptional factor in cholesterol metabolism. Factors interfering with the proper functioning of SREBP-2 potentially alter plasma lipid profiles. Phorbol 12-myristate 13-acetate (PMA), which is a common protein kinase C (PKC) activator, was shown to promote the post-translational processing and nuclear translocation of SREBP-2 in hepatic cells in the current study. Following SREBP-2 translocation, the transcripts of its target genes HMGCR and LDLR were upregulated as demonstrated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Electrophoretic mobility shift assays (EMSA) also demonstrated an induced DNA-binding activity on the sterol response element (SRE) domain under PMA treatment. The increase of activated Srebp-2 without the concurrent induced mRNA expression was also observed in an animal model. As the expression of SREBP-2 was not increased by PMA, the activation of PKC was the focus of investigation. Specific PKC isozyme inhibition and overexpression supported that PKCβ was responsible for the promoting effect. Further studies showed that the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not 5' adenosine monophosphate-activated protein kinase (AMPK), were the possible downstream signaling proteins of PKCβ. In conclusion, this study illustrated that PKCβ increased SREBP-2 nuclear translocation in a pathway mediated by MEK/ERK and JNK, rather than the one dictated by AMPK. These results revealed a novel signaling target of PKCβ in the liver cells. PMID:27032751

  2. 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...

  3. Treatment of mouse melanoma cells with phorbol 12-myristate 13-acetate counteracts mannosylerythritol lipid-induced growth arrest and apoptosis.

    PubMed

    Zhao, X; Geltinger, C; Kishikawa, S; Ohshima, K; Murata, T; Nomura, N; Nakahara, T; Yokoyama, K K

    2000-07-01

    Mannosylerythritol lipid (MEL), an extracellularglycolipid from yeast, induces the differentiation ofHL-60 promyelocytic leukemia cells towardsgranulocytes. We show here that MEL is also a potentinhibitor of the proliferation of mouse melanoma B16cells. Flow-cytometric analysis of the cell cycle ofMEL-treated B16 cells revealed the accumulation ofcells in the sub-G(0)/G(1) phase, which is a hallmark ofcells undergoing apoptosis. Treatment of B16 cellsfor 24 h with phorbol 12-myristate 13-acetate (PMA),an activator of protein kinase C (PKC), did notinterfere with the growth and survival of the cells,but it effectively counteracted the MEL-induced growtharrest and apoptosis. The activity of PKC was reducedin B16 cells treated with MEL at a concentration atwhich MEL induced apoptosis. However, incubation withPMA in addition to MEL reversed this reduction in theactivity of PKC. These results suggest thatconverging signaling pathways are triggeredindependently by MEL and PMA and that the signalsmight both be mediated by PKC. PMID:19002819

  4. 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.

  5. A Comparison Between Phorbol 12 Myristate 13 Acetate and Phorbol 12, 13 Dibutyrate in Human Melanocyte Culture

    PubMed Central

    Padma, Divya

    2016-01-01

    Introduction Melanocyte culture is an integral part of the studies of skin biology and cosmetic applications. After the introduction of selective medium for the culture of human melanocyte using Phorbol 12-myristate13-acetate (PMA) in 1982, a lot of methods of culturing were tried but till date PMA is a preferred mitogen because of its cost effectiveness compared to growth factors. We have tried to preliminarily evaluate the efficacy of another phorbol ester, Phorbol 12, 13-dibutyrate (PDBu) in melanocyte culture because of its less hydrophobic nature compared to PMA. This property minimizes the trace amount of mitogen in cell culture after washing off and hence does not interfere in other biological assays. Aim To evaluate the differences in the melanocyte survival rate, morphology and mitotic index when grown in media supplemented with PMA and PDBu. Materials and Methods Foreskins were collected from children undergoing circumcision. Epidermal cells were isolated from foreskin and cultured using PMA and PDBu. Melanocytes in culture were monitored for the better establishment and documented. In proliferative assay, melanocytes were treated with PMA and PDBu for 24, 48 and 72 hours and proliferation was measured using 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay method. Results When cultured, melanocytes acquired proliferative status and bipolar morphology quicker in PDBu medium than in PMA medium. Keratinocytes survived as contamination in PMA medium whereas PDBu medium had minimal keratinocytes. MTT assay showed that PDBu has higher proliferative induction capacity than PMA. In even lower concentration of PDBu in medium, melanocytes survived till 72 hours without significant cell loss in compared to PMA medium. Conclusion PDBu can be a valuable replacement for PMA in human melanocyte culture. Higher proliferation induction, unfavourable to keratinocyte survival and less hydrophobicity make PDBu a promising alternative for quicker

  6. 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. PMID:25518925

  7. 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

    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

    2015-09-01

    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

  8. 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.

  9. 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

  10. Effects of phorbol 12-myristate 13-acetate on triglyceride and cholesteryl ester synthesis in cultured coronary smooth muscle cells and macrophages.

    PubMed

    Moinat, M; Chevey, J M; Muzzin, P; Giacobino, J P; Kossovsky, M

    1990-02-01

    In cultured pig coronary smooth muscle cells phorbol 12-myristate 13-acetate (PMA) stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and the incorporation of [2-3H]glycerol into triglycerides 6.4- and 4.5-fold, respectively. The maximal effects occurred after 3 h of treatment and there was a return to basal values after 72 h. In the presence of 400 microM oleic acid, PMA stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and that of [2-3H]glycerol into triglycerides 5.3- and 2.3-fold, respectively. The stimulatory effects were more sustained (still significant after 72 h) and their maxima were delayed (peaks after 24 h). PMA was also found to increase 2-fold the amount of triglyceride that accumulated in the cells in the presence of oleic acid after 24 h. In macrophages IC-21, the effects of PMA were observed only in the presence of oleic acid. They consisted of a 1.9-fold stimulation in the conversion of [4-14C]cholesterol into cholesteryl esters after 72 h and of a 1.7-fold stimulation in the incorporation of [2-3H]glycerol into triglycerides after 24 h. PMA also increased the amount of triglyceride that accumulated in the cells 1.9-fold after 72 h. It is concluded that PMA, and possibly growth factors, may promote lipid storage in smooth muscle cells and that fatty acids favor long lasting effects of PMA in smooth muscle cells and are necessary for any effect of PMA in macrophages. PMID:2324651

  11. 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. PMID:26826276

  12. Phosphatidic acid mobilized by phospholipase D is involved in the phorbol 12-myristate 13-acetate-induced G2 delay of A431 cells.

    PubMed Central

    Kaszkin, M; Richards, J; Kinzel, V

    1996-01-01

    This study was aimed at gaining an understanding of metabolic events responsible for the inhibition of cells in G2 phase, a known physiological restriction site in the cell cycle of multicellular organisms. In an earlier study, phosphatidic acid was proposed as an inhibitory mediator in the epidermal growth factor (EGF)-induced inhibition of A431 cells in G2 phase via the phospholipase C pathway [Kaszkin, Richards and Kinzel (1992) Cancer Res. 52, 5627-5634]. We show here that the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces a reversible inhibition of the G2/M transition in A431 cells under conditions of phospholipase D-catalysed phosphatidic acid formation. Such PMA-induced inhibition in G2 phase is largely attenuated in the presence of 1-propanol (but not of 2-propanol). In this case the amount of phosphatidic acid is reduced to almost control levels, and instead phosphatidylpropanol is formed. In the case of EGF-induced activation of a phospholipase D the amount of phosphatidic acid is only slightly decreased in the presence of a primary alcohol. Under these conditions the EGF-induced G2 delay was not affected. The correlation between the formation of phosphatidic acid and the G2 delay induced by PMA, as well as by an exogenous bacterial phospholipase D (from Streptomyces chromofuscus), could be supported by using synchronized cells in order to increase the population of cells in G2 phase. This study indicates that the formation of substantial amounts of phosphatidic acid immediately before entry into mitosis seems to be important for establishing a delay in the cell cycle at the G2/M border by exogenous ligands. PMID:8660273

  13. 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.

  14. Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Galphaq promoter during megakaryocytic differentiation. Regulation by EGR-1 and MAP kinase pathway.

    PubMed

    Jalagadugula, Gauthami; Dhanasekaran, Danny N; Rao, A Koneti

    2008-11-01

    Galphaq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Galphaq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5' upstream region of the Galphaq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5' upstream of the Galphaq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Galphaq 5' upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt -1042/-1037 eliminated the difference. Gel-shift studies on Galphaq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence -1042/-1037. Silencing of endogenous EGR-1 inhibited Galphaq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence -1042/-1037 bp in Galphaq promoter mediates the induction of Galphaq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Galphaq promoter, and new insights into regulation of Galphaq gene by EGR-1. PMID:18989526

  15. Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

    PubMed Central

    Mykoniatis, Andreas; Shen, Le; Fedor-Chaiken, Mary; Tang, Jun; Tang, Xu; Worrell, Roger T.; Delpire, Eric; Turner, Jerrold R.; Matlin, Karl S.

    2010-01-01

    In secretory epithelial cells, the basolateral Na+-K+-2Cl− cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl− secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-β-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle “pinchase” dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, ∼80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and ∼40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway. PMID:19864322

  16. Roles of insulin, guanosine 5'-[gamma-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation.

    PubMed Central

    Todaka, M; Hayashi, H; Imanaka, T; Mitani, Y; Kamohara, S; Kishi, K; Tamaoka, K; Kanai, F; Shichiri, M; Morii, N; Narumiya, S; Ebina, Y

    1996-01-01

    Insulin, guanosine 5'-[gamma-thio]triphosphate (GTP[S] and phorbol 12-myristate 13-acetate (PMA) trigger the translocation of Gl UT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an intracellular pool to the cell surface. We have developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact 3T3-L1 adipocytes and Chinese hamster ovary (CHO) cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translocation in the CHO cell system. Among small molecular GTP-binding proteins, ras, rab3D, rad and rho seem to be candidates as signal transmitters of insulin-stimulated GLUT4 translocation. Overexpression of wild-type H-ras and the dominant negative mutant H-rass17N in our cell system respectively enhanced and blocked insulin-stimulated activation of mitogen-activated protein kinase, but did not affect insulin-stimulated GLUT4 translocation. Overexpression of rab3D or rad in the cells did not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, had no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely to be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent pathways. PMID:8645171

  17. Phorbol 12-myristate 13-acetate up-regulates the transcription of MUC2 intestinal mucin via Ras, ERK, and NF-kappa B.

    PubMed

    Lee, Hae-Wan; Ahn, Dae-Ho; Crawley, Suzanne C; Li, Jian-Dong; Gum, James R; Basbaum, Carol B; Fan, Nancy Q; Szymkowski, David E; Han, Sang-Young; Lee, Bong H; Sleisenger, Marvin H; Kim, Young S

    2002-09-01

    MUC2 is a secretory mucin normally expressed by goblet cells of the intestinal epithelium. It is overexpressed in mucinous type colorectal cancers but down-regulated in colorectal adenocarcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment of colon cancer cell lines increases MUC2 expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5'-flanking region of the MUC2 gene using stably and transiently transfected promoter reporter vectors. Protein kinase C inhibitors (bisindolylmaleimide, calphostin C) and inhibitors of mitogen-activated protein/extracellular signal regulated kinase kinase (MEK) (PD98059 and U0126) suppressed up-regulation of MUC2. Src tyrosine kinase inhibitor PP2, a protein kinase A inhibitor (KT5720), and a p38 inhibitor (SB 203580) did not affect transcription. Western blotting and reverse transcription-PCR analysis confirmed these results. In addition, co-transfections with mutants of Ras, Raf, and MEK showed that the induction of MUC2 promoter activity by PMA required these three signaling proteins. Our results demonstrate that PMA activates protein kinase C, stimulating MAP kinase through a Ras- and Raf-dependent mechanism. An important role for nuclear factor kappaB (NF-kappaB) was also demonstrated using the inhibitor caffeic acid phenethyl ester and electrophoretic mobility shift assays. Such identification of pathways involved in MUC2 up-regulation by PMA in the HM3 colon cancer cell line may serve as a model for the effects of cytokines and growth factors, which regulate MUC2 expression during the progression of colorectal cancer. PMID:12077118

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

    PubMed Central

    Choi, Hye-young; Choi, Bo-hyun; Kim, Sang-Tae; Heo, Tae-Hwe; Lee, Joo Young; Park, Pil-Hoon; Kwak, Mi-Kyoung

    2015-01-01

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

  20. 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).

  1. Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells.

    PubMed

    Ma, Wenbin; Galvin, Teresa A; Ma, Hailun; Ma, Yunkun; Muller, Jacqueline; Khan, Arifa S

    2011-05-01

    Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery. PMID:21470875

  2. Inhibitory Effects of 4'-Demethylnobiletin, a Metabolite of Nobiletin, on 12-O-Tetradecanoylphorbol-13-acetate (TPA)-Induced Inflammation in Mouse Ears.

    PubMed

    Wu, Xian; Song, Mingyue; Rakariyatham, Kanyasiri; Zheng, Jinkai; Wang, Minqi; Xu, Fei; Gao, Zili; Xiao, Hang

    2015-12-30

    Nobiletin (NOB) is major citrus flavonoid with many health-promoting benefits. We reported previously that 4'-demethylnobiletin (4DN), a major metabolite of NOB, significantly inhibited lipopolysaccharide (LPS)-stimulated inflammation in RAW 264.7 macrophages. In this study, we further studied the anti-inflammatory effects of 4DN in TPA-induced skin inflammation in mice. We demonstrated that topical application of 4DN decreased TPA-induced ear edema by >88 ± 4.77% in mice. This inhibitory effect was associated with inhibition on TPA-induced up-regulation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Immunoblotting results showed that 4DN resulted in profound effects on multiple proteins related with inflammation and carcinogenesis. 4DN significantly decreased the expression levels of iNOS, COX-2, and MMP-9, suppressed phosphorylation of PI3K/Akt and ERK, and increased the levels of HO-1 and NQO1 in TPA-treated mice. Overall, the results demonstrated that 4DN had strong anti-inflammatory effects in vivo, which provided a scientific basis for using NOB to inhibit inflammation-driven diseases. PMID:26651527

  3. Contact-stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: inhibition of proliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA)

    SciTech Connect

    Miller, D.R.; Hamby, K.M.; Slaga, T.J.

    1982-07-01

    Mouse epidermal cells can be subcultured at 31/sup 0/C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze-thawed A31 cells were ineffective. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, recently shown to interfere with contact-mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31-epidermal cell interaction is apparently not a typical mesenchymal-epithelial interaction, since the basement membrane would prevent this contact in intact skin.

  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. Effects of PMA (PHORBOL-12-MYRISTATE-13-ACETATE) on the Developing Rodent Brain.

    PubMed

    Dzietko, Mark; Hahnemann, Maria; Polley, Oliver; Sifringer, Marco; Felderhoff-Mueser, Ursula; Bührer, Christoph

    2015-01-01

    Perinatal infections have a negative impact on brain development. However, the underlying mechanisms leading to neurological impairment are not completely understood and reliable models of inflammation are urgently needed. Using phorbol-myristate-acetate as an activator of inflammation, we investigated the effect on the developing rodent brain. Neonatal rats and mice deficient in IL-18 or IRAK-4 were exposed to PMA. Brains were assessed for regulation of pro- and anti-inflammatory cytokines and cell death 24 hrs, 7 and 14 days after treatment. PMA induced an inflammatory response and caused widespread neurodegeneration in the brains of 3- and 7-day-old rats. In contrast, 14-day-old rats were resistant to the neurotoxic effect of PMA. Histological evaluation at the age of 14 and 21 days revealed a destruction of the cortical microstructure with decreased numerical density of neuronal cells. Mice deficient in IL-18 or IRAK-4 were protected against PMA induced brain injury. PMA treatment during a vulnerable period can alter brain development. IL-18 and IRAK-4 appear to be important for the development of PMA induced injury. PMID:25918710

  6. Curcumin relieves TPA-induced Th1 inflammation in K14-VEGF transgenic mice.

    PubMed

    Sun, Jun; Zhao, Yi; Jin, Hairong; Hu, Jinhong

    2015-04-01

    Curcumin has been confirmed to have anti-inflammatory properties in addition to the ability to decrease the expression of pro-inflammatory cytokines in keratinocytes. It was suggested that the interleukin-23 (IL-23)/IL-17A cytokine axis played a critical role in the pathogenesis of 12-O-tetradecanoyl phorbol 12-myristate 13-acetate (TPA)-induced K14-VEGF transgenic psoriasis-like mice model. Here, we report that topical use of a curcumin gel formulation inhibited TPA-induced Th1 inflammation in K14-VEGF transgenic mice ears but not Th17 inflammation as expected. Real-time PCR showed that mRNA levels of IL-23, IL-17A, IL-22, IL-6 and TNFα cytokines failed to increase after TPA-induction in K14-VEGF transgenic mice ear skin; but the mRNA level of IFNγ increased significantly at the same time. Furthermore, TPA-induction up-regulated the TCRγδ protein but failed to impact the CCR6 protein, which means that the proliferation of γδ T cells is incapable of IL-17A production. We find that curcumin is capable of relieving TPA-induced inflammation by directly down-regulating IFNγ production. In conclusion, curcumin inhibits TPA-induced Th1 inflammation in K14-VEGF transgenic mice which has not been previously described. PMID:25682767

  7. 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. PMID:10806212

  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

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

    2011-09-01

    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. 12-O-Tetradecanoyl phorbol-13-acetate (TPA)-induced growth arrest is increased by silibinin by the down-regulation of cyclin B1 and cdc2 and the up-regulation of p21 expression in MDA-MB231 human breast cancer cells.

    PubMed

    Kim, Sangmin; Lee, Hye Sook; Lee, Se-Kyung; Kim, Sung Hoon; Hur, Sung Mo; Kim, Jee Soo; Kim, Jung-Han; Choe, Jun-Ho; Shin, Incheol; Yang, Jung-Hyun; Lee, Jeong Eon; Nam, Seok Jin

    2010-12-01

    TPA is a potent regulator of cell growth, including cell proliferation and differentiation. In this study, we determined the effect of silibinin on TPA-induced growth arrest in breast cancer cells. Silibinin increased growth arrest of the G2/M phase in a dose-dependent fashion. Silibinin decreased the basal level of cyclin B1 and cdc2 expression, which is involved in S phase and G2/M transition. In addition, TPA-induced G2/M phase arrest was increased by silibinin. Under the same conditions, TPA-induced down-regulation of cyclin B1 and cdc2 was decreased by silibinin. In contrast, TPA-induced p21 expression was further increased by silibinin. To determine the regulatory mechanism of TPA-induced growth arrest, we pretreated cells with various inhibitors, such as UO126, SB203580, and LY294002. Interestingly, TPA-induced growth arrest was significantly increased by LY294002, but not by UO126 and SB203580. In addition, TPA-induced down-regulation of cyclin B1 was inhibited by LY294002; however, the basal level of p21 was increased by TPA and TPA-induced p21 expression was further increased by LY294002. Finally, adenoviral constitutively active-Akt (Ad-CA-Akt) overexpression regulated the up-regulation of cyclin B1 and the down-regulation of p21. Therefore, we have demonstrated that silibinin has an additive effect on TPA-induced growth arrest through the PI-3-kinase/Akt-dependent pathway. PMID:20554189

  10. EFFECT OF 12-0-TETRADECANOYLPHORBOL-13-ACETATE ON THE MORPHOLOGY AND GROWTH OF C3H/10TL/2 MOUSE EMBRYO CELLS

    EPA Science Inventory

    The effects of the tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) on the morphology and growth properties of C3H/10T1/2 clone 8 cells were examined. The morphology of these cells was changed within 30 min following treatment with 0.1 micrograms of TPA pe...

  11. 12-O-tetradecanoyl-phorbol-13-acetate down-regulates the Huntingtin promoter at Sp1 sites.

    PubMed

    Coles, R; Birdsall, M; Wyttenbach, A; Rubinsztein, D C

    2000-09-28

    We have studied the effects of the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on Huntington's disease (HD) gene transcription in neuronal and non-neuronal cell lines, to investigate pathways regulating HD gene expression. TPA reduced transcription from the HD gene promoter in SK-N-SH (neuroblastoma) and HeLa cells but not in JEG3 (choriocarcinoma) cells. In SK-N-SH cells, the responsible cis-acting promoter sequences comprise the tandemly duplicated Sp1 sites in the region from -213 to -174, relative to the translation start site. The TPA-down-regulating region in HeLa cells was mapped to the sequence from -141 to -126. In conclusion, this demonstrates that HD gene transcription can be down-regulated in vitro in a cell-specific manner. PMID:11043541

  12. Synthesis of seco-B-ring bryostatin analogue WN-1 via C-C bond-forming hydrogenation: critical contribution of the B-ring in determining bryostatin-like and phorbol 12-myristate 13-acetate-like properties.

    PubMed

    Andrews, Ian P; Ketcham, John M; Blumberg, Peter M; Kedei, Noemi; Lewin, Nancy E; Peach, Megan L; Krische, Michael J

    2014-09-24

    The seco-B-ring bryostatin analogue, macrodiolide WN-1, was prepared in 17 steps (longest linear sequence) and 30 total steps with three bonds formed via hydrogen-mediated C-C coupling. This synthetic route features a palladium-catalyzed alkoxycarbonylation of a C2-symmetric diol to form the C9-deoxygenated bryostatin A-ring. WN-1 binds to PKCα (Ki = 16.1 nM) and inhibits the growth of multiple leukemia cell lines. Although structural features of the WN-1 A-ring and C-ring are shared by analogues that display bryostatin-like behavior, WN-1 displays PMA-like behavior in U937 cell attachment and proliferation assays, as well as in K562 and MV-4-11 proliferation assays. Molecular modeling studies suggest the pattern of internal hydrogen bonds evident in bryostatin 1 is preserved in WN-1, and that upon docking WN-1 into the crystal structure of the C1b domain of PKCδ, the binding mode of bryostatin 1 is reproduced. The collective data emphasize the critical contribution of the B-ring to the function of the upper portion of the molecule in conferring a bryostatin-like pattern of biological activity. PMID:25207655

  13. Synthesis of seco-B-Ring Bryostatin Analogue WN-1 via C–C Bond-Forming Hydrogenation: Critical Contribution of the B-Ring in Determining Bryostatin-like and Phorbol 12-Myristate 13-Acetate-like Properties

    PubMed Central

    2015-01-01

    The seco-B-ring bryostatin analogue, macrodiolide WN-1, was prepared in 17 steps (longest linear sequence) and 30 total steps with three bonds formed via hydrogen-mediated C–C coupling. This synthetic route features a palladium-catalyzed alkoxycarbonylation of a C2-symmetric diol to form the C9-deoxygenated bryostatin A-ring. WN-1 binds to PKCα (Ki = 16.1 nM) and inhibits the growth of multiple leukemia cell lines. Although structural features of the WN-1 A-ring and C-ring are shared by analogues that display bryostatin-like behavior, WN-1 displays PMA-like behavior in U937 cell attachment and proliferation assays, as well as in K562 and MV-4-11 proliferation assays. Molecular modeling studies suggest the pattern of internal hydrogen bonds evident in bryostatin 1 is preserved in WN-1, and that upon docking WN-1 into the crystal structure of the C1b domain of PKCδ, the binding mode of bryostatin 1 is reproduced. The collective data emphasize the critical contribution of the B-ring to the function of the upper portion of the molecule in conferring a bryostatin-like pattern of biological activity. PMID:25207655

  14. Different effects of GPR120 and GPR40 on cellular functions stimulated by 12-O-tetradecanoylphorbol-13-acetate in melanoma cells.

    PubMed

    Fukushima, Kaori; Takahashi, Kaede; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

    2016-06-17

    G-protein-coupled receptor 120 (GPR120) and GPR40 exhibit a variety of biological responses by the binding of free fatty acids. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a tumor promoting agent of skin carcinogenesis. It is known that TPA treatment stimulates cell motile activity of cancer cells, including melanoma cells. In the present study, we investigated whether GRP120 and GPR40 are involved in regulation of cell motile activity induced by TPA in two melanoma cell lines. A375 and G361 cells were treated with TPA at a concentration of 10 nM for 24 h. The cell motile activity of A375 cells was significantly increased by TPA, correlating with GPR40 expression. In contrast, TPA suppressed the cell motile activity of G361 cells, while GPR120 and GPR40 expressions were increased. The cell motile activity of A375 cells treated with TPA was markedly increased by GPR120 knockdown. In addition, to assess roles of GPR120 and GPR40 in cellular functions of A375 cells by the long-term TPA treatment, cells were treated with TPA (1 nM) for at least 3 months. The long-term TPA treatment induced the high cell motile activity and elevated GPR120 and GPR40 expressions. The high cell motile activity of A375 cells stimulated by the long-term TPA treatment was enhanced by GPR120 knockdown. These results suggest that GPR120 negatively and GPR40 positively regulate cell motile activities induce by TPA in melanoma cells. PMID:27163640

  15. 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

  16. 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. PMID:25727244

  17. Okadaic acid: An additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter

    SciTech Connect

    Suganuma, Masami; Fujiki, Hirota; Suguri, Hiroko; Yoshizawa, Shigeru; Hirota, Mitsuru; Nakayasu, Michie ); Ojika, Makoto; Wakamatsu, Kazumasa; Yamada, Kiyoyuki ); Sugimura, Takashi )

    1988-03-01

    Okadaic acid is a polyether compound of a C{sub 38} fatty acid, isolated from a black sponge, Halichondria okadai. Previous studies showed that okadaic acid is a skin irritant and induces ornithine decarboxylase in mouse skin 4 hr after its application to the skin. This induction was strongly inhibited by pretreatment of the skin with 13-cis-retinoic acid. A two-stage carcinogenesis experiment in mouse skin initiated by a single application of 100 {mu}g of 7,12-dimethylbenz(a)anthracene (DMBA) and followed by application of 10 {mu}g of okadaic acid twice a week revealed that okadaic acid is a potent additional tumor promoter: tumors developed in 93% of the mice treated with DMBA and okadaic acid by week 16. In contrast, tumors were found in only one mouse each in the groups treated with DMBA alone or okadaic acid alone. An average of 2.6 tumors per mouse was found in week 30 in the group treated with DMBA and okadaic acid. Unlike phorbol 12-tetradecanoate 13-acetate (TPA), teleocidin, and aplysiatoxin, okadaic acid did not inhibit the specific binding of ({sup 3}H)TPA to a mouse skin particulate fraction when added up to 100 {mu}M or activate calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in vitro when added up to 1.2 {mu}M. Therefore, the actions of okadaic acid and phorbol ester may be mediated in different ways. These results show that okadaic acid is a non-TPA-type tumor promoter in mouse skin carcinogenesis.

  18. Combination of 12-O-tetradecanoylphorbol-13-acetate with diethyldithiocarbamate markedly inhibits pancreatic cancer cell growth in 3D culture and in immunodeficient mice

    PubMed Central

    HUANG, HUARONG; CAO, KAJIA; MALIK, SAQUIB; ZHANG, QIUYAN; LI, DONGLI; CHANG, RICHARD; WANG, HUAQIAN; LIN, WEIPING; VAN DOREN, JEREMIAH; ZHANG, KUN; DU, ZHIYUN; ZHENG, XI

    2015-01-01

    The aim of the present study was to determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC) alone or in combination on human pancreatic cancer cells cultured in vitro and grown as xenograft tumors in nude mice. Pancreatic cancer cells were treated with either DDTC or TPA alone, or in combination and the number of viable cells was then determined by trypan blue ecxlusion assay and the number of apoptotic cells was determined by morphological assessment by staining the cells with propidium idiode and examining them under a fluorescence microscope. Treatment with DDTC or TPA alone inhibited the growth and promoted the apoptosis of pancreatic cancer cells in a concentration-dependent manner. These effects were more prominent following treatment with TPA in combination with DDTC than following treatment with either agent alone in PANC-1 cells in monolayer cultures and in 3 dimensional (3D) cultures. The potent effects of the combination treatment on PANC-1 cells were associated with the inhibition of nuclear factor-κB (NF-κB) activation and the decreased expression of Bcl-2 induced by DDTC, as shown by NF-κB-dependent reporter gene expression assay and western blot analysis. Furthermore, treatment of nude mice with DDTC + TPA strongly inhibited the growth of PANC-1 xenograft tumors. The results of the present study indicate that the administration of TPA and DDTC in combination may be an effective strategy for inhibiting the growth of pancreatic cancer. PMID:25847449

  19. Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2

    PubMed Central

    Lee, Yun Yeong; Ryu, Min Sook; Kim, Hong Seok; Suganuma, Masami; Song, Kye Yong; Lim, In Kyoung

    2016-01-01

    The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS104 via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21WAF1 gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells. PMID:26912086

  20. Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2.

    PubMed

    Lee, Yun Yeong; Ryu, Min Sook; Kim, Hong Seok; Suganuma, Masami; Song, Kye Yong; Lim, In Kyoung

    2016-03-01

    The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS(104) via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21(WAF1) gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells. PMID:26912086

  1. Prostratin and 12-O-tetradecanoylphorbol 13-acetate are potent and selective inhibitors of Chikungunya virus replication.

    PubMed

    Bourjot, Mélanie; Delang, Leen; Nguyen, Van Hung; Neyts, Johan; Guéritte, Françoise; Leyssen, Pieter; Litaudon, Marc

    2012-12-28

    A chemical study of the Vietnamese plant species Trigonostemon howii led to the isolation of a new tigliane-type diterpenoid, trigowiin A (1), along with several known coumarins and phenylpropanoids. The planar structure and the relative configuration of compound 1 were elucidated based on spectroscopic analysis, including 1D- and 2D-NMR experiments, mass spectrometry, and comparison with literature data. Trigowiin A (1) exhibited moderate antiviral activity in a virus-cell-based assay for Chikungunya virus (CHIKV). Since the structure of compound 1 is closely related to those of well-known tigliane diterpenoids such as prostratin (2), phorbol (3), 12-O-tetradecanoylphorbol 13-acetate (TPA) (4), and 4α-TPA (5), the antiviral activity of the latter compounds was also evaluated against CHIKV, as well as in virus-cell-based assays of two additional members of the genus Alphavirus (Sindbis virus, SINV, and Semliki forest virus, SFV). Whereas prostratin inhibited CHIKV replication with a moderate EC(50) of 2.6 μM and a selectivity index (SI) approximating 30, compound 4 proved to be an extremely potent inhibitor, with an EC(50) of ∼3 nM and a SI near 2000. Interestingly, no or very little activity was observed on the replication of SINV and SFV. PMID:23215460

  2. 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

  3. 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.

  4. HTLV-1 tax-induced NF-kappaB activation is synergistically enhanced by 12-O-tetradecanoylphorbol-13-acetate: mechanism and implications for Tax oncogenicity.

    PubMed

    Azran-Shaish, Inbal; Tabakin-Fix, Yulia; Huleihel, Mahmoud; Bakhanashvili, Mary; Aboud, Mordechai

    2008-07-01

    Nuclear factor kappa B (NF-kappaB) factors regulate a wide range of physiological and oncogenic processes. Normally, these factors are transiently activated by specific external signals which induce their dissociation from inhibitors of kappaB (IkappaB) and subsequent translocation to the nucleus where p65 links to the cyclic adenosine monophosphate response element binding protein (CBP)-p300 and P/CAF coactivators that are essential for its transcriptional activity. The pathogenic potential of human T-cell leukemia virus type 1 (HTLV-1) Tax protein is partly ascribed to its capacity to constitutively activate NF-kappaB factors because constitutive activity of these factors play an important role in the pathophysiology of adult T-cell leukemia (ATL) and tropical spastic paraparesis-HTLV-1 associated myelopathy (TSP-HAM). In assessing the possibility of modulating Tax pathogenic potential by external factors, we focused here on 12-O -tetradecanoylphorbol-13-acetate (TPA) which is a potent protein kinase C (PKC) activator. There are conflicting reports regarding the effect of TPA and PKC on NF-kappaB. Therefore, we reassessed this issue and also investigated their influence on Tax-mediated activation of these factors. We found that TPA promoted NF-kappaB nuclear translocation and the DNA binding of p65 dimers through PKC activation. However, both TPA and ectopically expressed PKC had only a marginal effect on the transcriptional competence of these dimers, indicating that the DNA binding of such dimers is insufficient by itself for gene activation. Notably, however, both TPA and the ectopic PKC displayed strong synergistic enhancement of the Tax-induced activation of the NF-kappaB transcriptional function. In contrast, TPA and the ectopic PKC only slightly elevated the low activation of the NF-kappaB transcriptional capacity by cytoplasmic Tax mutants, indicating that the nuclear translocation of Tax was essential for this synergism. Subsequent experiments suggested

  5. 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.

  6. Inhibitory effect of euphol, a triterpene alcohol from the roots of Euphorbia kansui, on tumour promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin.

    PubMed

    Yasukawa, K; Akihisa, T; Yoshida, Z Y; Takido, M

    2000-01-01

    The anti-inflammatory activity of euphol, twelve other triterpene alcohols and sitosterol-beta-D-glucopyranoside, isolated from the dichloromethane extract of the roots of Euphorbia kansui, has been evaluated in mice with inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA (1.7 nmol; 1.0 microg/ear) was dissolved in acetone and 10 microL delivered to the inner and outer surfaces of the right ear of ICR mice. A triterpene alcohol, sterol glucoside or vehicle (20 microL; chloroform-methanol 1:1), was applied topically approximately 30 min before each TPA treatment. The ear thickness was measured before treatment and then oedema was measured 6 h after TPA treatment. For the two-stage carcinogenesis experiment, initiation was accomplished by administration of a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA; 195 nmol; 50 microg/mouse) to the shaved backs of mice. Promotion was with 1.7 nmol (1.0 microg) TPA, applied twice weekly to the same shaved area, begun one week after the initiation. Euphol (2.0 micromol; 853 microg), or its vehicle (acetone-dimethylsulphoxide, 9:1; 100 microL), was applied topically 30 min before each TPA treatment. The number and diameter of skin tumours were measured every other week for 20 weeks. All the compounds were found to possess marked inhibitory activity and their 50% inhibitory dose for TPA-induced inflammation was 0.2-1.0 mg/ear. Topical application of euphol (2.0 micromol; 853 microg/mouse) markedly suppressed the tumour-promoting effect of TPA (1.7 nmol; 1.0 microg/mouse) in mouse skin initiated with DMBA. PMID:10716613

  7. Hexahydro-β-acids potently inhibit 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation and tumor promotion in mice.

    PubMed

    Hsu, Chung-Huei; Ho, Yuan-Soon; Lai, Ching-Shu; Hsieh, Shu-Chen; Chen, Li-Hua; Lin, Edwin; Ho, Chi-Tang; Pan, Min-Hsiung

    2013-11-27

    We previously reported that hexahydro-beta-acids (HBAs), reduced derivatives of beta-acids (BA) from hop (Humulus lupulus L.), displayed more potent anti-inflammatory activity than BA in lipopolysaccharide-stimulated murine macrophages. In this study, we investigated the effects and underlying molecular mechanisms of hexahydro-β-acids (HBAs) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated mouse skin inflammation and in the two-stage carcinogenesis model. Female ICR mice pretreated with HBA at 1 and 10 μg significantly reduced ear edema, epidermal hyperplasia, and infiltration of inflammatory cells caused by TPA. Molecular analysis exhibited that HBA suppressed iNOS, COX-2, and ornithine decarboxylase (ODC) protein and gene expression through interfering with mitogen-activated protein kinases (MAPKs) and phosphatidylinositiol 3-kinase (PI3K)/Akt signaling as well as the activation of transcription factor NF-κB. Furthermore, application of HBA (1 and 10 μg) prior to each TPA treatment (17.2 ± 0.9 tumors/mouse) resulted in the significant reduction of tumor multiplicity (5.1 ± 1.2, P < 0.01 and 2.3 ± 1.2, P < 0.001, respectively) in 7,12-dimethyl-benzanthracene (DMBA)-initiated mouse skin. The tumor incidence was significantly lowered to 75% (P < 0.05) and 58.7% (P < 0.01) by HBA pretreatment, respectively, and significantly reduced the tumor weight (0.34 ± 0.14 g, P < 0.01 and 0.09 ± 0.10 g, P < 0.001, respectively) as compared to DMBA/TPA-induced tumors (0.76 ± 0.04 g). PMID:24206127

  8. Differential carcinogenic effects of intraperitoneal initiation with 7,12-dimethylbenz(a)anthracene or urethane and topical promotion with 12-O-tetradecanoylphorbol-13-acetate in skin and internal tissues of female SENCAR and BALB/c mice

    SciTech Connect

    Ward, J.M.; Rehm, S.; Devor, D.; Hennings, H.; Wenk, M.L.

    1986-09-01

    Groups of female SENCAR or BALB/c mice were initiated once intraperitoneally with 300 ..mu..g/mouse of 7,12-dimethylbenz(a) anthracene (DMBA) or 20 mg/mouse of urethane at 7 weeks of age. Beginning one week later, mice received topically applied acetone or 12-O-tetradecanoylphorbol-13-acetate (TPA), once weekly, at 2.5 ..mu..g/mouse for weeks 1 through 6 and 1.25 ..mu..g/mouse for weeks 7 through 52. The skin lesions were evaluated clinically. A complete necropsy was performed on all mice at week 52. SENCAR mice exposed to DMBA/TPA and urethane/TPA had more skin tumors than SENCAR mice exposed to DMBA or urethane alone and more than BALB/c mice in any treatment group. Of all skin carcinomas diagnosed histologically in DMBA/TPA-exposed mice, less than one-third had been identified clinically while the mice were alive. Most of the carcinomas arose within papillomas. BALB/c mice developed more vascular and uterine tumors than did SENCAR mice injected with DMBA and more lung and vascular tumors than did SENCAR mice injected with urethane. TPA exposure after treatment with either initiator had no significant effect on internal tumor development in either SENCAR or BALB/c mice.

  9. 12-O-Tetradecanoylphorbol-13-Acetate Induces Up-Regulated Transcription of Variant 1 but Not Variant 2 of VIL2 in Esophageal Squamous Cell Carcinoma Cells via ERK1/2/AP-1/Sp1 Signaling

    PubMed Central

    Zhang, Xiao-Dan; Xie, Jian-Jun; Liao, Lian-Di; Long, Lin; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

    2015-01-01

    The membrane-cytoskeleton link organizer ezrin may be the most “dramatic” tumor marker, being strongly over-expressed in nearly one-third of human malignancies. However, the molecular mechanisms of aberrant ezrin expression still need to be clarified. Ezrin, encoded by the VIL2 gene, has two transcript variants that differ in the transcriptional start site (TSS): V1 and V2. Both V1 and V2 encode the same protein. Here, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) induced over-expression of human VIL2 in esophageal squamous cell carcinoma (ESCC) cells. Furthermore, VIL2 V1 but not V2 was up-regulated after TPA stimulation in a time-dependent manner. AP-1 and Sp1 binding sites within the promoter region of VIL2 V1 acted not only as basal transcriptional elements but also as a composite TPA-responsive element (TRE) for the transcription of VIL2 V1. TPA stimulation enhanced c-Jun and Sp1 binding to the TRE via activation of the ERK1/2 pathway and increased protein levels of c-Jun, c-Fos, and Sp1, resulting in over-expression of VIL2 V1, whereas the MEK1/2 inhibitor U0126 blocked these events. Finally, we showed that TPA promoted the migration of ESCC cells whereas MEK1/2 inhibitor or ezrin silencing could partially inverse this alteration. Taken together, these results suggest that TPA is able to induce VIL2 V1 over-expression in ESCC cells by activating MEK/ERK1/2 signaling and increasing binding of Sp1 and c-Jun to the TRE of the VIL2 V1 promoter, and that VIL2 is an important TPA-induced effector. PMID:25915860

  10. 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

  11. Effect of the promoter 12-O-tetradecanolyphorbol-13- acetate on the evolution of carcinogen-altered cell populations in tracheas initiated with 7,12-dimethylbenz(a)anthracene

    SciTech Connect

    Terzaghi, M.; Klein-Szanto, A.; Nettesheim, P.

    1983-04-01

    The aim of these studies was to investigate the effect(s) of the promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) on the evolution of different types of 7,12-dimethylbenz(a)anthracene (DMBA)-initiated rat tracheal epithelial cells in vivo. In the present study, tracheal transplants were exposed in vivo to 35 ..mu..g DMBA for 2 weeks and subsequently to 100 ..mu..g TPA. Controls were exposed to DMBA alone, TPA alone, or blank pellets alone. Tracheal cells were harvested by enzymatic procedures at 0, 3, 6, 12, or 18 months after the end of exposure to DMBA and at the same time points after the beginning of exposure to TPA or control pellets and were assayed in vitro with the epithelial focus (EF) assay for the frequency of different types of EF-forming units. Control tracheas yielded <1 EF/10/sub 4/ viable cells harvested. Exposure to TPA alone did not increase the yield of EF, EF/sub s/, or EF/sub s,ag+/ above control levels. Carcinogen exposure resulted in a 6- to 20-fold increase in yield of EF, a 2- to 3-fold increase in EF/sub s/, and a greater than or equal to 15-fold increase in yield of EF/sub s,ag+/ above control levels. Neither the yield of EF nor the yield of EF/sub s/ was affected by subsequent TPA. In contrast, there was a marked effect of subsequent TPA exposure on the maintenance and size of the cell pool giving rise to anchorage-independent offspring (EF/sub s,ag+/). In summary, it appears that initiation can be viewed as a series of complex cellular changes. With time, some of these changes are reversible. Exposure to TPA of cell populations initiated with low doses of DMBA results in the persistence of altered cell populations in the intact tissue. Without TPA treatment, some phenotypically altered cells appear to revert to a more normal state and/or fail to replicate. 26 references, 1 figure, 1 table.

  12. myo-Inositol 1,3-acetals as early intermediates during the synthesis of cyclitol derivatives.

    PubMed

    Gurale, Bharat P; Sardessai, Richa S; Shashidhar, Mysore S

    2014-11-18

    Synthetic sequences starting from commercially available myo-inositol necessarily involve protection-deprotection strategies of its six hydroxyl groups. Several strategies have been developed/attempted over the last several decades leading to the synthesis of naturally occurring phosphoinositols, their analogs, and cyclitol derivatives. Of late, myo-inositol 1,3-acetals, which can be obtained by the reductive cleavage of myo-inositol orthoesters have emerged as early intermediates for the synthesis of phosphorylated and other inositol derivatives. This mini-review is an attempt to illustrate the economy and convenience of using myo-inositol 1,3-acetals as early intermediates during syntheses from myo-inositol. PMID:25216930

  13. Liganded Thyroid Hormone Receptor Inhibits Phorbol 12-O-Tetradecanoate-13-Acetate-Induced Enhancer Activity via Firefly Luciferase cDNA

    PubMed Central

    Misawa, Hiroko; Sasaki, Shigekazu; Matsushita, Akio; Ohba, Kenji; Iwaki, Hiroyuki; Matsunaga, Hideyuki; Suzuki, Shingo; Ishizuka, Keiko; Oki, Yutaka; Nakamura, Hirotoshi

    2012-01-01

    Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity

  14. 12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G.

    PubMed

    Welgus, H G; Connolly, N L; Senior, R M

    1986-05-01

    Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is

  15. Aronia melanocarpa Concentrate Ameliorates Pro-Inflammatory Responses in HaCaT Keratinocytes and 12-O-Tetradecanoylphorbol-13-Acetate-Induced Ear Edema in Mice.

    PubMed

    Goh, Ah Ra; Youn, Gi Soo; Yoo, Ki-Yeon; Won, Moo Ho; Han, Sang-Zin; Lim, Soon Sung; Lee, Keun Wook; Choi, Soo Young; Park, Jinseu

    2016-07-01

    Abnormal expression of pro-inflammatory mediators such as cell adhesion molecules and cytokines has been implicated in various inflammatory skin diseases, including atopic dermatitis. In this study, we investigated the anti-inflammatory activity of Aronia melanocarpa concentrate (AC) and its action mechanisms using in vivo and in vitro skin inflammation models. Topical application of AC on mouse ears significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema formation, as judged by measuring ear thickness and weight, and histological analysis. Topical administration of AC also reduced the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in TPA-stimulated mouse ears. Pretreatment with AC suppressed TNF-α-induced ICAM-I expression and subsequent monocyte adhesiveness in human keratinocyte cell line HaCaT. In addition, AC significantly decreased intracellular reactive oxygen species (ROS) generation as well as mitogen-activated protein kinase (MAPK) activation in TNF-α-stimulated HaCaT cells. AC and its constituent cyanidin 3-glucoside also attenuated TNF-α-induced IKK activation, IκB degradation, p65 phosphorylation/nuclear translocation, and p65 DNA binding activity in HaCaT cells. Overall, our results indicate that AC exerts anti-inflammatory activities by inhibiting expression of pro-inflammatory mediators in vitro and in vivo possibly through suppression of ROS-MAPK-NF-κB signaling pathways. Therefore, AC may be developed as a therapeutic agent to treat various inflammatory skin diseases. PMID:27331630

  16. Glycoprotein isolated from Solanum nigrum L. modulates the apoptotic-related signals in 12-O-tetradecanoylphorbol 13-acetate-stimulated MCF-7 cells.

    PubMed

    Heo, Kyung-Sun; Lim, Kye-Taek

    2005-01-01

    Solanum nigrum L. (SNL) has been used in folk medicine for its anti-inflammatory activity. We isolated only the SNL glycoprotein from SNL and found that it was cytotoxic at low concentration. With respect to cytotoxicity, we investigated whether purified SNL glycoprotein is able to regulate protein kinase C (PKC) alpha activation and nuclear factor (NF)- kappaB activities in 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced tumor promotion, and whether it has an apoptosis-inducing effect in MCF-7 cells using western blot analysis. In addition, to elucidate the relationship between PKCalpha and NF-kappaB, inhibitory studies were performed with staurosporine (an inhibitor of phospholipid/calcium-dependent protein kinase) and pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB activation). To verify induction of apoptosis by the SNL glycoprotein, we performed DNA fragmentation and nuclear staining assays using ethidium bromide and bisbenzamide H33342. The results in this study indicated that SNL glycoprotein induces apoptosis through modulation of PKCalpha and NF-kappaB activity in MCF-7 cells. In fact, SNL glycoprotein interfered with PKCalpha membrane translocation and inhibited NF-kappaB (p50) protein activity in MCF-7 cells stimulated with TPA (61.68 ng/mL, 100 nM) dose-dependently. Regarding the apoptotic-inducing effect, nucleosomal DNA fragmentation and nuclear staining by SNL glycoprotein in MCF-7 cells were shown. Collectively, the data demonstrate that SNL glycoprotein is a potential natural anticancer agent because of its ability to induce apoptosis in MCF-7 cells. PMID:15857213

  17. 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

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

    SciTech Connect

    Graem, N.

    1986-01-01

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

  19. The mechanism of the action of IFN-gamma and TPA on the modulation of epidermal growth factor receptors of human amnion cells.

    PubMed

    Katoh, T; Higashi, K; Karasaki, Y

    1992-06-01

    We have examined the mechanism of synergistic action occurring between interferon (IFN)-gamma and 12-0-tetradecanoylphorbol-13-acetate (TPA) with respect to the reduction of 125I-epidermal growth factor (125I-EGF) binding to human amnion (WISH) cells [Karasaki Y et al (1989) J Biol Chem 264: 6158-6163]. The cells were treated with protein kinase C (PKC) inhibitors (H7, staurosporine) to investigate the role of PKC in the synergism between IFN-gamma and TPA, since TPA is a strong activator of PKC. The combined effect of IFN-gamma and TPA was blocked by the PKC inhibitor, suggesting that PKC plays an important role in the synergistic action of TPA and IFN-gamma on the inhibition of EGF binding to the cells. The prolonged incubation (24 h) of the cells with TPA resulted in the restoration of EGF binding to the cells. A 24 h treatment of WISH cells with both IFN-gamma and TPA, however, still exhibited greater than 50% inhibition of EGF binding. No PKC activity, however, was observed in the WISH cells treated with both IFN-gamma and TPA for 24 h as well as with TPA alone for 24 h, indicating that IFN-gamma may synergize with the second mediator induced by PKC rather than PKC itself in the reduction of EGF binding to WISH cells. In addition, IFN-gamma showed the synergistic action with calcium ionophores on the reduction of EGF binding to the cells, suggesting that Ca2+ may be one of the second mediators which was induced by TPA and which cooperated with IFN-gamma. PMID:1621011

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

    PubMed

    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

  2. Surfactin suppresses TPA-induced breast cancer cell invasion through the inhibition of MMP-9 expression.

    PubMed

    Park, Sun Young; Kim, Ji-Hee; Lee, Young Ji; Lee, Sang Joon; Kim, Younghee

    2013-01-01

    Metastasis is the main cause of cancer mortality. In this study, we investigated the effects of surfactin, a cyclic lipopeptide produced by Bacillus subtilis, on cancer metastasis in vitro and the underlying molecular mechanisms involved. Surfactin inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced invasion, migration and colony formation of human breast carcinoma cells. Western blot analysis, gelatin zymography and reverse transcription-PCR analysis revealed that matrix metalloproteinase-9 (MMP-9) expression and activation was significantly suppressed by surfactin in a dose-dependent manner. Surfactin attenuated TPA-induced nuclear translocation and activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Furthermore, surfactin strongly repressed the TPA-induced phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Treatment with specific inhibitors of Akt and ERK suppressed MMP-9 expression and activation. These results suggest that the surfactin-mediated inhibition of breast cancer cell invasion and MMP-9 expression involves the suppression of the NF-κB, AP-1, phosphatidylinositol 3-kinase (PI-3K)/Akt and the ERK signaling pathways. Thus surfactin may have potential value in therapeutic strategies for the treatment of breast cancer metastasis. PMID:23151889

  3. Heme oxygenase-1-mediated anti-inflammatory effects of tussilagonone on macrophages and 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation in mice.

    PubMed

    Lee, Joohee; Kang, Unwoo; Seo, Eun Kyoung; Kim, Yeong Shik

    2016-05-01

    The dried flower buds of Tussilago farfara L. have been used in traditional medicine, mainly as an antitussive in the treatment of cough and other respiratory problems. In the present study, we investigated the anti-inflammatory signaling pathway via the upregulation of heme oxygenase-1 (HO-1) in response to tussilagonone (TGN), a sesquiterpene compound isolated from T. farfara. TGN induced HO-1 expression and nuclear factor-E2-related factor 2 (Nrf2) activation in RAW 264.7 cells. Nuclear translocation of Nrf2 by TGN also increased in a time- and dose-dependent manner, indicating that TGN induced HO-1 via the Nrf2 pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, TGN suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and reduced the mRNA expression of proinflammatory cytokines, as well as nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. TGN inhibited the phosphorylation and degradation of inhibitory κB-α (IκB-α) and the nuclear translocation of nuclear factor (NF)-κB. However, a specific inhibitor of HO-1 reversed the TGN-mediated suppression of NO production and knockdown of HO-1 by small interfering RNA abrogated inhibitory effects of TGN on iNOS and COX-2 protein expression and NF-κB nuclear translocation. Furthermore, TGN reduced iNOS and COX-2 expression in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation mouse model. Taken together, these findings suggest an important role for TGN-induced HO-1 activation in regulating inflammatory responses. Moreover, TGN is a potent therapeutic candidate for targeting the crosstalk between Nrf2/HO-1 and the NF-κB signaling pathway in the prevention or treatment of inflammation-associated diseases. PMID:26950613

  4. Participation of mitogen-activated protein kinase in thapsigargin- and TPA-induced histamine production in murine macrophage RAW 264.7 cells

    PubMed Central

    Shiraishi, Muneshige; Hirasawa, Noriyasu; Kobayashi, Yuriko; Oikawa, Shinji; Murakami, Akira; Ohuchi, Kazuo

    2000-01-01

    Stimulation of the murine macrophage cell line RAW 264.7 with thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induced histamine production in a time- and concentration-dependent manner. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), also enhanced histamine production. α-Fluoromethylhistidine, a suicide substrate of L-histidine decarboxylase (HDC), suppressed the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production. Both thapsigargin (30 nM) and TPA (30 nM) induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059, a specific inhibitor of MEK-1 which phosphorylates p44/p42 MAP kinase, strongly suppressed both the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production, whereas SB203580, a specific inhibitor of p38 MAP kinase, inhibited them only partially. The other MEK-1 inhibitor, U-0126, also inhibited both the thapsigargin- and TPA-induced histamine production in a concentration-dependent manner. Thapsigargin (30 nM) and TPA (30 nM) increased the levels of HDC mRNA at 4 h, but PD98059 suppressed both the thapsigargin- and TPA-induced increases in the HDC mRNA level. These findings indicate that thapsigargin and TPA induce histamine production in RAW 264.7 cells by increasing the level of HDC mRNA, and that both the thapsigargin- and TPA-induced histamine production are regulated largely by p44/p42 MAP kinase and partially by p38 MAP kinase.. PMID:10711350

  5. Sulforaphane controls TPA-induced MMP-9 expression through the NF-κB signaling pathway, but not AP-1, in MCF-7 breast cancer cells

    PubMed Central

    Lee, Young-Rae; Noh, Eun-Mi; Han, Ji-Hey; Kim, Jeong-Mi; Hwang, Bo-Mi; Kim, Byeong-Soo; Lee, Sung-Ho; Jung, Sung Hoo; Youn, Hyun Jo; Chung, Eun Yong; Kim, Jong-Suk

    2013-01-01

    Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells. [BMB Reports 2013; 46(4): 201-206] PMID:23615261

  6. 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.

  7. Naringenin suppresses TPA-induced tumor invasion by suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells.

    PubMed

    Yen, Hung-Rong; Liu, Ching-Ju; Yeh, Chia-Chou

    2015-06-25

    Naringenin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong anti-proliferative properties and the ability to induce apoptosis in hepatoma cell lines. However, there are no reports describing its effects on the invasion and metastasis of hepatoma cell lines, and the detailed molecular mechanisms of its effects are still unclear. In this study, we investigated the mechanisms underlying naringenin-mediated inhibition of 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced cell invasion and inhibition of secreted and cytosolic MMP-9 production in human hepatoma cells (HepG2, Huh-7, and HA22T) and murine embryonic liver cells (BNL CL2). Naringenin suppressed MMP-9 transcription by inhibiting activator protein (AP)-1 and nuclear factor-κB (NF-κB) activity. It suppressed TPA-induced AP-1 activity through inhibiting the phosphorylation of the extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and it suppressed TPA-induced inhibition of NF-κB nuclear translocation through IκB. Additionally, it suppressed TPA-induced activation of ERK/phosphatidylinositol 3-kinase/Akt upstream of NF-κB and AP-1. These data suggest that naringenin suppresses the invasiveness and metastatic potential of hepatocellular carcinoma (HCC) by inhibiting multiple signal transduction pathways. PMID:25866363

  8. Effect of phorbol derivatives and staurosporine on gravitropic response of primary root of maize

    SciTech Connect

    Mulkey, T.J.; Kim, S.Y. ); Lee, J.S. )

    1991-05-01

    Time-lapse videography and computer-based, video image digitization were used to examine the effects of phorbol derivatives (phorbol 12-myristate 13-acetate, TPA; phorbol 12-myristate 13-acetate 4-O-methyl ether, mTPA) and staurosporine on the kinetics of gravicurvature of primary roots of maize (Zea mays L., Pioneer 3343 and Golden Cross Bantam). Pretreatment of roots with TPA (3 hr, 1 {mu}M) decreases the time lag prior to induction of positive gravicurvature in horizontally-oriented roots by > 60%. The rate of curvature is not significantly different than the rate observed in control roots. Wrongway curvature which is observed in 30-40% of control roots is not observed in TPA-pretreated roots. Oscillatory movements observed in control roots after completion of gravitropic reorientation is completely dampened in TPA-pretreated roots. Pretreatment of roots with mTPA(3hr,1{mu}M), the inactive analog of TPA, does not significantly alter the kinetics of gravicurvature of primary roots of maize. Staurosporine (10{sup {minus}8}M), a microbial alkaloid which has been reported to have antifungal activity and to inhibit phospholipid/Ca{sup ++} dependent protein kinase, completely inhibits TPA-induced alteration of the kinetics of gravitropism. DAG (1-oleoyl-2-acetyl-rac-glycerol), a synthetic diglyceride activator of protein kinase C, exhibits similar activity to TPA. TPA-induced alterations in tissue response to auxin are presented.

  9. 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.

  10. The role of PKC/ERK1/2 signaling in the anti-inflammatory effect of tetracyclic triterpene euphol on TPA-induced skin inflammation in mice.

    PubMed

    Passos, Giselle F; Medeiros, Rodrigo; Marcon, Rodrigo; Nascimento, Andrey F Z; Calixto, João B; Pianowski, Luiz F

    2013-01-01

    Inflammation underlies the development and progression of a number of skin disorders including psoriasis, atopic dermatitis and cancer. Therefore, novel antiinflammatory agents are of great clinical interest for prevention and treatment of these conditions. Herein, we demonstrated the underlying molecular mechanisms of the antiinflammatory activity of euphol, a tetracyclic triterpene isolated from the sap of Euphorbia tirucalli, in skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice. Topical application of euphol (100 μg/ear) significantly inhibited TPA-induced ear edema and leukocyte influx through the reduction of keratinocyte-derived chemokine (CXCL1/KC) and macrophage inflammatory protein (MIP)-2 levels. At the intracellular level, euphol reduced TPA-induced extracellular signal-regulated protein kinase (ERK) activation and cyclooxygenase-2 (COX-2) upregulation. These effects were associated with euphol's ability to prevent TPA-induced protein kinase C (PKC) activation, namely PKCα and PKCδ isozymes. Our data indicate that topical application of euphol markedly inhibits the inflammatory response induced by TPA. Thus, euphol represents a promising agent for the management of skin diseases with an inflammatory component. PMID:23099255

  11. Dehydroglyasperin C suppresses TPA-induced cell transformation through direct inhibition of MKK4 and PI3K.

    PubMed

    Lee, Ji Hoon; Kim, Jong-Eun; Jang, Young Jin; Lee, Charles C; Lim, Tae-Gyu; Jung, Sung Keun; Lee, Eunjung; Lim, Soon Sung; Heo, Yong Seok; Seo, Sang Gwon; Son, Joe Eun; Kim, Jong Rhan; Lee, Chang Yong; Lee, Hyong Joo; Lee, Ki Won

    2016-05-01

    Bioactive natural compounds from plant-derived sources have received substantial interest due to their potential therapeutic and preventive effects toward various human diseases. Licorice (Glycyrrhiza), a frequently-used component in traditional oriental medicines, has been incorporated into recipes not only to enhance taste, but also to treat various conditions including inflammation, chronic fatigue syndrome, and even cancer. Dehydroglyasperin C (DGC) is a major isoflavone found in the root of licorice. In the present study, we investigated the cancer chemopreventive effect of DGC and the underlying molecular mechanisms involved, by analyzing its effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation and cyclooxygenase (COX)-2 expression in JB6 P+ mouse epidermal cells. DGC treatment attenuated TPA-induced activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) transcriptional activation, two major regulators of TPA-induced cell transformation, and COX-2 expression. TPA-induced phosphorylation of p38, JNK1/2 and Akt was also suppressed by DGC. Kinase assay data revealed that DGC inhibited the kinase activity of MKK4 and PI3K and this outcome was due to direct physical binding with DGC. Notably, DGC bound directly to MKK4 and PI3K in an ATP-competitive manner. Taken together, these results suggest that DGC exhibits cancer chemopreventive potential via its inhibitory effect on TPA-induced neoplastic cell transformation and COX-2 modulation through regulation of the MKK4 and PI3K pathways. © 2015 Wiley Periodicals, Inc. PMID:25787879

  12. Relationship between exposure to TPA and appearance of transformed cells in MNNG-initiated transformation of BALB/c 3T3 cells.

    PubMed

    Tsuchiya, T; Umeda, M

    1997-10-01

    In the BALB/c-3T3-cell transformation system, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure on the appearance of transformed cells was examined in order to investigate the mechanisms of in vitro tumor promotion. Optimal duration of TPA exposure on N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-initiated cells was at least 11 days. To investigate the effect of transformation frequencies of altering inoculating cell density at the replating of MNNG-exposed cells and of altering the time of starting TPA exposure, MNNG-exposed cells were replated at various inoculum sizes. With lower inoculum sizes (1 x 10(3) to 3 x 10(4) cells/dish), maximum TPA-induced transformation occurred for TPA commencement at confluence, while with higher inoculum size (1 x 10(5) cells/dish), maximum transformation frequency was observed when TPA exposure was started on day 7 after replating, being some 2 days after confluence. This may suggest that there are different mechanisms involved, depending on inoculum size, and that these may involve cell-cell interactions (at lower inoculum) and mutation expression periods (at higher inoculum). By means of redispersion experiments, it was demonstrated that the appearance of transformed cells begins on about day 7 after replating at a cell density of 1 x 10(4) cells/dish. These results suggest the usefulness of the replating method for optimizing transformation in the BALB/c-3T3-cell transformation assay, and provide insight into the time frame of expression of MNNG-initiated transformants and TPA-induced expansion of these transformants. PMID:9335454

  13. Hispolon inhibits TPA-induced invasion by reducing MMP-9 expression through the NF-κB signaling pathway in MDA-MB-231 human breast cancer cells

    PubMed Central

    SUN, YI-SHENG; ZHAO, ZHAO; ZHU, HAN-PING

    2015-01-01

    Hispolon has been demonstrated to possess analgesic, anti-inflammatory and anticancer activities. However, whether hispolon prevents the invasion of breast carcinoma cells and the underlying mechanisms of its action remain unknown. In the present study, various assays, including a matrigel-based Transwell invasion assay and electrophoretic mobility shift assay, were used to investigate the anti-invasion effect of hispolon and explore its mechanism of action. The results revealed that hispolon inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced migration and invasion of MDA-MB-231 human breast cancer cells at non-toxic concentrations. Hispolon also prevented the TPA-induced secretion of matrix metalloproteinase-9 (MMP-9) and reduced its expression at the transcriptional and translational levels. Furthermore, the phosphorylation of IκBα was reduced by hispolon, which resulted in the suppression of nuclear factor-κB (NF-κB), and p65 phosphorylation and nuclear translocation. An electrophoretic mobility shift assay demonstrated that NF-κB DNA-binding activity was induced by TPA and inhibited by hispolon. In addition, Bay 11–7082, which is a specific inhibitor of NF-κB, functioned in a similar manner as hispolon and blocked the secretion and expression of MMP-9. In conclusion, the results of the present study indicated that hispolon inhibited TPA-induced migration and invasion of MDA-MB-231 cells by reducing the secretion and expression of MMP-9 through the NF-κB signaling pathway. PMID:26171065

  14. 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.

  15. 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,…

  16. 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

  17. Synchronous fluorescence spectroscopic characterization of DMBA-TPA-induced squamous cell carcinoma in mice

    NASA Astrophysics Data System (ADS)

    Diagaradjane, Parmeswaran; Yaseen, Mohammad A.; Yu, Jie; Wong, Michael S.; Anvari, Bahman

    2006-01-01

    While initially confined to the epidermis, squamous cell carcinoma can eventually penetrate into the underlying tissue if not diagnosed early and treated. The noninvasive early detection of the carcinoma is important to achieve a complete treatment of the disease. Of the various non-invasive optical techniques, the synchronous fluorescence (SF) technique is considered to provide a simplified spectral profile with more sharp spectral signatures of the endogenous fluorophores in complex systems. The potential use of the SF technique in the characterization of the sequential tissue transformation in 7,12-dimethylbenz(a)anthracene-12-O-tetradecanoylphorbol-13-acetate (DMBA-TPA)-induced mouse skin tumor model in conjunction with simple statistical analysis is explored. The SF spectra show distinct differences during the earlier weeks of the tumor-induction period. Intensity ratio variables are calculated and used in three discriminant analyses. All the discriminant analyses show better classification results with accuracy greater than 80%. From the observed differences in the spectral characteristics and the ratio variables that resulted in better classification between groups, it is concluded that tryptophan, collagen, and NADH are the key fluorophores that undergo changes during tissue transformation process and hence they can be targeted as tumor markers to diagnose normal from abnormal tissues using the SF technique.

  18. IL-13 but not IL-4 signaling via IL-4Rα protects mice from papilloma formation during DMBA/TPA two-step skin carcinogenesis

    PubMed Central

    Rothe, Michael; Quarcoo, David; Chashchina, Anna A; Bozrova, Svetlana V; Qin, Zhihai; Nedospasov, Sergei A; Blankenstein, Thomas; Kammertoens, Thomas; Drutskaya, Marina S

    2013-01-01

    Interleukin 4 (IL-4) was shown to be tumor-promoting in full carcinogenesis studies using 3-methylcholanthrene (MCA). Because heretofore the role of IL-4 in DMBA/TPA (9,10-dimethyl-1,2-benz-anthracene/12-O-tetradecanoylphorbol-13-acetate) two-stage carcinogenesis was not studied, we performed such experiments using either IL-4−/− or IL-4Rα−/− mice. We found that IL-4Rα−/− but not IL-4−/− mice have enhanced papilloma formation, suggesting that IL-13 may be involved. Indeed, IL-13−/− mice developed more papillomas after exposure to DMBA/TPA than their heterozygous IL-13-competent littermate controls. However, when tested in a full carcinogenesis experiment, exposure of mice to 25 μg of MCA, both IL-13−/− and IL-13+/− mice led to the same incidence of tumors. While IL-4 enhances MCA carcinogenesis, it does not play a measurable role in our DMBA/TPA carcinogenesis experiments. Conversely, IL-13 does not affect MCA carcinogenesis but protects mice from DMBA/TPA carcinogenesis. One possible explanation is that IL-4 and IL-13, although they share a common IL-4Rα chain, regulate signaling in target cells differently by employing distinct JAK/STAT-mediated signaling pathways downstream of IL-13 or IL-4 receptor complexes, resulting in different inflammatory transcriptional programs. Taken together, our results indicate that the course of DMBA/TPA- and MCA-induced carcinogenesis is affected differently by IL-4 versus IL-13-mediated inflammatory cascades. PMID:24403255

  19. 3'-untranslated region of SP-B mRNA mediates inhibitory effects of TPA and TNF-alpha on SP-B expression.

    PubMed

    Pryhuber, G S; Church, S L; Kroft, T; Panchal, A; Whitsett, J A

    1994-07-01

    Surfactant protein-B (SP-B) is a small hydrophobic polypeptide that enhances spreading and stability of surfactant phospholipids in the alveolus of the lung. Decreased expression of SP-B is associated with respiratory failure in premature infants and in adult patients with acute respiratory distress syndrome (ARDS). Tumor necrosis factor-alpha (TNF-alpha) and 12-O-tetradecanoylphorbol-13 acetate (TPA) cause ARDS-like lung injury in vivo. Inhibitory effects of TPA and TNF-alpha on SP-B mRNA expression in vitro were mediated by decreased SP-B mRNA stability rather than by decreased rate of SP-B gene transcription. In the present study, a human pulmonary adenocarcinoma cell line, NCI H441-4, was stably transfected with expression vectors consisting of the thymidine kinase (TK) promotor and human growth hormone (hGH) gene, in which the hGH 3'-untranslated region (3'-UTR) was replaced by the 2.0-kb human SP-B cDNA [pTKGH(SP-B2.0)] or the 837-bp human SP-B 3'-UTR [pTKGH(SP-B.837)]. The mRNAs and cellular growth hormone protein generated from the chimeric TKGH(SP-B2.0) and TKGH(SP-B.837) genes were each inhibited by approximately 50% by TPA and TNF-alpha. Dexamethasone decreased the inhibitory effects of TPA and TNF-alpha. The inhibition of steady-state hGH-SP-B mRNA by TPA and TNF-alpha was mediated by a cis-active element located in the 3-UTR region of SP-B mRNA. PMID:8048538

  20. The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice.

    PubMed

    Kowalczyk, Piotr; Junco, Jacob J; Kowalczyk, Magdalena C; Sosnowska, Renata; Tolstykh, Olga; Walaszek, Zbigniew; Hanausek, Margaret; Slaga, Thomas J

    2014-06-01

    Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin. PMID:23852815

  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. 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

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

    2016-02-16

    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 ≥4 mm/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

  3. Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK1 cells.

    PubMed Central

    Lee, J S; von der Ahe, D; Kiefer, B; Nagamine, Y

    1993-01-01

    Urokinase-type plasminogen activator (uPA) is an extracellular protease and expressed in various cells that exhibit dynamic changes in cell morphology, suggesting a link between cytoskeletal reorganization (CSR) and uPA expression. CSR can be induced by pharmacological agents, such as by colchicine for microtubule cytoskeleton and by cytochalasin for microfilament cytoskeleton. Using these agents, we previously showed that CSR induced the uPA gene in LLC-PK1 cells independently of the protein kinase C and cAMP-dependent protein kinase. Here we show that the induction of the uPA gene by CSR is mediated by the activation of c-Jun which interacts with an AP-1-like site located 2 kb upstream of the uPA gene. 12-O-tetradecanoylphorbol 13-acetate (TPA) induces the uPA gene through the same elements, but additionally utilizes an adjacent PEA3 element and induces c-fos. Furthermore, CSR induces a greater accumulation and a more pronounced phosphorylation of c-Jun than TPA induction. AP-1 is a positive regulator of growth and oncogenesis, and CSR is an integral part of these processes. Our results provide a view how CSR and AP-1 could be coupled in these processes. We also show that TPA and CSR act synergistically, suggesting a model where an initial activation signal could be amplified by CSR. Images PMID:8346015

  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. 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.

  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. PMID:17012771

  7. 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.

  8. Ultraviolet Radiation and 12-O-Tetradecanoylphorbol-13-Acetate-Induced Interaction of Mouse Epidermal Protein Kinase Cε With Stat3 Involve Integration With Erk1/2

    PubMed Central

    Sand, Jordan Marshall; Hafeez, Bilal Bin; Aziz, Moammir Hasan; Siebers, Emily Marie; Dreckschmidt, Nancy Ellen; Verma, Ajit Kumar

    2012-01-01

    We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present severa novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m2/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention. PMID:21480396

  9. Ultraviolet radiation and 12-O-tetradecanoylphorbol-13-acetate-induced interaction of mouse epidermal protein kinase Cε with Stat3 involve integration with ERK1/2.

    PubMed

    Sand, Jordan Marshall; Bin Hafeez, Bilal; Aziz, Moammir Hasan; Siebers, Emily Marie; Dreckschmidt, Nancy Ellen; Verma, Ajit Kumar

    2012-04-01

    We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present several novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m(2)/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention. PMID:21480396

  10. EFFECTS OF CHRONIC TOPICAL APPLICATION OF 12-0-TETRADECANOYLPHORBOL-13-ACETATE ON THE SKIN AND INTERNAL ORGANS OF SENCAR MICE

    EPA Science Inventory

    Repetitive topical applications of 2 micrograms TPA twice weekly for 37 to 52 weeks induced a sustained epidermal hyperplasia, hyperplasia of hair follicles, and increased dermal cellularity in SENCAR mice. In addition, after 52 weeks of protracted promoter treatment most animals...

  11. 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. PMID:18579711

  12. 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. PMID:21480506

  13. 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. PMID:26717978

  14. Luteolin 8-C-β-fucopyranoside inhibits invasion and suppresses TPA-induced MMP-9 and IL-8 via ERK/AP-1 and ERK/NF-κB signaling in MCF-7 breast cancer cells.

    PubMed

    Park, Su-Ho; Kim, Jung-Hee; Lee, Dong-Hun; Kang, Jeong-Woo; Song, Hyuk-Hwan; Oh, Sei-Ryang; Yoon, Do-Young

    2013-11-01

    Matrix metalloproteinase 9 (MMP-9) and interleukin-8 (IL-8) play major roles in tumor progression and invasion of breast cancer cells. The present study was undertaken to investigate the inhibitory mechanism of cell invasion by luteolin 8-C-β-fucopyranoside (named as LU8C-FP), a C-glycosylflavone, in human breast cancer cells. We investigated whether LU8C-FP would inhibit MMP-9 activation and IL-8 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 breast cancer cells. LU8C-FP suppressed TPA-induced MMP-9 and IL-8 secretion and mRNA expression via inhibition of the MAPK signaling pathway and down-regulation of nuclear AP-1 and NF-κB. TPA-induced phosphorylation of ERK 1/2 was suppressed by LU8C-FP, whereas JNK and p38 MAPK phosphorylation were unaffected. In addition, LU8C-FP blocked the ERK 1/2 pathways following expression of MMP-9 and IL-8. These results suggest LU8C-FP may function to suppress invasion of breast cancer cells through the ERK/AP-1 and ERK/NF-κB signaling cascades. PMID:23933110

  15. 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…

  16. 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…

  17. 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…

  18. Genome sequence and characterization of the Tsukamurella bacteriophage TPA2.

    PubMed

    Petrovski, Steve; Seviour, Robert J; Tillett, Daniel

    2011-02-01

    The formation of stable foam in activated sludge plants is a global problem for which control is difficult. These foams are often stabilized by hydrophobic mycolic acid-synthesizing Actinobacteria, among which are Tsukamurella spp. This paper describes the isolation from activated sludge of the novel double-stranded DNA phage TPA2. This polyvalent Siphoviridae family phage is lytic for most Tsukamurella species. Whole-genome sequencing reveals that the TPA2 genome is circularly permuted (61,440 bp) and that 70% of its sequence is novel. We have identified 78 putative open reading frames, 95 pairs of inverted repeats, and 6 palindromes. The TPA2 genome has a modular gene structure that shares some similarity to those of Mycobacterium phages. A number of the genes display a mosaic architecture, suggesting that the TPA2 genome has evolved at least in part from genetic recombination events. The genome sequence reveals many novel genes that should inform any future discussion on Tsukamurella phage evolution. PMID:21183635

  19. Characterization of an ExoS Type III Translocation-Resistant Cell Line

    PubMed Central

    Rucks, Elizabeth A.; Olson, Joan C.

    2005-01-01

    Pseudomonas aeruginosa ExoS is a type III-secreted type III-secreted, bifunctional protein that causes diverse effects on eukaryotic cell function. The coculture of P. aeruginosa strains expressing ExoS with HL-60 myeloid cells revealed the cell line to be resistant to the toxic effects of ExoS. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (TPA) rendered the cell line sensitive to ExoS. To understand the cellular basis for the alteration in sensitivity, undifferentiated and TPA-differentiated HL-60 cells were compared for differences in bacterial adherence, type III secretion induction, and ExoS translocation. These comparisons found that ExoS was translocated more efficiently in TPA-differentiated HL-60 cells than in undifferentiated cells. The studies support the ability of eukaryotic cells to influence P. aeruginosa TTS at the level of membrane translocation. PMID:15618208

  20. 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. PMID:25381260

  1. A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair*

    PubMed Central

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

    2014-01-01

    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. PMID:25381260

  2. 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

  3. Shock Hugoniot measurements on Ta to 0. 78 TPa

    SciTech Connect

    Froeschner, K.E.; Lee, R.S.; Chau, H.H.; Weingart, R.C.

    1983-08-18

    Symmetric impact shock Hugoniot measurements have been made on Ta with an electrically exploded foil gun system. The results obtained to date for the Hugoniot of Ta cover the range 0.19 to 0.78 TPa (impact velocities from 4.0 to 9.7 km/s) and agree with data obtained by other researchers to within 2.7% rms. Recent improvements in the system include electromagnetic shielding of impactor and target, continuous measurement of impactor velocity with a Fabry-Perot interferometer and computer-aided analysis of shot film. Conservative extrapolation from current operating conditions indicate that pressures of 1.1 to 1.5 TPa could be achieved with little difficulty.

  4. Spermine synthase overexpression in vivo does not increase susceptibility to DMBA/TPA skin carcinogenesis or Min-Apc intestinal tumorigenesis.

    PubMed

    Welsh, Patricia A; Sass-Kuhn, Suzanne; Prakashagowda, Chethana; McCloskey, Diane; Feith, David

    2012-04-01

    Numerous studies have demonstrated a link between elevated polyamine biosynthesis and neoplastic growth, but the specific contribution of spermine synthase to epithelial tumor development has never been explored in vivo. Mice with widespread overexpression of spermine synthase (CAG-SpmS) exhibit decreased spermidine levels, increased spermine and a significant rise in tissue spermine:spermidine ratio. We characterized the response of CAG-SpmS mice to two-stage skin chemical carcinogenesis as well as spontaneous intestinal carcinogenesis induced by loss of the Apc tumor suppressor in Apc (Min) (/+) (Min) mice. CAG-SpmS mice maintained the canonical increases in ornithine decarboxylase (ODC) activity, polyamine content and epidermal thickness in response to tumor promoter treatment of the skin. The induction of S-adenosylmethionine decarboxylase (AdoMetDC) activity and its product decarboxylated AdoMet were impaired in CAG-SpmS mice, and the spermine:spermidine ratio was increased 3-fold in both untreated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated skin. The susceptibility to 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis was not altered in CAG-SpmS mice, and SpmS overexpression did not modify the previously described tumor resistance of mice with targeted antizyme expression or the enhanced tumor response in mice with targeted spermidine/spermine-N ( 1) -acetyltransferase expression. CAG-SpmS/Min mice also exhibited elevated spermine:spermidine ratios in the small intestine and colon, yet their tumor multiplicity and size was similar to Min mice. Therefore, studies in two of the most widely used tumorigenesis models demonstrate that increased spermine synthase activity and the resulting elevation of the spermine:spermidine ratio does not alter susceptibility to tumor development initiated by c-Ha-Ras mutation or Apc loss. PMID:22258329

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

    PubMed

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

    2015-12-01

    Ellagic acid has previously been reported to induce reduced proliferation and activation of apoptosis in several tumor cell lines including our own previous data from non-differentiated human neuroblastoma SH-SY5Y cells. The aim of this study was now to investigate if in vitro differentiation with the phorbol ester 12-O- tetradecanoylphorbol-13-acetate or the vitamin A derivative all-trans retinoic acid altered the sensitivity to ellagic acid in SH-SY5Y cells. The methods used were cell counting and LDH-assay for evaluation of cell number and cell death, flow cytometric analysis of SubG1- and TUNEL-analysis for apoptosis and western blot for expression of apoptosis-associated proteins. In vitro differentiation was shown to reduce the sensitivity to ellagic acid with respect to cell detachment, loss of viability and activation of apoptosis. The protective effect was phenotype-specific and most prominent in all-trans retinoic acid-differentiated cultures. Differentiation-dependent up-regulation of Bcl-2 and integrin expression is introduced as possible protective mechanisms. The presented data also point to a positive correlation between proliferative activity and sensitivity to ellagic-acid-induced cell detachment. In conclusion, the presented data emphasize the need to consider degree of neuronal differentiation and phenotype of neuroblastoma cells when discussing a potential pharmaceutical application of ellagic acid in tumor treatment. PMID:26653548

  6. 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

    2014-01-01

    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. PMID:25029073

  7. Poly-γ-Glutamic Acid Induces Apoptosis via Reduction of COX-2 Expression in TPA-Induced HT-29 Human Colorectal Cancer Cells

    PubMed Central

    Shin, Eun Ju; Sung, Mi Jeong; Park, Jae Ho; Yang, Hye Jeong; Kim, Myung Sunny; Hur, Haeng Jeon; Hwang, Jin-Taek

    2015-01-01

    Poly-γ-glutamic acid (PGA) is one of the bioactive compounds found in cheonggukjang, a fast-fermented soybean paste widely utilized in Korean cooking. PGA is reported to have a number of beneficial health effects, and interestingly, it has been identified as a possible anti-cancer compound through its ability to promote apoptosis in cancer cells, although the precise molecular mechanisms remain unclear. Our findings demonstrate that PGA inhibits the pro-proliferative functions of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a known chemical carcinogen in HT-29 human colorectal cancer cells. This inhibition was accompanied by hallmark apoptotic phenotypes, including DNA fragmentation and the cleavage of poly (ADP-ribose) polymerase (PARP) and caspase 3. In addition, PGA treatment reduced the expression of genes known to be overexpressed in colorectal cancer cells, including cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). Lastly, PGA promoted activation of 5' adenosine monophosphate-activated protein (AMPK) in HT-29 cells. Taken together, our results suggest that PGA treatment enhances apoptosis in colorectal cancer cells, in part by modulating the activity of the COX-2 and AMPK signaling pathways. These anti-cancer functions of PGA make it a promising compound for future study. PMID:25854428

  8. Dodecafluoropentane Emulsion Extends Window for tPA Therapy in a Rabbit Stroke Model

    PubMed Central

    Brown, A. T.; Lowery, J. D.; Arthur, M. C.; Roberson, P. K.; Skinner, R. D.

    2016-01-01

    Dodecafluoropentane emulsion (DDFPe) nanodroplets are exceptional oxygen transporters and can protect ischemic brain in stroke models 24 h without reperfusion. Current stroke therapy usually fails to reach patients because of delays following stroke onset. We tested using DDFPe to extend the time window for tissue plasminogen activator (tPA). Longer treatment windows will allow more patients more complete stroke recovery. We test DDFPe to safely extend the time window for tPA thrombolysis to 9 h after stroke. With IACUC approval, randomized New Zealand white rabbits (3.4–4.7 kg, n=30) received angiography and 4-mm blood clot in the internal carotid artery for flow-directed middle cerebral artery occlusion. Seven failed and were discarded. Groups were IV tPA (n=11), DDFPe + tPA (n=7), and no therapy controls (n=5). DDFPe (0.3 ml/kg, 2 % emulsion) IV dosing began at 1 h and continued at 90 min intervals for 6 doses in one test group; the other received saline injections. Both got standard IV tPA (0.9 mg/kg) therapy starting 9 h post stroke. At 24 h, neurological assessment scores (NAS, 0–18) were determined. Following brain removal percent stroke volume (%SV) was measured. Outcomes were compared with Kruskal-Wallis analysis. For NAS, DDFPe + tPA was improved overall, p=0.0015, and vs. tPA alone, p=0.0052. For %SV, DDFPe + tPA was improved overall, p=0.0003 and vs. tPA alone, p=0.0018. NAS controls and tPA alone were not different but %SV was, p=0.0078. With delayed reperfusion, DDFPe + tPAwas more effective than tPA alone in preserving functioning brain after stroke. DDFPe significantly extends the time window for tPA therapy. PMID:26055229

  9. t-PA activity in peripheral blood obtained from pregnant women.

    PubMed

    Ishii, A; Yamada, S; Yamada, R; Hamada, H

    1994-01-01

    Concentrations of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were measured in blood obtained from pregnant women to elucidate the fluctuations in the fibrinolytic system which occur during the course of pregnancy. The t-PA activity was measured with a modified bioimmunoassay using anti-t-PA monoclonal antibody (SP-322) against a single chain of recombinant t-PA. The t-PA antigen was measured by ELISA using the same antibody. PAI activity was determined with a competitive inhibition assay of t-PA activity. In early pregnancy, t-PA activity was found to be close to the standard range seen in nonpregnant women, and gradually decreased during the course of pregnancy, then recovered to rise to the normal range within 48 hours after delivery. The t-PA antigen and PAI activity levels rose slowly during the course of pregnancy, and fell promptly after delivery. t-PA activity and t-PA antigen in levels in umbilical cord blood were higher after vaginal delivery than after cesarean section. These findings suggest that there may be an important physiological balance of the fibrinolytic system between mother and fetus during the course of pregnancy and the puerperium. PMID:7965540

  10. tPA promotes cortical neuron survival via mTOR-dependent mechanisms.

    PubMed

    Grummisch, Julia A; Jadavji, Nafisa M; Smith, Patrice D

    2016-07-01

    Tissue plasminogen activator (tPA) is a thrombolytic agent commonly used in the treatment of ischemic stroke. While the thrombolytic effects of tPA have been well established, the impact of this blood-brain barrier (BBB) crossing drug on neurons is not known. Given the widespread use of tPA in the clinical setting and the strict therapeutic window established for effective use of the drug, we examined the molecular mechanisms mediating the impact of tPA on postnatal cortical neurons isolated from the mouse brain. Dissociated postnatal primary cortical neurons were treated with tPA and the effects on neuron survival were evaluated. Pharmacological inhibitors of several signaling pathways previously implicated in neuroprotection (mTOR, JAK/STAT, MAPK and PKA-dependent mechanisms) were used to pinpoint the mechanistic effectors of tPA on neuron survival in vitro. We report here that tPA treatment results in a time-dependent neuroprotective effect on postnatal cortical neurons that relies predominantly on Janus kinase (JAK) and mammalian target of rapamycin (mTOR) signaling mechanisms. Taken together, these data suggest that tPA promotes neuroprotection in a temporally-regulated manner and that both JAK and mTOR signaling effectors are critical mediators of this neuroprotective effect. The results suggest the possibility of targeting these defined mechanisms to potentially expand the therapeutic window for tPA. PMID:26995507

  11. Activation of protein kinase C potentiates postsynaptic acetylcholine response at developing neuromuscular synapses.

    PubMed Central

    Fu, W. M.; Lin, J. L.

    1993-01-01

    1. Phorbol 12-myristate 13-acetate (TPA, 1 microM) and phorbol 12,13-dibutyrate (PDBu, 2 microM), activators of protein kinase C (PKC), increased the mean amplitude and decay time of the spontaneous synaptic currents of Xenopus nerve-muscle coculture, whereas, 4 alpha-phorbol (2 microM) which is an inactive phorbol analogue had no effect. 2. Staurosporine (0.5 microM) and H-7 (10 microM), inhibitors of PKC, inhibited the potentiation effects of TPA on the spontaneous synaptic currents. 3. Effects of TPA on the postsynaptic acetylcholine (ACh) sensitivity were examined by iontophoresis of ACh to the surface of embryonic muscle cells of 1-day-old Xenopus cultures. TPA increased both the amplitude and decay time of ACh-induced whole-cell currents in isolated myocytes. 4. TPA concentration-dependently increased the mean open time of low-conductance ACh channels but did not affect those of high-conductance ACh channels. PDBu but not 4 alpha-phorbol exhibited similar effects to TPA. Staurosporine and H-7 inhibited the increasing effects of TPA. 5. These results suggest that activation of PKC might be involved in synaptogenesis at developing neuromuscular synapses by the postsynaptic potentiation of ACh sensitivity. PMID:7694757

  12. Application of several advanced oxidation processes for the destruction of terephthalic acid (TPA).

    PubMed

    Thiruvenkatachari, Ramesh; Kwon, Tae Ouk; Jun, Jung Chul; Balaji, Subramanian; Matheswaran, Manickam; Moon, Il Shik

    2007-04-01

    Terephthalic acid (TPA) is widely applied as a raw material in making polyester fiber, polyethylene terephthalate (PET) bottles, polyester films, etc. TPA is toxic and is known to act as endocrine disruptor. TPA wastewater is traditionally treated by biological process and this study aims to evaluate the effectiveness of several advanced oxidation processes on TPA removal. The oxidation processes studied were: UV-TiO(2), UV-H(2)O(2), UV-H(2)O(2)-Fe, O(3), O(3)/Fe, O(3)/TiO(2), UV-O(3)-H(2)O(2)-Fe and UV-O(3)-H(2)O(2)-Fe-TiO(2). The results indicate that the time required for the complete destruction of 50 ppm of TPA can be minimized from 10h using UV-TiO(2) system, to less than 10 min by UV-H(2)O(2)-Fe-O(3) system. Some of the likely organic intermediates identified during TPA destruction include, benzoquinone, benzene, maleic acid and oxalic acid. Possible destruction pathway of TPA has been proposed. TPA degradation by various systems was also analyzed based on the reaction kinetics and operating costs. PMID:17023113

  13. 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…

  14. "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…

  15. Photon upconversion: from two-photon absorption (TPA) to triplet-triplet annihilation (TTA).

    PubMed

    Ye, Changqing; Zhou, Liwei; Wang, Xiaomei; Liang, Zuoqin

    2016-04-20

    Organic upconversion is a unique process in which low-energy light (usually NIR light) is converted to high-energy light through either the two-photon absorption (TPA) mechanism or the triplet-triplet annihilation (TTA) mechanism. Both TPA upconversion (TPA-UC) and TTA upconversion (TTA-UC) have been actively investigated in recent years due to their intriguing applications in optics, biophotonics, and solar energy utilization. Although they show some similarity (i.e., belonging to the nonlinear two-quantum process and needing focused excitation light), TPA-UC and TTA-UC are very different, such as in mechanism, characteristics involved, molecular design and potential applications. Here, we collectively reviewed these two kinds of upconversion processes and compared their respective characteristics and merits. We also present recent advances made in the areas of TPA- and TTA-UC, the remaining challenges and opportunities, with particular emphasis on molecular engineering of these two active upconversion materials. PMID:26843136

  16. 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

  17. 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.

  18. Production of human tissue plasminogen activator (tPA) in Cucumis sativus.

    PubMed

    Asgari, Mishaneh; Javaran, Mokhtar Jalali; Moieni, Ahmad; Masoumiasl, Asad; Abdolinasab, Maryam

    2014-01-01

    Tissue plasminogen activator (tPA) as a serine protease with 72 kD molecular mass and 527 amino acids plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The collective production of this drug in plants such as cucumber, one of the most important vegetables in the world, could reduce its production costs. In this study, after scrutiny of the appropriate regeneration of cucumber plant (Isfahan variety) on MS medium with naphthalene acetic acid hormone (NAA; 0/1 mg L⁻¹) and benzyl amino purine hormone (BAP; 3 mg L⁻¹) hormones, the cloned human tPA gene under the CaMV 35S promoter and NOS terminator into pBI121 plasmid was transferred into cotyledon explants by Agrobacterium tumefaciens strain LBA4404. Subsequent to the regeneration of inoculated explants on the selective medium, the persistence of tPA gene in recombinant plants was confirmed by polymerase chain reaction (PCR) with specific primers. To evaluate the tPA gene expression in transgenic plants, RNA was extracted and the tPA gene transcription was confirmed by reverse-transcription (RT) PCR. Followed the extraction of protein from the leaves of transgenic plants, the presence of tPA protein was confirmed by dot blot and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis in order to survey the production of recombinant tPA protein. The enzyme-linked immunosorbent assay (ELISA) test was used for recombinant tPA protein level in transgenic cucumber plants. It was counted between 0.8 and 1%, and based on this, it was concluded that the presence of three expressions of regulatory factors (CaMV 35S, Kozak, NOS) and KDEL signal in the construct caused the increase of the tPA gene expression in cucumber plants. PMID:24152103

  19. Effect of Fagonia arabica on thrombin induced release of t-PA and complex of PAI-1 tPA in cultured HUVE cells.

    PubMed

    Aloni, Prutha D; Nayak, Amit R; Chaurasia, Sweta R; Deopujari, Jayant Y; Chourasia, Chhaya; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

    2016-07-01

    Fagonia arabica (FA) possesses a thrombolytic property which has been earlier reported in our laboratory. Current study was undertaken to investigate the effect of aqueous extract of FA on thrombin-induced tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) release from cultured human umbilical vein endothelial cell line (HUVE) for studying its clot lytic activity. For this, establishment of cell line model has been done by isolating the cells from human umbilical cord. Cell toxicity was evaluated using XTT assay. Estimation of t-PA and PAI-1 t-PA complex were done using ELISA technique. Thrombin treatment induces the t-PA and PAI-1 release from HUVE cell line, and FA treatment was found to antagonize the thrombin induced t-PA and PAI-1 release. Our preliminary results suggest that FA may be used as an alternative to thrombolytic drug. However, study demands further experiments using animal model of thrombosis to establish the role of FA as a novel thrombolytic drug. PMID:27419084

  20. 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.

  1. Purification of a peptide from seahorse, that inhibits TPA-induced MMP, iNOS and COX-2 expression through MAPK and NF-kappaB activation, and induces human osteoblastic and chondrocytic differentiation.

    PubMed

    Ryu, BoMi; Qian, Zhong-Ji; Kim, Se-Kwon

    2010-03-30

    Ongoing efforts to search for naturally occurring, bioactive substances for the amelioration of arthritis have led to the discovery of natural products with substantial bioactive properties. The seahorse (Hippocampus kuda Bleeler), a telelost fish, is one source of known beneficial products, yet has not been utilized for arthritis research. In the present work, we have purified and characterized a bioactive peptide from seahorse hydrolysis. Among the hydrolysates tested, pronase E-derived hydrolysate exhibited the highest alkaline phosphatase (ALP) activity, a phenotype marker of osteoblast and chondrocyte differentiation. After its separation from the hydrolysate by several purification steps, the peptide responsible for the ALP activity was isolated and its sequence was identified as LEDPFDKDDWDNWK (1821Da). We have shown that the isolated peptide induces differentiation of osteoblastic MG-63 and chondrocytic SW-1353 cells by measuring ALP activity, mineralization and collagen synthesis. Our results indicate that the peptide acts during early to late stages of differentiation in MG-63 and SW-1353 cells. We also assessed the concentration dependence of the peptide's inhibition of MMP (-1, -3 and -13), iNOS and COX-2 expression after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a common form of phorbol ester. The peptide also inhibited NO production in MG-63 and SW-1353 cells. To elucidate the mechanisms by which the peptide acted, we examined its effects on TPA-induced MAPKs/NF-kappaB activation and determined that the peptide treatment significantly reduced p38 kinase/NF-kappaB in MG-63 cells and MAPKs/NF-kappaB in SW-1353 cells. PMID:20004183

  2. Pulsed High–Intensity-focused US and Tissue Plasminogen Activator (TPA) Versus TPA Alone for Thrombolysis of Occluded Bypass Graft in Swine

    PubMed Central

    Abi-Jaoudeh, Nadine; Pritchard, William F.; Amalou, Hayet; Linguraru, Marius; Chiesa, Oscar A.; Adams, Joshua D.; Gacchina, Carmen; Wesley, Robert; Maruvada, Subha; McDowell, Briana; Frenkel, Victor; Karanian, John W.; Wood, Bradford J.

    2012-01-01

    Purpose Prosthetic arteriovenous or arterial-arterial bypass grafts can thrombose and be resistant to revascularization. A thrombosed bypass graft model was created to evaluate the potential therapeutic enhancement and safety profile of pulsed high-intensity-focused ultrasound (pHIFU) on pharmaceutical thrombolysis. Materials and Methods In swine, a right carotid-carotid expanded polytetrafluoroethylene bypass graft was surgically constructed, containing a 40% stenosis at its distal end to induce graft thrombosis. The revascularization procedure was performed 7 days after surgery. After model development and dose response experiments (n = 11), two cohorts were studied: pHIFU with tissue plasminogen activator (TPA; n = 4) and sham pHIFU with TPA (n = 3). The experiments were identical in both groups except no energy was delivered in the sham pHIFU group. Serial angiograms were obtained in all cases. The area of graft opacified by contrast medium on angiograms was quantified with digital image processing software. A blinded reviewer calculated the change in the graft area opacified by contrast medium and expressed it as a percentage, representing percentage of thrombolysis. Results Combining pHIFU with 0.5 mg of TPA resulted in a 52% ± 4% increase in thrombolysis on angiograms obtained at 30 minutes, compared with a 9% ± 14% increase with sham pHIFU and 0.5 mg TPA (P = .003). Histopathologic examination demonstrated no differences between the groups. Conclusions Thrombolysis of occluded bypass grafts was significantly increased when combining pHIFU and TPA versus sham pHIFU and TPA. These results suggest that application of pHIFU may augment thrombolysis with a reduced time and dose. PMID:22609287

  3. 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.

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

    DOE PAGESBeta

    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.; et al

    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

  5. tPA Deficiency in Mice Leads to Rearrangement in the Cerebrovascular Tree and Cerebroventricular Malformations

    PubMed Central

    Stefanitsch, Christina; Lawrence, Anna-Lisa E.; Olverling, Anna; Nilsson, Ingrid; Fredriksson, Linda

    2015-01-01

    The serine protease tissue-type plasminogen activator (tPA) is used as a thrombolytic agent in the management of ischemic stroke, but concerns for hemorrhagic conversion greatly limits the number of patients that receive this treatment. It has been suggested that the bleeding complications associated with thrombolytic tPA may be due to unanticipated roles of tPA in the brain. Recent work has suggested tPA regulation of neurovascular barrier integrity, mediated via platelet derived growth factor (PDGF)-C/PDGF receptor-α (PDGFRα) signaling, as a possible molecular mechanism affecting the outcome of stroke. To better understand the role of tPA in neurovascular regulation we conducted a detailed analysis of the cerebrovasculature in brains from adult tPA deficient (tPA−/−) mice. Our analysis demonstrates that life-long deficiency of tPA is associated with rearrangements in the cerebrovascular tree, including a reduction in the number of vascular smooth-muscle cell covered, large diameter, vessels and a decrease in vessel-associated PDGFRα expression as compared to wild-type (WT) littermate controls. In addition, we found that ablation of tPA results in an increased number of ERG-positive endothelial cells and increased junctional localization of the tight junction protein ZO1. This is intriguing since ERG is an endothelial transcription factor implicated in regulation of vascular integrity. Based on these results, we propose that the protection of barrier properties seen utilizing these tPA−/− mice might be due, at least in part, to these cerebrovascular rearrangements. In addition, we found that tPA−/− mice displayed mild cerebral ventricular malformations, a feature previously associated with ablation of PDGF-C, thereby providing an in vivo link between tPA and PDGF signaling in central nervous system (CNS) development. Taken together, the data presented here will advance our understanding of the role of tPA within the CNS and in regulation of

  6. Evidence for impairment of behavioural inhibition in performance of operant tasks in tPA-/- mice.

    PubMed

    Ripley, T L; Horwood, J M; Stephens, D N

    2001-11-01

    We have previously shown that mice that lack the serine protease, tissue plasminogen activator (tPA), show over-responding on the active lever during time-out periods in an I.V. cocaine self-administration task. To investigate this effect further, tPA knockout mice (tPA-/-) were tested in a number of operant paradigms for a liquid food reinforcer. tPA-/- and wild-type (WT) control mice acquired a fixed ratio (FR) and a fixed interval (FI) task equally. However, extinction from the FR schedule resulted in a significant decrease in responses on the active and inactive levers in the WT mice whilst responding on the inactive lever remained high in the tPA-/- animals. In a differential reinforcement of low rate (DRL) task, tPA-/- mice acquired the task at a slower rate than WT animals. This was characterised by high levels of responding on the active lever during the first 15 sessions in the tPA-/- mice. Burst responding on the active lever (lever press rate with an inter-response time of less than 3 s) was especially high in these animals. This behaviour pattern resulted in the animals obtaining less reinforcers than the WT controls. Acute cocaine dose-dependently shifted the pattern of behaviour on the active lever towards shorter inter-response times. However, there was no difference between the tPA-/- and WT mice in their sensitivity to cocaine on this task. Repeated administration of a low dose of cocaine did not alter performance on this task in either set of animals. When the DRL task was modified to allow the tPA-/- and WT mice an equal number of reinforced trials per session there was no difference in the ability of the animals to perform the task. This would suggest that the tPA-/- mice have a tendency to over-respond but that this can be overcome when the task is modified to allow equal opportunity to learn. PMID:11682113

  7. 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.

  8. 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.

  9. 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

  10. 1,25-Dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca(2+)-dependent and Ca(2+)-independent isoforms of protein kinase C in rat colonocytes.

    PubMed Central

    Bissonnette, M; Wali, R K; Hartmann, S C; Niedziela, S M; Roy, H K; Tien, X Y; Sitrin, M D; Brasitus, T A

    1995-01-01

    Considerable evidence that alterations in protein kinase C (PKC) are intimately involved in important physiologic and pathologic processes in many cells, including colonic epithelial cells, has accumulated. In this regard, phorbol esters, a class of potent PKC activators, have been found to induce a number of cellular events in normal or transformed colonocytes. In addition, our laboratory has demonstrated that the major active metabolite of vitamin D3, 1,25(OH)2D3, also rapidly (seconds-minutes) activated PKC and increased intracellular calcium in isolated rat colonocytes. These acute responses, however, were lost in vitamin D deficiency and partially restored with the in vivo repletion of 1,25(OH)2D3. The Ca(2+)-independent or novel isoforms of PKC expressed in the rat colon and the isoform-specific responses of PKC to acute treatment with phorbol esters or 1,25(OH)2D3 have not been previously characterized. Moreover, the effects of vitamin D status on PKC isoform expression, distribution, and response to agonists are also unknown. In the present experiments, in addition to PKC-alpha, rat colonocytes were found to express the novel isoforms delta, epsilon, and zeta by Western blotting using isoform-specific PKC antibodies. The tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, caused time- and concentration-dependent translocations of all these isoforms except PKC-zeta. In vitamin D deficiency, there were no alterations in colonic PKC isoform expression but significant changes in the subcellular distribution of PKC-alpha, -delta, and -zeta. Acute treatment of colonocytes from D-sufficient, but not D-deficient, rats with 1,25(OH)2D3 caused a rapid transient redistribution of only PKC-alpha from the soluble to the particulate fraction. The alterations in PKC isoform distribution and PKC-alpha responsiveness to 1,25(OH)2D3 in vitamin D deficiency were partially, but significantly, restored with 5-7 d in vivo repletion of this secosteroid. Both 12

  11. 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…

  12. 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…

  13. Novel Ru (II) complex with TPA derivatives as a donor for dye-sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Kwon, Dong Yuel; Chang, Dong Min; Kim, Young Sik

    2015-01-01

    Novel heteroleptic ruthenium(II) complex [Ru(CF3-ppyd-TPA)(tctpy)]+ (ppyd = 2-phenyl-6-(pyridin-2-yl)pyridine, TPA = triphenylamine, and tctpy = 4,4',4″-tricarboxy-2,2':6',2″-terpyridine) was designed and investigated to increase its molar absorptivity compared to [Ru(ppd)(tctpy)]+ (ppd = 2-(3-(pyridin-2-yl)phenyl)pyridine). Density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations were performed to gain insight into the factors responsible for the photovoltaic properties of a dye sensitizer. [Ru(CF3-ppyd-TPA)(tctpy)]+ showed a broad absorption spectrum and enhanced the molar extinction coefficient. Significant improvements to light absorption were exhibited by enhancing the metal-to-ligand charge transfer (MLCT) characteristics through the addition of the electron-withdrawing group-CF3 para to the organometallic bond and by increasing the transition dipole moment through the addition of TPA as an electron-donating group compared to the [Ru(ppd)(tctpy)]+. This study suggests that a ruthenium-based dye sensitizer would show improved photovoltaic performance in conversion efficiency for DSSCs by adding electron-donating and electron-withdrawing groups.

  14. 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…

  15. 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…

  16. 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…

  17. Current perspectives on the use of intravenous recombinant tissue plasminogen activator (tPA) for treatment of acute ischemic stroke

    PubMed Central

    Chapman, Sherita N; Mehndiratta, Prachi; Johansen, Michelle C; McMurry, Timothy L; Johnston, Karen C; Southerland, Andrew M

    2014-01-01

    In 1995, the NINDS (National Institute of Neurological Disorders and Stroke) tPA (tissue plasminogen activator) Stroke Study Group published the results of a large multicenter clinical trial demonstrating efficacy of intravenous tPA by revealing a 30% relative risk reduction (absolute risk reduction 11%–15%) compared with placebo at 90 days in the likelihood of having minimal or no disability. Since approval in 1996, tPA remains the only drug treatment for acute ischemic stroke approved by the US Food and Drug Administration. Over the years, an abundance of research and clinical data has supported the safe and efficacious use of intravenous tPA in all eligible patients. Despite such supporting data, it remains substantially underutilized. Challenges to the utilization of tPA include narrow eligibility and treatment windows, risk of symptomatic intracerebral hemorrhage, perceived lack of efficacy in certain high-risk subgroups, and a limited pool of neurological and stroke expertise in the community. With recent US census data suggesting annual stroke incidence will more than double by 2050, better education and consensus among both the medical and lay public are necessary to optimize the use of tPA for all eligible stroke patients. Ongoing and future research should continue to improve upon the efficacy of tPA through more rapid stroke diagnosis and treatment, refinement of advanced neuroimaging and stroke biomarkers, and successful demonstration of alternative means of reperfusion. PMID:24591838

  18. Tissue plasminogen activator (tPA) and matrix metalloproteinases in the pathogenesis of stroke: therapeutic strategies.

    PubMed

    Adibhatla, Rao Muralikrishna; Hatcher, James F

    2008-06-01

    Today there exists only one FDA-approved treatment for ischemic stroke; i.e., the serine protease tissue-type plasminogen activator (tPA). In the aftermath of the failed stroke clinical trials with the nitrone spin trap/radical scavenger, NXY-059, a number of articles raised the question: are we doing the right thing? Is the animal research truly translational in identifying new agents for stroke treatment? This review summarizes the current state of affairs with plasminogen activators in thrombolytic therapy. In addition to therapeutic value, potential side effects of tPA also exist that aggravate stroke injury and offset the benefits provided by reperfusion of the occluded artery. Thus, combinational options (ultrasound alone or with microspheres/nanobubbles, mechanical dissociation of clot, activated protein C (APC), plasminogen activator inhibitor-1 (PAI-1), neuroserpin and CDP-choline) that could offset tPA toxic side effects and improve efficacy are also discussed here. Desmoteplase, a plasminogen activator derived from the saliva of Desmodus rotundus vampire bat, antagonizes vascular tPA-induced neurotoxicity by competitively binding to low-density lipoprotein related-receptors (LPR) at the blood-brain barrier (BBB) interface, minimizing the tPA uptake into brain parenchyma. tPA can also activate matrix metalloproteinases (MMPs), a family of endopeptidases comprised of 24 mammalian enzymes that primarily catalyze the turnover and degradation of the extracellular matrix (ECM). MMPs have been implicated in BBB breakdown and neuronal injury in the early times after stroke, but also contribute to vascular remodeling, angiogenesis, neurogenesis and axonal regeneration during the later repair phase after stroke. tPA, directly or by activation of MMP-9, could have beneficial effects on recovery after stroke by promoting neurovascular repair through vascular endothelial growth factor (VEGF). However, any treatment regimen directed at MMPs must consider their

  19. Inhibition of PAI-1 Antiproteolytic Activity Against tPA by RNA Aptamers

    PubMed Central

    Damare, Jared; Brandal, Stephanie

    2014-01-01

    Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) inhibits the plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease. Pharmacologically suppressing PAI-1 might prevent, or successfully treat PAI-1 related vascular diseases. This can potentially be accomplished by using small RNA molecules (aptamers). This study's goal is to develop RNA aptamers to a region of PAI-1 that will prevent the ability of PAI-1 to interact with the plasminogen activators. The aptamers were generated through a systematic evolution of ligands via exponential enrichment approach that ensures the creation of RNA molecules that bind to our target protein, PAI-1. In vitro assays were used to determine the effect of these aptamers on PAI-1's inhibitory activity. Three aptamers that bind to PAI-1 with affinities in the nanomolar range were isolated. The aptamer clones R10-4 and R10-2 inhibited PAI-1's antiproteolytic activity against tPA and disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations correlated positively with an increase in cleaved PAI-1. To the best of our knowledge, this is the first report of RNA molecules that inhibit the antiproteolytic activity of PAI-1. PMID:24922319

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

    NASA Astrophysics Data System (ADS)

    Lazicki, Amy; Rygg, Ryan; Coppari, Federica; Smith, Ray; Fratanduono, Dayne; Braun, Dave; Kraus, Richard; Swift, Damian; Collins, Gilbert; Eggert, Jon

    2015-06-01

    We present x-ray diffraction studies of solid crystal structure at the highest stress state where such measurements have ever been performed. Using laser-driven ramp compression methods coupled with angle-resolved powder x-ray diffraction at the Omega laser facility, we explore the phase diagram of tin below the melting curve between 0.1 and 1.2 terapascals (TPa). We demonstrate that, at dynamic-compression rates on the order of 107 s-1, tin transforms from the ambient tetragonal beta-Sn phase to the stable high pressure body-centered cubic (bcc) phase with densities consistent with static-compression measurements. Above 0.16 TPa our experiments identify a new feature in the phase diagram: a crystal structure clearly inconsistent with the hexagonal-close-packed (hcp) phase identified at these conditions by ambient-temperature static-compression measurements and by zero-kelvin density functional theory structure predictions. Our results suggest that the bcc phase is stabilized relative to hcp at high temperature, analogous to the heavier group IV metal Pb and numerous other elemental metals, and retains this phase during ramp compression to 1.2 TPa. This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

  1. tPA-S481A prevents neurotoxicity of endogenous tPA in traumatic brain injury.

    PubMed

    Armstead, William M; Riley, John; Yarovoi, Serge; Cines, Douglas B; Smith, Douglas H; Higazi, Abd Al-Roof

    2012-06-10

    Traumatic brain injury (TBI) is associated with loss of autoregulation due to impaired responsiveness to cerebrovascular dilator stimuli, which leads to cerebral hypoperfusion and neuronal impairment or death. Upregulation of tissue plasminogen activator (tPA) post-TBI exacerbates loss of cerebral autoregulation and NMDA-receptor-mediated impairment of cerebral hemodynamics, and enhances excitotoxic neuronal death. However, the relationship between NMDA-receptor activation, loss of autoregulation, and neurological dysfunction is unclear. Here, we evaluated the potential therapeutic efficacy of a catalytically inactive tPA variant, tPA S481A, that acts by competing with wild-type tPA for binding, cleavage, and activation of NMDA receptors. Lateral fluid percussion brain injury was produced in anesthetized piglets. Pial artery reactivity was measured via a closed cranial window, and cerebrospinal fluid (CSF) extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) was quantified by enzyme-linked immunosorbent assay (ELISA). tPA-S481A prevented impairment of cerebral autoregulation and reduced histopathologic changes after TBI by inhibiting upregulation of the ERK isoform of MAPK. Treatment with this tPA variant provides a novel approach for limiting neuronal toxicity caused by untoward NMDA-receptor activation mediated by increased tPA and glutamate following TBI. PMID:22435890

  2. 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. PMID:22940786

  3. MRI evaluation of BBB disruption after adjuvant AcSDKP treatment of stroke with tPA in rat.

    PubMed

    Ding, G; Zhang, Z; Chopp, M; Li, L; Zhang, L; Li, Q; Wei, M; Jiang, Q

    2014-06-20

    The primary limitation of thrombolytic treatment of ischemic stroke with tissue plasminogen activator (tPA) is the hemorrhagic risk. We tested AcSDKP (N-acetyl-seryl-aspartyl-lysyl-proline), as an auxiliary therapeutic agent, to reduce blood-brain barrier (BBB) disruption in a combination tPA thrombolytic treatment of stroke. Wistar rats subjected to embolic stroke were randomly assigned to either the tPA monotherapy group (n=9) or combination of tPA and AcSDKP treatment group (n=9) initiated at 4 h after ischemia. Magnetic resonance imaging (MRI) measurements were performed before and after the treatments. Immunohistochemical staining and measurements were performed to confirm MRI findings. Longitudinal MRI permeability measurements with gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) demonstrated that combination treatment of acute embolic stroke with AcSDKP and tPA significantly reduced BBB leakage, compared to tPA monotherapy, at 3 and 6 days (18.3±9.8 mm3 vs. 65.0±21.0 mm3, p<0.001) after the onset of stroke, although BBB leakage was comparable between the two groups prior to the treatments (6.8±4.4 mm3 vs. 4.3±3.3 mm3, p>0.18). The substantial reduction of BBB leakage observed in the combination treatment group was closely associated with reduced ischemic lesions measured by T2 maps (113.6±24.9 mm3 vs. 188.1±60.8 mm3, p<0.04 at 6 days). Histopathological analysis of the same population of rats showed that the combination treatment significantly reduced parenchymal fibrin deposition (0.063±0.059 mm2 vs. 0.172±0.103 mm2, p<0.03) and infarct volume (146.7±35.9 mm3 vs. 199.3±60.4 mm3, p<0.05) compared to the tPA monotherapy at 6days after stroke. MRI provides biological insight into the therapeutic benefit of combination treatment of stroke with tPA and AcSDKP 4h after onset, and demonstrates significantly improved cerebrovascular integrity with neuroprotective effects compared with tPA monotherapy. PMID:24769225

  4. Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR

    PubMed Central

    Bertrand, T; Lesept, F; Chevilley, A; Lenoir, S; Aimable, M; Briens, A; Hommet, Y; Bardou, I; Parcq, J; Vivien, D

    2015-01-01

    Tissue-type plasminogen activator (tPA) is a pleiotropic serine protease of the central nervous system (CNS) with reported neurotrophic and neurotoxic functions. Produced and released under its single chain form (sc), the sc-tPA can be cleaved by plasmin or kallikrein in a two chain form, tc-tPA. Although both sc-tPA and tc-tPA display a similar fibrinolytic activity, we postulated here that these two conformations of tPA (sc-tPA and tc-tPA) could differentially control the effects of tPA on neuronal survival. Using primary cultures of mouse cortical neurons, our present study reveals that sc-tPA is the only one capable to promote N-methyl-D-aspartate receptor (NMDAR)-induced calcium influx and subsequent excitotoxicity. In contrast, both sc-tPA and tc-tPA are capable to activate epidermal growth factor receptors (EGFRs), a mechanism mediating the antiapoptotic effects of tPA. Interestingly, we revealed a tPA dependent crosstalk between EGFR and NMDAR in which a tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and to subsequent neurotrophic effects. PMID:26469972

  5. 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.

  6. tPA promotes ADAMTS-4-induced CSPG degradation, thereby enhancing neuroplasticity following spinal cord injury.

    PubMed

    Lemarchant, Sighild; Pruvost, Mathilde; Hébert, Marie; Gauberti, Maxime; Hommet, Yannick; Briens, Aurélien; Maubert, Eric; Gueye, Yatma; Féron, François; Petite, Didier; Mersel, Marcel; do Rego, Jean-Claude; Vaudry, Hubert; Koistinaho, Jari; Ali, Carine; Agin, Véronique; Emery, Evelyne; Vivien, Denis

    2014-06-01

    Although tissue plasminogen activator (tPA) is known to promote neuronal remodeling in the CNS, no mechanism of how this plastic function takes place has been reported so far. We provide here in vitro and in vivo demonstrations that this serine protease neutralizes inhibitory chondroitin sulfate proteoglycans (CSPGs) by promoting their degradation via the direct activation of endogenous type 4 disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4). Accordingly, in a model of compression-induced spinal cord injury (SCI) in rats, we found that administration of either tPA or its downstream effector ADAMTS-4 restores the tPA-dependent activity lost after the SCI and thereby, reduces content of CSPGs in the spinal cord, a cascade of events leading to an improved axonal regeneration/sprouting and eventually long term functional recovery. This is the first study to reveal a tPA-ADAMTS-4 axis and its function in the CNS. It also raises the prospect of exploiting such cooperation as a therapeutic tool for enhancing recovery after acute CNS injuries. PMID:24576594

  7. Anti-tumour promoting activity and antioxidant properties of girinimbine isolated from the stem bark of Murraya koenigii S.

    PubMed

    Kok, Yih Yih; Mooi, Lim Yang; Ahmad, Kartini; Sukari, Mohd Aspollah; Mat, Nashriyah; Rahmani, Mawardi; Ali, Abdul Manaf

    2012-01-01

    Girinimbine, a carbazole alkaloid isolated from the stem bark of Murraya koenigii was tested for the in vitro anti-tumour promoting and antioxidant activities. Anti-tumour promoting activity was determined by assaying the capability of this compound to inhibit the expression of early antigen of Epstein-Barr virus (EA-EBV) in Raji cells that was induced by the tumour promoter, phorbol 12-myristate 13-acetate. The concentration of this compound that gave an inhibition rate at fifty percent was 6.0 µg/mL and was not cytotoxic to the cells. Immunoblotting analysis of the expression of EA-EBV showed that girinimbine was able to suppress restricted early antigen (EA-R). However, diffused early antigen (EA-D) was partially suppressed when used at 32.0 µg/mL. Girinimbine exhibited a very strong antioxidant activity as compared to a-tocopherol and was able to inhibit superoxide generation in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiated premyelocytic HL-60 cells more than 95%, when treated with the compound at 5.3 and 26.3 µg/mL, respectively. However girinimbine failed to scavenge the stable diphenyl picryl hydrazyl (DPPH)-free radical. PMID:22522395

  8. Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA

    PubMed Central

    Larsson, Pia; Magnusson, Mia; Karlsson, Lena; Bergh, Niklas; Jern, Sverker

    2016-01-01

    Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5. PMID:26966581

  9. Crystal structure searching by free energy surface trekking: application to carbon at 1 TPa

    NASA Astrophysics Data System (ADS)

    Ishikawa, T.; Suzuki, N.; Shimizu, K.

    2014-05-01

    We have developed an ab-initio crystal structure searching method, free energy surface trekking (FEST). This method consists of an ascent-run and a descent-run. First, the system is forced to climb up a free energy surface following by the inversion of the restoring forces acting on the simulation cell (ascent-run). Then, the system climbs down the surface toward neighboring local minima according to the release from the constraint of the inversion immediately after the system crosses the ridges of the surface (descent-run). We have applied the FEST simulations to carbon at terapascal pressures and obtained a BC8-like structure with a tetragonal I41 in addition to the earlier-predicted BC8, R8, and simple cubic structures. This structure is mechanically stable in the pressure range of at least 0.5-3.5 TPa, and has a potential to survive as a metastable structure in carbon at terapascal pressures.

  10. 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.

  11. t-PA, but not desmoteplase, induces plasmin-dependent opening of a blood-brain barrier model under normoxic and ischaemic conditions.

    PubMed

    Freeman, Roxann; Niego, Be'eri; Croucher, David R; Pedersen, Lars O; Medcalf, Robert L

    2014-05-27

    Tissue-type plasminogen activator (t-PA) is the only thrombolytic treatment available for patients with acute ischaemic stroke. However, t-PA can increase permeability of the blood-brain barrier (BBB). Desmoteplase is a plasminogen activator derived from the common vampire bat, currently under clinical development for ischaemic stroke. We compared how t-PA and desmoteplase influenced BBB permeability using a human in vitro model where primary brain endothelial cells (BEC) and astrocytes are co-cultured on the opposite sides of a porous membrane. Permeability changes were evaluated 6 or 24h post-stimulation by passage of fluorescent albumin across the membrane. Under normoxic conditions, t-PA, but not desmoteplase, increased BBB permeability. Surprisingly, the ability of t-PA to affect the barrier was lost under conditions of oxygen-glucose deprivation (OGD). Addition of plasminogen re-sensitised the BBB to the action of t-PA under both normoxia and OGD, but did not affect the inert behaviour of desmoteplase, even when digested fibrinogen was added to ensure optimal plasmin generation. These observations coincided with plasmin-dependent changes in astrocyte and BEC morphology and disruption of tight junction proteins in BECs, specifically initiated by t-PA but not by desmoteplase. Finally, inhibition of plasmin post-stimulation with t-PA and plasminogen, especially within 2h, protected the BBB against t-PA-mediated barrier opening. Hence t-PA, but not desmoteplase, increases BBB permeability under both normoxic and OGD conditions in a reversible, plasmin-dependent process. The inability of desmoteplase to increase permeability despite its capacity to generate plasmin provides further support for its use as thrombolytic in patients with ischaemic stroke. PMID:24675027

  12. 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. PMID:22097098

  13. Crystal Structure Searching by Free Energy Surface Trekking: Application to Carbon above 1 TPa

    NASA Astrophysics Data System (ADS)

    Ishikawa, Takahiro; Suzuki, Naoshi; Shimizu, Katsuya

    2013-06-01

    Crystal structure determination of materials under extreme conditions has been one of grand challenges in high-pressure materials science. In computer simulations, the crystal structure searching is carried out by exploring Gibbs free energy surface (GFES) at given pressures and temperatures. Here, we propose a new crystal structure searching technique named as free energy surface trekking (FEST). FEST is based on a very simple idea and consists of an ascent-run and a descent-run. In the ascent-run, the system is forced to ascend GFES from a starting local minimum by following the inversion of the driving force acting on the simulation cell. Then, the system descends it toward a neighboring local minimum by flipping the inverted force at the ridge of GFES. The details of GFES around the starting local minimum are more correctly obtained by more investigating different trekking routes. We have applied FEST to carbon at 1.2 TPa and at 300 K, and successfully obtained the transition from the cubic diamond phase to the previously predicted BC8 phase. In this transition, 3 cell-angles concurrently increase from 90° to 101° in the ascent-run and become 109° through the descent-run, in which the activation energy is approximately 0.17 Ry/atom.

  14. 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

    2016-07-01

    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. PMID:27305044

  15. Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis

    PubMed Central

    Goldstein, Jill; Roth, Eve; Roberts, Natalie; Zwick, Rachel; Lin, Samantha; Fletcher, Sean; Tadeu, Ana; Wu, Christine; Beck, Amanda; Zeiss, Caroline; Suárez-Fariñas, Mayte; Horsley, Valerie

    2015-01-01

    Immunosuppressive therapies using calcineurin inhibitors, such as cyclosporine A, are associated with a higher incidence of squamous cell carcinoma formation in mice and humans. Calcineurin is believed to suppress tumorigenesis in part through Nfatc1, a transcription factor expressed primarily in hair follicle bulge stem cells in mice. However, mice overexpressing a constitutively active Nfatc1 isoform in the skin epithelium developed increased spontaneous skin squamous cell carcinomas. Because follicular stem cells can contribute to skin tumorigenesis, whether the endogenous expression of Nfatc1 inhibits or enhances skin tumorigenesis is unclear. Here we show that loss of the endogenous expression of Nfatc1 suppresses the rate of DMBA/TPA-induced skin tumorigenesis. Inducible deletion of Nfatc1 in follicular stem cells before tumor initiation significantly reduces the rate of tumorigenesis and the contribution of follicular stem cells to skin tumors. We find that skin tumors from mice lacking Nfatc1 display reduced Hras codon 61 mutations. Furthermore, Nfatc1 enhances the expression of genes involved in DMBA metabolism and increases DMBA-induced DNA damage in keratinocytes. Together these data implicate Nfatc1 in the regulation of skin stem cell–initiated tumorigenesis via the regulation of DMBA metabolism. PMID:26310443

  16. Cloning and characterization of the goadsporin biosynthetic gene cluster from Streptomyces sp. TP-A0584.

    PubMed

    Onaka, Hiroyasu; Nakaho, Mizuho; Hayashi, Keiko; Igarashi, Yasuhiro; Furumai, Tamotsu

    2005-12-01

    The biosynthetic gene cluster of goadsporin, a polypeptide antibiotic containing thiazole and oxazole rings, was cloned from Streptomyces sp. TP-A0584. The cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity and transcriptional regulation. Although the gene organization is similar to typical bacteriocin biosynthetic gene clusters, each goadsporin biosynthetic gene shows low homology to these genes. Goadsporin biosynthesis is initiated by the translation of godA, and the subsequent cyclization, dehydration and acetylation are probably catalysed by godD, godE, godF, godG and godH gene products. godI shows high similarity to the 54 kDa subunit of the signal recognition particle and plays an important role in goadsporin immunity. Furthermore, four goadsporin analogues were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is used for the heterocyclization of peptides. PMID:16339937

  17. Design, synthesis, and characterization of TPA-thiophene-based amide or imine functionalized molecule for potential optoelectronic devices

    NASA Astrophysics Data System (ADS)

    Sarswat, Prashant K.; Sathyapalan, Amarchand; Zhu, Yakun; Free, Michael L.

    2013-01-01

    New sets of molecules containing tri-phenyl-amine (TPA) core and thiophene unit with amide and imine functional groups are designed, synthesized, characterized, and compared. These are solution processable small molecules with high mobility. The newly designed molecules have better solubility due to the C=N (imine) and CONH2 (amide) moiety as compared to the established molecules with CH=CH (methine) for optoelectronic applications. They have an optimal energy band gap, which indicates their potential utility in a variety of optoelectronic applications. These molecules also show efficient intermolecular charge transfer mechanisms similar to conventional organic semiconducting molecules as evidenced by optical measurements. Density functional theory simulation results show that the localization of the frontier highest occupied molecular orbital is around the TPA core for molecules coupled with imine and amide, and is reasonably stable.

  18. Effects of Early Post-Ischemic Reperfusion and tPA on Cerebrovascular Function and Nitrosative Stress in Female Rats.

    PubMed

    Ahnstedt, Hilda; Sweet, Julie; Cruden, Patrick; Bishop, Nicole; Cipolla, Marilyn J

    2016-06-01

    Stroke is a major health issue in women. Our previous studies in male rats showed decreased myogenic tone in middle cerebral arteries (MCAs) after ischemia and reperfusion (I/R), while tone in parenchymal arterioles (PAs) was increased. This vascular response may aggravate stroke damage in males by limiting reperfusion; however, the effect in females is not known. The current study investigated the effect of I/R and tissue plasminogen activator (tPA) on myogenic tone and reactivity of MCAs and PAs in female rats. Nitrosative stress by peroxynitrite and recruitment of inflammatory neutrophils to the microvasculature were also studied. Female rats were subjected to 2-h MCA filament occlusion (n = 16) or sham surgery (n = 17) and given tPA (1 mg/kg, i.v) or vehicle followed by 30-min reperfusion. Myogenic tone and reactivity were measured in isolated and pressurized MCAs and PAs from the same animals. Cerebrovascular F-actin, 3-nitrotyrosine (3-NT, peroxynitrite marker), and intravascular neutrophils were quantified. Myogenic tone and constriction to the nitric oxide synthase inhibitor Nω-nitro-L-arginine were decreased in MCAs but unchanged in PAs after I/R with no effect of tPA. F-actin and 3-NT expression were unaffected by I/R or tPA. Our study showed that MCAs from females, similar to what has been seen in males, are dilated after I/R and have decreased myogenic tone while tone in PAs was unchanged. Increased small vessel resistance may contribute to decreased reperfusion and worse outcome after stroke. PMID:27125535

  19. Pulsed-high intensity focused ultrasound enhanced tPA mediated thrombolysis in a novel in vivo clot model, a pilot study

    PubMed Central

    Stone, Michael J.; Frenkel, Victor; Dromi, Sergio; Thomas, Peter; Lewis, Ryan P.; Li, King CP; Horne, McDonald; Wood, Bradford J.

    2007-01-01

    Introduction Thrombotic disease continues to account for significant morbidity and mortality. Ultrasound energy has been investigated as a potential primary and adjunctive treatment for thrombotic disease. We have previously shown that pulsed-high intensity focused ultrasound (HIFU) enhances thrombolysis induced by tissue plasminogen activator (tPA) in vitro, including describing the non-destructive mechanism by which tPA availability and consequent activity is increased. In this study we aimed to determined if the same effects could be achieved in vivo. Materials and Methods In this study, pulsed-HIFU exposures combined with tPA boluses was compared to treatment with tPA alone, HIFU alone and control in a novel in vivo clot model. Clots were formed in the rabbit marginal ear vein and verified using venography and infrared imaging. The efficacy of thrombolytic treatment was monitored via high resolution ultrasonography for five hours post treatment. The cross-sectional area of clots at 4 points along the vein was measured and normalized to the pre-treatment size. Results At five hours the complete recanalization of clots treated with pulsed-HIFU and tPA was significantly different from the partial recanalization seen with tPA treatment alone. tPA treatment alone showed a significant decrease in clot versus control, where HIFU was not significantly different than control. Histological analysis of the vessel walls in the treated veins showed no apparent irreversible damage to endothelial cells or extravascular tissue. Conclusions This study demonstrates that tPA mediated thrombolysis can be significantly enhanced when combined with non-invasive pulsed-HIFU exposures. PMID:17481699

  20. 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

  1. 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.

  2. Circulating t-PA antigen predicts major adverse coronary events in patients with stable coronary artery disease--a 13-year follow-up.

    PubMed

    Niessner, Alexander; Graf, Senta; Nikfardjam, Mariam; Speidl, Walter S; Huber-Beckmann, Renate; Zorn, Gerlinde; Wojta, Johann; Huber, Kurt

    2003-08-01

    Thrombus formation after rupture of an atherosclerotic plaque plays a crucial role in coronary artery disease (CAD). A decreased endogenous fibrinolytic system and prothrombotic factors are supposed to influence coronary thrombosis. It was our aim to investigate the predictive value of tissue plasminogen activator (t-PA) antigen, von Willebrand Factor, Lipoprotein (a) and anti-cardiolipin antibodies for major adverse coronary events in patients with stable CAD in a prospective cohort study of more than 10 years. We observed 141 patients with angiographically proven CAD for a median follow-up period of 13 years. t-PA antigen was the only marker predicting coronary events (logistic regression, p = 0.044) with a poor prognosis for patients in the 5th quintile with an odds ratio of 7.3 (compared to the 1st quintile). The odds ratio even increased to 10.0 for coronary events associated with the "natural course" of CAD excluding events due to restenosis. t-PA antigen had a slightly higher prognostic power (ROC curve; AUC = 0.69) than fasting glucose (AUC = 0.68) and cholesterol (AUC = 0.67). Triglycerides influenced plasma levels of t-PA antigen (regression, p < 0.001). The predictive value of t-PA antigen remained significant after adjustment for inflammation (logistic regression, p = 0.013) and extent of CAD (p = 0.045) but disappeared adjusting for insulin resistance (p = 0.12). In conclusion t-PA antigen predicted coronary events during a very long-term follow-up with a comparable prognostic power to established cardiovascular risk factors. Markers of insulin resistance influenced t-PA antigen and its predictive value. PMID:12888883

  3. 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. PMID:24646324

  4. Treatment of a Class II Division 2 Patient with Severe Skeletal Discrepancy by Using a Custom Made TPA Proclination Spring

    PubMed Central

    Paduano, Sergio; Spagnuolo, Gianrico; Biase, Giuseppe di; Cioffi, Iacopo

    2013-01-01

    This case report describes the orthodontic treatment of a boy, aged 15.3 years, with permanent dentition, mesofacial typology, affected with a severe sagittal skeletal Class II division 2 malocclusion, due to a mandibular retrusion. His chief compliant was the position of the maxillary incisors, displaced too palatally, and an impaired facial profile. Herbst and multi-bracket straightwire fixed appliances, together with a custom made modified transpalatal arch (i.e. TPA proclination spring), were used to correct the sagittal discrepancy and to improve the attractiveness of the impaired facial profile. PMID:24155800

  5. 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

    2013-10-01

    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. PMID:24116573

  6. Systemic Inflammatory Response Syndrome in tPA Treated Patients Is Associated with Worse Short-term Functional Outcome

    PubMed Central

    Boehme, Amelia K.; Kapoor, Niren; Albright, Karen C.; Lyerly, Michael J.; Rawal, Pawan V.; Shahripour, Reza Bavarsad; Alvi, Muhammad; Houston, J. Thomas; Sisson, April; Beasley, T. Mark; Alexandrov, Anne W.; Alexandrov, Andrei V.; Miller, David W.

    2013-01-01

    Background and Purpose Systemic Inflammatory Response (SIRS) is a generalized inflammatory state. The primary goal of the study was to determine if differences exist in outcomes in SIRS and non-SIRS IV tPA treated patients. Methods Consecutive patients were retrospectively reviewed for evidence of SIRS during their admission. SIRS was defined as the presence of two or more: body temperature <36° C or >38° C, HR >90, respiratory rate >20 and WBC <4,000/mm or >12,000mm or >10% bands. Patients diagnosed with infection (via positive culture) were excluded. Results Out of 241 patients, 44 had evidence of SIRS (18%). Adjusting for pre-tPA NIHSS, age, and race, SIRS remained a predictor of poor functional outcome at discharge (OR= 2.58, 95% CI, 1.16 – 5.73, p=0.0197). Conclusion In our sample of tPA treated patients, almost 1 out of 5 patients developed SIRS. Further, we found the presence of SIRS to be associated with poor short-term functional outcomes and prolonged length of stay. PMID:23704110

  7. 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

  8. A family of uranyl-aromatic dicarboxylate (pht-, ipa-, tpa-) framework hybrid materials: photoluminescence, surface photovoltage and dye adsorption.

    PubMed

    Gao, Xue; Wang, Che; Shi, Zhong-Feng; Song, Jian; Bai, Feng-Ying; Wang, Ji-Xiao; Xing, Yong-Heng

    2015-07-01

    Four uranyl complexes [(UO2)(pht)H2O]·H2O (pht = phthalic acid) (1), (UO2)2(Hipa)4(H2O)2 (Hipa = isophthalic acid) (2), (UO2)(tpa)(DMF)2 (tpa = terephthalic acid) (3) and (UO2)(box)2 (box = benzoic acid) (4) were synthesized by the reaction of UO2(CH3COO)2·2H2O as the metal source and phthalic acid, isophthalic acid, terephthalic acid or benzoic acid as the ligand. They were characterized by elemental analyses, IR, UV-Vis, XRD, single crystal X-ray diffraction analysis and thermal gravimetric analysis. The structural analysis reveals that complex 1 exhibits a one-dimensional chain structure constructed by the building unit [(UO2)2(pht)4(H2O)2] and further extends the chain into a 2D supramolecular architecture by hydrogen bonding interactions. Complex 2 is a discrete [(UO2)2(Hipa)4(H2O)2] structure, and by the hydrogen bonding interaction, forms a 3D supramolecular structure. In complexes 3 and 4, adjacent uranyl polyhedra form a 1D chain through bridging terephthalic acid and benzoic acid, respectively. In order to extend their functional properties, their photoluminescence, surface photovoltage and dye adsorption properties have been studied. PMID:26038888

  9. Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes.

    PubMed

    Jardon, Mario A; Sattha, Beheroze; Braasch, Katrin; Leung, Amy O; Côté, Hélène C F; Butler, Michael; Gorski, Sharon M; Piret, James M

    2012-05-01

    Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production. PMID:22125188

  10. 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

  11. Examination of percutaneous application in a 26-week carcinogenicity test in CB6F1-TG rasH2 mice.

    PubMed

    Urano, Koji; Suzuki, Shuzo; Machida, Kazuhiko; Eguchi, Natsuko; Sawa, Nobuko; Kikuchi, Koji; Hattori, Yuji; Usui, Toshimi

    2007-10-01

    We examined the possibility of expanding applications of rasH2 mice, which are genetically manipulated mice for short-term carcinogenicity tests, to percutaneous application. A 26-week short-term carcinogenicity study was performed on a total of 300 mice including 75 male and female rasH2 mice each, and 75 male and female non-Tg mice each from the same litter as the rasH2 mice divided into untreated group, an ethanol group, a white Vaseline group, an acetone group, and a phorbol 12-myristate 13-acetate (TPA) group. Only shaving of dorsal skin was performed on the untreated mice. As a positive control, TPA was administered percutaneously at a dose of 2.5 microg/kg and 3 times/week for 26 weeks based on the protocol for Tg.AC mice in the ILSI/HESI international validation study. In the ethanol, white Vaseline, and acetone groups, no tumorous changes were observed on the skin at the administration site. In the TPA group, nodular changes at the administration site were observed from seven weeks after the start of administration in rasH2 mice, and the incidence in males and females was 50.0% (7/14) and 53.3% (8/15), respectively. In a pathological examination, nodules in 21.4% (3/14) of males and 46.7% (7/15) of females were diagnosed as skin papilloma or keratoacanthoma, and the rest as squamous cell hyperplasia. In the non-Tg mice, no nodules or tumorigenic changes were observed at the administration site. These findings show that percutaneous application in rasH2 mice is possible in 26-week carcinogenicity tests. PMID:17965551

  12. Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

    PubMed

    Montejo-López, Wilber; Rivera-Ramírez, Nayeli; Escamilla-Sánchez, Juan; García-Hernández, Ubaldo; Arias-Montaño, José-Antonio

    2016-09-01

    Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation. PMID:27350581

  13. The inhibitory effects of boldine, glaucine, and probucol on TPA-induced down regulation of gap junction function. Relationships to intracellular peroxides, protein kinase C translocation, and connexin 43 phosphorylation.

    PubMed

    Hu, J; Speisky, H; Cotgreave, I A

    1995-11-01

    The naturally occurring antioxidant boldine and its di-methoxy analogue glucine, as well as the drug antioxidant probucol, all inhibit TPA-induced downregulation of gap junctional intercellular communication in WB-F344 rat liver epithelial cells in dose-dependent manners. The compounds were essentially 100% inhibitory to the effect of TPA (10 nM) at 50 microM each. Analysis of the mechanism of the antitumor promotive action of these agents in vitro revealed that boldine and probucol (both at 10 microM) totally inhibited the TPA-induced accumulation of intracellular oxidants. Additionally, boldine, glaucine, and probucol, each at 50 microM, inhibited TPA-induced translocation of protein kinase C (PKC) to the particulate fraction of the cells, with concomitant inhibition of TPA-induced hyperphosphorylation of gap junctional connexin 43 (cx43) and TPA-induced internalisation of cx43 protein from the plasma membrane of the cells. None of the compounds inhibited the binding of (3H)-PDBu to TPA-specific binding sites in the cells. The results indicate that antioxidant molecules, irrespective of structure, possess common antitumor promotive potential in this model of gap junctional intercellular communication. The data also indicate that the compounds may interfere with the promotive function of TPA, at least in part, by the destruction of oxidants within the cells. Xanthine oxidase was excluded as a major source of such intracellular oxidants because allopurinol (50 microM) did not significantly affect either the accumulation of oxidants in the cells or the downregulation of gap junctional communication in response to TPA. Taken together, these data also suggest that TPA-induced oxidants play a role in the translocation of PKC to cellular membranes and it is at this level where the antioxidants may interfere in TPA-induced downregulation of gap junctional function. PMID:7503766

  14. A [4+2] mixed ligand approach to ruthenium DNA metallointercalators [Ru(tpa)(N-N)](PF(6))(2) using a tris(2-pyridylmethyl)amine (tpa) capping ligand.

    PubMed

    Kraft, Sabine Seeberg née; Bischof, Caroline; Loos, Annette; Braun, Sebastian; Jafarova, Nigar; Schatzschneider, Ulrich

    2009-08-01

    A series of five tris(2-pyridylmethyl)amine (tpa) ruthenium complexes [Ru(tpa)(N-N)](PF(6))(2) with N-N=bpy (2,2'-bipyridine), phen (1,10-phenanthroline), dpq (dipyrido[3,2-d:2',3'-f]quinoxaline), dppz (dipyrido[3,2-a;2',3'-c]phenazine), and dppn (4,5,9,16-tetraazadibenzo[a,c]naphthacene) was prepared and characterized by NMR, UV-Visible (UV/Vis), and fluorescence spectroscopy as well as cyclic voltammetry. Structures optimized with density functional theory methods (DFT, BP86, TZVP) without constraints show C(1) symmetry while in solution, the (1)H and (13)C NMR spectra are in accordance with an average C(s) symmetry. This is thought to be due to a low energy barrier for flipping of the equatorial pyridine ring from one side of the N-N plane to the other. The electronic structure of the compounds was studied with DFT and a change in the highest occupied molecular orbital (HOMO) character from Ru t(2g) for the bpy, phen, and dpq to N-N ligand-based for the dppz and dppn complexes was found. TDDFT calculations showed dominant N-N-based intra-ligand charge transfer (ILCT) transitions in the latter two complexes mixed with metal-to-ligand charge transfer (MLCT) bands found for all five compounds. DNA binding of the complexes was studied with UV/Vis titrations, the fluorescent ethidium bromide displacement assay, and CD spectroscopy. The affinity increases with the aromatic surface area of of the bidentate N-N ligand in the order bpy

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

    PubMed

    Akram, Muhammad; Shin, Iljin; Kim, Kyeong-A; Noh, Dabi; Baek, Seung-Hoon; Chang, Sun-Young; Kim, Hyoungsu; Bae, Ok-Nam

    2016-09-15

    Impaired immune responses in skin play a pivotal role in the development and progression of chemical-associated inflammatory skin disorders. In this study, we synthesized new flavonoid derivatives from macakurzin C, and identified in vitro and in vivo efficacy of a potent anti-inflammatory flavonoid, Compound 14 (CPD 14), with its underlying mechanisms. In lipopolysaccharide (LPS)-stimulated murine macrophages and IFN-γ/TNF-α-stimulated human keratinocytes, CPD 14 significantly inhibited the release of inflammatory mediators including nitric oxide (NO), prostaglandins, and cytokines (IC50 for NO inhibition in macrophages: 4.61μM). Attenuated NF-κB signaling and activated Nrf2/HO-1 pathway were responsible for the anti-inflammatory effects of CPD 14. The in vivo relevance was examined in phorbol 12-myristate 13-acetate (TPA)-induced acute skin inflammation and oxazolone-induced atopic dermatitis models. Topically applied CPD 14 significantly protected both irritation- and sensitization-associated skin inflammation by suppressing the expression of inflammatory mediators. In summary, we demonstrated that a newly synthesized flavonoid, CPD 14, has potent inhibitory effects on skin inflammation, suggesting it is a potential therapeutic candidate to treat skin disorders associated with excessive inflammation. PMID:27450019

  16. Dermal inflammation in primates, mice, and guinea pigs: attenuation by second-generation leukotriene B4 receptor antagonist, SC-53228.

    PubMed

    Fretland, D J; Gokhale, R; Mathur, L; Baron, D A; Paulson, S K; Stolzenbach, J

    1995-06-01

    Granulocyte infiltration is a prominent feature of human psoriasis. Psoriatic lesional skin contains abnormally high amounts of immunoreactive leukotriene B4 (LTB4), a potent granulocyte chemotaxin in vivo and in vitro. SC-53228 [(+)-(S)-7-(3-}2-(cyclopropylmethyl)-3-methoxy-4- [(methylamino)carbonyl]phenoxy}propoxy}-3,4-dihydro-8-propyl-2H-1- benzopyran-2-propanoic acid], a second-generation LTB4 receptor antagonist, was tested topically and orally in phorbol ester-induced dermal inflammation in three species. Skin inflammation was induced by topical application of phorbol-12-myristate-13-acetate-(PMA/TPA) and assessed by ear thickness, levels of the neutrophil marker enzyme myeloperoxidase (MPO) and histological examination. In mice, SC-53228 inhibited inflammation with a topical ED50 value of 200 +/- 18 micrograms. When applied to guinea pigs, SC-53228 (100 micrograms) inhibited the MPO increase by 86%, while 1000 micrograms abrogated inflammation in rhesus macaques with no plasma accumulation of the drug. A 1% gel formulation was also efficacious in guinea pig PMA-induced epidermal inflammation. Furthermore, single oral dose administration to mice was efficacious (ED50 < 2.5 mg/kg) as was multidose administration to rhesus macaques. PMA-induced skin inflammation possesses some of the attributes of human psoriasis and an agent such as SC-53228 may have utility in the medical management of this condition. PMID:7628862

  17. Are G-protein-coupled receptors involved in mediating larval settlement and metamorphosis of coral planulae?

    PubMed

    Tran, Cawa; Hadfield, Michael G

    2012-04-01

    Larvae of the scleractinian coral Pocillopora damicornis are induced to settle and metamorphose by the presence of marine bacterial biofilms, and the larvae of Montipora capitata respond to a combination of filamentous and crustose coralline algae. The primary goal of this study was to better understand metamorphosis of cnidarian larvae by determining what types of receptors and signal-transduction pathways are involved during stimulation of metamorphosis of P. damicornis and M. capitata. Evidence from studies on larvae of hydrozoans suggests that G-protein-coupled receptors (GPCRs) are good candidates. Settlement experiments were conducted in which competent larvae were exposed to neuropharmacological agents that affect GPCRs and their associated signal-transduction pathways, AC/cAMP and PI/DAG/PKC. On the basis of the results of these experiments, we conclude that GPCRs and these pathways do not mediate settlement and metamorphosis in either coral species. Two compounds that had an effect on both species, forskolin and phorbol-12-myristate-13-acetate (TPA), may be acting on other cellular processes not related to GPCRs. This study strengthens our understanding of the underlying physiological mechanisms that regulate metamorphosis in coral larvae. PMID:22589403

  18. Topical Apigenin Alleviates Cutaneous Inflammation in Murine Models

    PubMed Central

    Man, Mao-Qiang; Hupe, Melanie; Sun, Richard; Man, George; Mauro, Theodora M.; Elias, Peter M.

    2012-01-01

    Herbal medicines have been used in preventing and treating skin disorders for centuries. It has been demonstrated that systemic administration of chrysanthemum extract exhibits anti-inflammatory properties. However, whether topical applications of apigenin, a constituent of chrysanthemum extract, influence cutaneous inflammation is still unclear. In the present study, we first tested whether topical applications of apigenin alleviate cutaneous inflammation in murine models of acute dermatitis. The murine models of acute allergic contact dermatitis and acute irritant contact dermatitis were established by topical application of oxazolone and phorbol 12-myristate 13-acetate (TPA), respectively. Inflammation was assessed in both dermatitis models by measuring ear thickness. Additionally, the effect of apigenin on stratum corneum function in a murine subacute allergic contact dermatitis model was assessed with an MPA5 physiology monitor. Our results demonstrate that topical applications of apigenin exhibit therapeutic effects in both acute irritant contact dermatitis and allergic contact dermatitis models. Moreover, in comparison with the vehicle treatment, topical apigenin treatment significantly reduced transepidermal water loss, lowered skin surface pH, and increased stratum corneum hydration in a subacute murine allergic contact dermatitis model. Together, these results suggest that topical application of apigenin could provide an alternative regimen for the treatment of dermatitis. PMID:23304222

  19. A TPA-caged precursor of (imino)coumarin for "turn-on" fluorogenic detection of Cu(.).

    PubMed

    Hu, Zhangjun; Hu, Jiwen; Wang, Hui; Zhang, Qiong; Zhao, Meng; Brommesson, Caroline; Tian, Yupeng; Gao, Hongwen; Zhang, Xuanjun; Uvdal, Kajsa

    2016-08-24

    We strategize to utilize the precursors of (imino)coumarin fluorophores to deliver novel reactive Cu(+) probes, where tris[(2-pyridyl)-methyl] amine (TPA) works as a reactive receptor towards Cu(+). To verify this strategy, CP1, a representative probe and relevant sensing behaviors towards Cu(+) are presented here. CP1 features good solubility and fast response for monitoring labile copper in aqueous solution and live cells. The sensing mechanism of CP1 is determined by HPLC titration and mass spectrometric analysis. The probe CP1 exhibits a 60-fold fluorescence enhancement and a detection limitation of 10.8 nM upon the detection of Cu(+). CP1 is further applied for imaging labile copper in live cells. This work provides a starting point for future development of Cu(+) probes, based on in situ formation of (imino)coumarin scaffolds, as well as their further investigations of copper signaling and biological events. PMID:27497012

  20. 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

  1. 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…

  2. Vitros 5600 Syphilis TPA assay: evaluation of an automated chemiluminescence assay for detection of Treponema pallidum antibodies in a high prevalence setting.

    PubMed

    Van den Bossche, Dorien; Florence, Eric; Kenyon, Christopher; Van Esbroeck, Marjan

    2014-11-01

    The performance of the Syphilis TPA assay (Ortho-Clinical Diagnostics) on Vitros 5600 Integrated System was evaluated and demonstrated excellent results. Our data support the use of this assay for test confirmation in the traditional algorithm and for screening for syphilis in a routine automated laboratory setting when using the reverse algorithm. PMID:25299416

  3. Loss of CRABP-II Characterizes Human Skin Poorly Differentiated Squamous Cell Carcinomas and Favors DMBA/TPA-Induced Carcinogenesis.

    PubMed

    Passeri, Daniela; Doldo, Elena; Tarquini, Chiara; Costanza, Gaetana; Mazzaglia, Donatella; Agostinelli, Sara; Campione, Elena; Di Stefani, Alessandro; Giunta, Alessandro; Bianchi, Luca; Orlandi, Augusto

    2016-06-01

    Retinol and its derivatives play an important role in epidermal growth and differentiation and represent chemopreventive agents in nonmelanoma skin cancer. Retinoic acid binding protein II (CRABP-II) is a cytoplasmic receptor that critically regulates all-trans-retinoic acid (ATRA) trafficking. We documented the marked reduced expression of CRABP-II and its promoter methylation in human poorly differentiated squamous cell carcinomas. To investigate the role of CRABP-II in skin carcinogenesis we used skin lesion induction by dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate in CRABP-II-knockout C57BL/6 mice. We observed earlier and more diffuse epidermal dysplasia, greater incidence and severity of tumors, reduced expression of cytokeratin 1/cytokeratin 10 and involucrin, increased proliferation, and impaired ATRA inhibition of tumor promotion compared with wild-type animals. CRABP-II-transfected HaCaT, FaDu, and A431 cells showed expression of differentiation markers, retinoic acid receptor-β/-γ signaling, ATRA sensitivity, and suppression of EGFR/v-akt murine thymoma viral oncogene homolog 1 (AKT) pathways in a fatty acid binding protein 5/peroxisome proliferator-activated receptor-β/-δ-independent manner. The opposite was true in keratinocytes isolated from CRABP-II-knockout mice. Finally, CRABP-II accumulation induced ubiquitination-associated reduction of EGFR. Our results showed reduced CRABP-II expression in human poorly differentiated squamous cell carcinomas, and its gene deletion favored experimental skin carcinogenesis and impaired ATRA antitumor efficacy, likely modulating EGFR/AKT pathways and retinoic acid receptor-β/-γ signaling. Therapeutic interventions aimed at restoring CRABP-II-mediated signaling may amplify therapeutic retinoid efficacy in nonmelanoma skin cancer. PMID:26945879

  4. Lattice stability and high-pressure melting mechanism of dense hydrogen up to 1.5 TPa

    NASA Astrophysics Data System (ADS)

    Geng, Hua Y.; Hoffmann, R.; Wu, Q.

    2015-09-01

    Lattice stability and metastability, as well as melting, are important features of the physics and chemistry of dense hydrogen. Using ab initio molecular dynamics (AIMD), the classical superheating limit and melting line of metallic hydrogen are investigated up to 1.5 TPa. The computations show that the classical superheating degree is about 100 K, and the classical melting curve becomes flat at a level of 350 K when beyond 500 GPa. This information allows us to estimate the well depth and the potential barriers that must be overcome when the crystal melts. Inclusion of nuclear quantum effects (NQE) using path integral molecular dynamics (PIMD) predicts that both superheating limit and melting temperature are lowered to below room temperature, but the latter never reaches absolute zero. Detailed analysis indicates that the melting is thermally activated, rather than driven by pure zero-point motion (ZPM). This argument was further supported by extensive PIMD simulations, demonstrating the stability of Fddd structure against liquefaction at low temperatures.

  5. Absolute equation of state and opacity measurements of CH plastic to 40 TPa using the National Ignition Facility

    NASA Astrophysics Data System (ADS)

    Doeppner, T.; Swift, D.; Hawreliak, J.; Kritcher, A.; Collins, G.; Glenzer, S.; Rothman, S.; Chapman, D.; Gaffney, J.; Rose, S.; Falcone, R.

    2013-06-01

    We have developed an experimental configuration using a hohlraum-driven spherically-convergent shock to induce pressures into the gigabar range, measuring the Hugoniot radiographically. The shock pressure increases with convergence, so a range of Hugoniot states is obtained from a single experiment. The opacity along the Hugoniot is also deduced, which is essential in gigabar experiments as it changes significantly from its initial value. We are focusing initially on plastics, as we can reliably obtain spherical samples with the desired design of ablator, and the radiographic signal is reasonable. Our initial measurements on NIF used a conservative timing of the x-ray backlighter to allow for uncertainty in the EOS, and probed only part of the pressure range. The shock speed and compression, obtained from radiographic analysis, gave absolute Hugoniot states from 12-41 TPa, which is an order of magnitude greater than previously measured in CH. The measured EOS locus was consistent with the previous measurements, and significantly stiffer than the theoretical EOS used for comparison. Our analysis also gave the variation of opacity along the Hugoniot, which showed a decrease of an order of magnitude, as expected from atomic physics calculations. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

  6. Atrazine represses S100A4 gene expression and TPA-induced motility in HepG2 cells.

    PubMed

    Peyre, Ludovic; Zucchini-Pascal, Nathalie; Rahmani, Roger

    2014-03-01

    Atrazine (ATZ) is probably the most widely used herbicide in the world. However there are still many controversies regarding its impacts on human health. Our investigations on the role of pesticides in liver dysfunctions have led us to detect an inhibition of FSP1 expression of 70% at 50μm and around 95% at 500μM of ATZ (p<0.01). This gene encodes the protein S100a4 and is a clinical biomarker of epithelial-mesenchymal transition (EMT), a key step in the metastatic process. Here we investigated the possible effect of ATZ on cell migration and noticed that it prevents the EMT and motility of the HepG2 cells induced by the phorbol ester TPA. ATZ decreases Fak pathway activation but has no effect on the Erk1/2 pathway known to be involved in metastasis in this cell line. These results suggest that ATZ could be involved in cell homeostasis perturbation, potentially through a S100a4-dependant mechanism. PMID:24211529

  7. Optimization of alternate-strand triple helix formation at the 5"-TpA-3" and 5"-ApT-3" junctions.

    PubMed Central

    Brodin, P; Sun, J S; Mouscadet, J F; Auclair, C

    1999-01-01

    Alternate-strand triple helix formation was optimized at the two junction steps, the 5"-TpA-3" and 5"-ApT-3" junctions. Footprint experiments, gel retardation assays and thermal denaturation measures on a sequence appropriately designed with two adjacent alternate-strand polypurine tracts points out that the addition of an adenine residue and the removal of one nucleotide should facilitate the crossing strands at the 5"-TpA-3" junction and at the 5"-ApT-3" junction, respectively. These results provide a 'switch code' for the construction of alternate-strand triple helix forming oligonucleotides which open new possibilities for extending the range of applications of antigene strategy. PMID:10454596

  8. Provider perceptions of barriers to the emergency use of tPA for Acute Ischemic Stroke: A qualitative study

    PubMed Central

    2011-01-01

    Background Only 1-3% of ischemic stroke patients receive thrombolytic therapy. Provider barriers to adhering with guidelines recommending tPA delivery in acute stroke are not well known. The main objective of this study was to describe barriers to thrombolytic use in acute stroke care. Methods Twenty-four hospitals were randomly selected and matched into 12 pairs. Barrier assessment occurred at intervention sites only, and utilized focus groups and structured interviews. A pre-specified taxonomy was employed to characterize barriers. Two investigators independently assigned themes to transcribed responses. Seven facilitators (three emergency physicians, two nurses, and two study coordinators) conducted focus groups and interviews of emergency physicians (65), nurses (62), neurologists (15), radiologists (12), hospital administrators (12), and three others (hospitalists and pharmacist). Results The following themes represented the most important external barriers: environmental and patient factors. Important barriers internal to the clinician included familiarity with and motivation to adhere to the guidelines, lack of self-efficacy and outcome expectancy. The following themes were not substantial barriers: lack of awareness of the existence of acute stroke guidelines, presence of conflicting guidelines, and lack of agreement with the guidelines. Conclusions Healthcare providers perceive environmental and patient-related factors as the primary barriers to adherence with acute stroke treatment guidelines. Interventions focused on increasing physician familiarity with and motivation to follow guidelines may be of highest yield in improving adherence. Improving self-efficacy in performing guideline concordant care may also be useful. Trial Registration ClinicalTrials.gov identifier: NCT00349479 PMID:21548943

  9. Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022.

    PubMed

    Kohnert, U; Rudolph, R; Verheijen, J H; Weening-Verhoeff, E J; Stern, A; Opitz, U; Martin, U; Lill, H; Prinz, H; Lechner, M

    1992-01-01

    BM 06.022 is a t-PA deletion variant which comprises the kringle 2 and the protease domain. Production of BM 06.022 in Escherichia coli leads to the formation of inactive inclusion bodies, which have to be refolded by an in vitro refolding process to achieve activity and proper structure of the domains. We analysed the biochemical properties of BM 06.022 to obtain some information about the structure of kringle 2 and the protease as compared with the structure of these domains in the intact t-PA molecule. The kinetic analysis of the amidolytic activity of BM 06.022 and CHO-t-PA yielded similar values for kcat (13.9 s-1 and 11.4 s-1 for the single chain forms and 33.9 s-1 and 27.1 s-1 for the two chain forms of BM 06.022 and CHO-t-PA, respectively) and for Km (2.5 mM and 2.1 mM for the single chains forms and 0.5 mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA, respectively). BM 06.022 and CHO-t-PA have the same plasminogenolytic activity in the absence of CNBr fragments of fibrinogen. However, BM 06.022 has a lower plasminogenolytic activity in the presence of CNBr fragments of fibrinogen and a lower affinity to fibrin as compared with CHO-t-PA. The affinity of BM 06.022 for fibrin is completely suppressed by 0.3 mM epsilon-aminocaproic acid, while the intact t-PA has a residual affinity of approximately 30%. The dissociation constants for the interaction with the lysine analogue epsilon-aminocaproic acid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA, respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibited by PAI-1 in a similar manner. PMID:1321420

  10. 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.

  11. 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

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

    NASA Astrophysics Data System (ADS)

    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.

    2016-05-01

    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.

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

    PubMed

    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

  14. 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. PMID:27435387

  15. Absolute Hugoniot measurements of polystyrene between 3 and 12 TPa using radiography of a converging shock at the National Ignition Facility

    NASA Astrophysics Data System (ADS)

    Doeppner, T.; Kritcher, A. L.; Swift, D. C.; Bachmann, B.; Hawreliak, J.; Colllins, G. W.; Glenzer, S.; Rothman, S. D.; Kraus, D.; Falcone, R. W.

    2015-06-01

    A converging shock was induced with hohlraum-driven soft x-ray radiation on a solid, spherical sample of poly alpha-methyl styrene. The time-history of density profiles through the sample was measured by x-ray radiography using a laser-heated backlighter and a streak camera, viewing a diameter across the sample through slots in the hohlraum wall. Profile-matching in radius and time was used to increase the accuracy of density inferred from the transmission. The speed and compression of the shock were measured from the density profiles. The shock pressure increased with convergence, so a range of Hugoniot states was obtained from a single experiment. Using a laser power based on the early part of a ``high foot'' pulse from ignition experiments, the low end of the pressure range was brought down to 2 TPa, overlapping states accessible by experiments in plane geometry, and ensuring that the opacity of the compressed sample was the same as for unshocked material, simplifying the analysis. Shock states were measured up to 12 TPa, when the shock was close to the center of the sample. This is several times higher than has been obtained by other methods and is an absolute measurement. Performed under the auspices of the US DOE by LLNL under Contract DE-AC52-07NA27344.

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

    DOE PAGESBeta

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

    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

  17. Cerebroprotective effects of TAK-937, a novel cannabinoid receptor agonist, in permanent and thrombotic focal cerebral ischemia in rats: therapeutic time window, combination with t-PA and efficacy in aged rats.

    PubMed

    Murakami, Koji; Suzuki, Motohisa; Suzuki, Noriko; Hamajo, Kazuhiro; Tsukamoto, Tetsuya; Shimojo, Masato

    2013-08-14

    Some occluded arteries of acute ischemic stroke (AIS) patients are not recanalized, even if thrombolytic therapy is performed. Considering such clinical settings, we examined the potential cerebroprotective efficacy of TAK-937, a novel cannabinoid receptor agonist, in young adult and aged rats with a permanent middle cerebral artery occlusion (MCAO) model and conducted a combination study with TAK-937 and tissue type plasminogen activator (t-PA) in a rat thrombotic MCAO model. TAK-937 significantly reduced infarct volume when it was administered 3 and 5h after permanent MCAO in young adult rats. A thrombotic MCAO was induced by photo-irradiation of the middle cerebral artery with Rose Bengal administration and a permanent MCAO was produced by thermoelectric coagulation of occluded arteries. TAK-937 (10, 30 and 100μg/kg/h) was intravenously infused 1, 3, 5, or 8-24h after MCAO. t-PA (3 or 10mg/kg) was intravenously administered 1, 1.5 or 2h after MCAO. Infarct volume was determined using a 2,3,5-triphenyltetrazolium chloride staining method 24 or 48h after MCAO. The combined treatment of TAK-937 with t-PA significantly reduced the cerebral infarction compared with t-PA treatment alone in a rat thrombotic MCAO model. TAK-937 reduced infarct volume of aged rats as well, when it was administered 1h after permanent MCAO. These results suggest that TAK-937 exerts protective effects regardless of age and has a wide therapeutic time window in permanent occlusion. Furthermore, combined treatment of TAK-937 with t-PA would provide more therapeutic efficacy compared to t-PA treatment alone. PMID:23791950

  18. 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

  19. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-01-01

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway. PMID:27598116

  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. 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.

  2. Anti-tissue plasminogen activator (tPA) as an effective therapy of neonatal hypoxia-ischemia with and without inflammation.

    PubMed

    Yang, Dianer; Kuan, Chia-Yi

    2015-04-01

    Hypoxic-ischemic brain injury is an important cause of neurodevelopmental deficits in neonates. Intrauterine infection and the ensuing fetal inflammatory responses augment hypoxic-ischemic brain injury and attenuate the efficacy of therapeutic hypothermia. Here, we review evidences from preclinical studies suggesting that the induction of brain parenchymal tissue-type plasminogen activator (tPA) plays an important pathogenic role in these conditions. Moreover, administration of a stable-mutant form of plasminogen activator inhibitor-1 called CPAI confers potent protection against hypoxic-ischemic injury with and without inflammation via different mechanisms. Besides intracerebroventricular injection, CPAI can also be administered into the brain using a noninvasive intranasal delivery strategy, adding to its applicability in clinical use. In sum, the therapeutic potential of CPAI in neonatal care merits further investigation with large-animal models of hypoxia-ischemia and cerebral palsy. PMID:25475942

  3. Table-top Generation and Spectroscopic Study of ~10 TPa High-Energy Density Materials with C60 Hypervelocity (v ~ 100 km/s) Impact

    NASA Astrophysics Data System (ADS)

    Bae, Young

    2013-06-01

    Intense bursts of soft x-rays were discovered by Bae et al. in hypervelocity (v ~ 100 km/s) impact of bio and water nanoparticles at the Brookhaven National Lab (BNL) in 1994. In the experiment, the nanoparticles were directly impacted on and detected by Si particle detectors that also detected the soft x-rays. Energy deposition measurements through thin films revealed that the impact generated pressures were ~10 TPa, and the photon energies in the range of 75-100 eV for Si targets. The conversion efficiency from the kinetic energy to the radiation energy was unexpectedly high, ~38%, which was attributed to Dicke Superradiance of collective quantum states in High-Energy Density Materials (HEDM), Metastable Innershell Molecular States (MIMS). This talk presents recent experimental results obtained in a table-top apparatus completely different from and orders of magnitude smaller than that at BNL. In the new setup, hypervelocity (v 100 km/s) C60+ ions impacted on Al targets, and the impact generated soft x-rays were detected off-axis and analyzed using three Si photodiode detectors with selective energy response curves. The photon energy was determined to be ~70 eV with the kinetic-energy to photon-energy conversion efficiency of ~35% in confirmation of the results by Bae et al. at BNL. The present results demonstrate a new way of generation and spectroscopic study of HEDM with pressures exceeding 10 TPa, and show the pathway to scaling up the soft x-ray generation method for a wide range of applications from lithography to inertial fusion. This work was supported by DTRA under the contract HDTRA1-12-C-0094.

  4. Adjunctive treatment with ticagrelor, but not clopidogrel, added to tPA enables sustained coronary artery recanalisation with recovery of myocardium perfusion in a canine coronary thrombosis model.

    PubMed

    Wang, Kai; Zhou, Xiaorong; Huang, Yanming; Khalil, Mazen; Wiktor, Dominik; van Giezen, J J J; Penn, Marc S

    2010-09-01

    Reperfusion therapy for myocardial infarction is limited by significant re-occlusion rates and less-than-optimal myocardial tissue perfusion. It was the objective of this study to assess and compare the effect of ticagrelor, the first reversibly binding oral P2Y12 receptor antagonist, with that of clopidogrel, in conjunction with thrombolytic therapy, on platelet aggregation, thrombus formation, and myocardial perfusion in a canine model. Thrombus formation was induced by electrolytic injury and blood flow was measured with a Doppler ultrasonic flowmeter. All animals received tissue plasminogen activator (tPA) (1 mg/kg over 20 min); 10 animals received clopidogrel (10 mg/kg IV bolus over 5 min), 10 animals received ticagrelor initiated with a 1-min bolus (75 microg/kg/min), followed by continuous infusion (10 microg/kg/min) for 2 h, and 10 animals received IV saline. Re-occlusion rate and cyclic flow variation decreased with ticagrelor compared to saline groups (p<0.05). Adenosine phosphate (ADP)-induced platelet aggregation decreased with ticagrelor (1.9% +/- 2.67) and clopidogrel (1.11% +/- 2.0) vs. saline (26.3% +/- 23.5, p<0.05) at the end of adjunctive therapy. Bleeding time increased in the clopidogrel compared to the ticagrelor group (p=0.01). Infarct size was reduced with ticagrelor compared to the clopidogrel and saline groups (p<0.05). Blood flow remained significantly below baseline values at 20 min after tPA administration in the saline and clopidogrel groups but not in the ticagrelor group. In conclusion, in a dog coronary thrombosis model, ticagrelor blocks ADP-induced platelet activation and aggregation; prevents platelet-mediated thrombosis; prolongs reperfusion time and reduces re-occlusion and cyclic flow variation; and significantly decreases infarct size and rapidly restores myocardial tissue perfusion. PMID:20694285

  5. Regulation of viral and cellular gene expression by Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA.

    PubMed

    Rossetto, Cyprian C; Tarrant-Elorza, Margaret; Verma, Subhash; Purushothaman, Pravinkumar; Pari, Gregory S

    2013-05-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphoma. In cell culture, KSHV results in a latent infection, and lytic reactivation is usually induced with the expression of K-Rta or by treatment with phorbol 12-myristate 13-acetate (TPA) and/or n-butyrate. Lytic infection is marked by the activation of the entire viral genomic transcription cascade and the production of infectious virus. KSHV-infected cells express a highly abundant, long, noncoding transcript referred to as polyadenylated nuclear RNA (PAN RNA). PAN RNA interacts with specific demethylases and physically binds to the KSHV genome to mediate activation of viral gene expression. A recombinant BACmid lacking the PAN RNA locus fails to express K-Rta and does not produce virus. We now show that the lack of PAN RNA expression results in the failure of the initiation of the entire KSHV transcription program. In addition to previous findings of an interaction with demethylases, we show that PAN RNA binds to protein components of Polycomb repression complex 2 (PRC2). RNA-Seq analysis using cell lines that express PAN RNA shows that transcription involving the expression of proteins involved in cell cycle, immune response, and inflammation is dysregulated. Expression of PAN RNA in various cell types results in an enhanced growth phenotype, higher cell densities, and increased survival compared to control cells. Also, PAN RNA expression mediates a decrease in the production of inflammatory cytokines. These data support a role for PAN RNA as a major global regulator of viral and cellular gene expression. PMID:23468496

  6. Suppression of A549 cell proliferation and metastasis by calycosin via inhibition of the PKC-α/ERK1/2 pathway: An in vitro investigation

    PubMed Central

    CHENG, XU-DONG; GU, JUN-FEI; YUAN, JIA-RUI; FENG, LIANG; JIA, XIAO-BIN

    2015-01-01

    The migration and invasion of lung cancer cells into the extracellular matrix contributes to the high mortality rates of lung cancer. The protein kinase C (PKC) and downstream signaling pathways are important in the invasion and migration of lung cancer cells. Calycosin (Cal), an effector chemical from Astragalus has been reported to affect the recurrence and metastasis of cancer cells via the regulation of the protein expression of matrix metalloproteinases (MMPs). The inhibition of Cal on the migration and invasion of A549 cells was investigated in the present study. Cell viability and apoptosis assays were performed using MTT and flow cytometric analyses. A wound healing assay and Transwell invasion assay were performed to evaluate the effect of Cal on A549 cell migration and invasion. Invasion-associated proteins, including MMP-2, MMP-9, E-cadherin (E-cad), integrin β1, PKC-α and extracellular signal-regulated kinase 1/2 (ERK1/2) were detected using western blotting. In addition, PKC-α inhibitor, AEB071, and ERK1/2 inhibitor, PD98059, were used to determine the association between the suppression of PKC-α/ERK1/2 and invasion, MMP-2, MMP-9, E-cad and integrin β1. Cal was observed to suppress cell proliferation and induce apoptosis. There were significant differences between the phorbol-12-myristate-13-acetate (TPA)-induced A549 cells treated with Cal and the untreated cells in the rates of migration and invasion. The levels of MMP-2, MMP-9, E-cad and integrin β1 in the TPA-induced A549 cells changed markedly, compared with the untreated cells. In addition, the suppression of Cal was affected by the PKC inhibitor, AEB071, an ERK1/2 inhibitor, PD98059. The results of the present study indicated that Cal inhibited the proliferation, adhesion, migration and invasion of the TPA-induced A549 cells. The Cal-induced repression of PKC-α/ERK1/2, increased the expression of E-Cad and inhibited the expression levels of MMP-2, MMP-9 and integrin β1, which possibly

  7. 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.

  8. Surface membrane antigen expression changes induced in vitro by exogenous growth factors in chronic lymphocytic leukemia cells.

    PubMed

    Vilpo, J; Hulkkonen, J; Hurme, M; Vilpo, L

    2002-09-01

    The factors determining the growth and survival of cells in B chronic lymphocytic leukemia (CLL) have remained poorly understood. We investigated the effects of optimal mitogen combinations (OMCs) on the expression of 26 surface membrane antigens among 33 CLL patients. The seven OMCs used were selected after pre-testing 14 combinations of (1) S. aureus Cowan I (SAC), (2) interleukin-2 (IL-2), (3) tumor necrosis factor alpha (TNF-alpha) and (4) 12-O-tetradecanoylphorbol 13-acetate (TPA; also known as phorbol 12-myristate 13-acetate or PMA). In flow cytometry we revealed that OMCs induced statistically highly significant upregulation of the expression of CD5, CD11c, CD19, CD22, CD23, CD25, CD38, CD40, CD45, CD45RO, CD95, CD126, CD130 and FMC7, and downregulation of CD20 and CD124 expression. Interestingly, the expression of CD27, CD45RA, CD79b, CD80, CD122 and that of the immunoglobulin gene superfamily members CD21, Ig-kappa, Ig-lambda, Ig-delta and Ig-micro were not significantly affected under similar conditions. The expression of several antigens was co-regulated, suggesting common regulatory pathways. These antigens include CD11c/CD5, CD11c/CD22, CD11c/CD126, CD11c/FMC7 as well as CD27/CD45, CD27/CD45RA and CD27/CD79b. Upregulation of surface antigen expression, induced by OMCs, should be applicable in antibody therapy in vitro and in vivo, and in negative stem cell selection for autotransplantation. Furthermore, the current strategy to enhance cell surface antigen expression may be a versatile tool to raise humoral and cell-mediated host defense against CLL cells. Upregulation of proteins mediating positive growth signals (eg CD25, CD40) and negative signals or apoptosis (eg CD95) may be used to sensitize cells to chemotherapy and programmed cell death. PMID:12200683

  9. Calculated distortions of duplex DNA by a cis, syn cyclobutane thymine dimer are unaffected by a 3' TpA step.

    PubMed Central

    Cooney, M G; Miller, J H

    1997-01-01

    Molecular dynamics simulations were performed on the duplex DNA dodecamers d(CGCGAA TT CGCG): d(CGCGAATTCGCG) and d(GCACGAA TT AAG): d(CTTAATTCGTGC), where TT denotes a cis, syn cyclobutane thymine dimer. The constant temperature and pressure algorithm of the AMBER 4.1 molecular-modeling package was used with explicit water and counterions, periodic boundary conditions and electrostatic interactions evaluated by the particle-mesh Ewald method. Results were analyzed by the CURVES algorithm and its implementation in DIALS and WINDOWS. Calculated distortions of DNA structure by the thymine dimer were qualitatively and quantitatively similar for the two sequences. Despite the enhanced flexibility of the native TpA dinucleotide step, major deviations from the B-DNA values of helicoidal parameters were found only at the Ap and p dinucleotide steps in both sequences. Only the AT base pairs of the two sequences that contain the 5' thymine of the dimers exhibited weakened Watson-Crick hydrogen bonds and anomalous stretching. Hence, we conclude that the pattern of structural perturbations responsible for recognition of cis, syn thymine dimers by repair enzymes is not sensitive to their sequence context. PMID:9060440

  10. Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators.

    PubMed

    Krstic, Dimitrije; Rodriguez, Myriam; Knuesel, Irene

    2012-01-01

    The extracellular signaling protein Reelin, indispensable for proper neuronal migration and cortical layering during development, is also expressed in the adult brain where it modulates synaptic functions. It has been shown that proteolytic processing of Reelin decreases its signaling activity and promotes Reelin aggregation in vitro, and that proteolytic processing is affected in various neurological disorders, including Alzheimer's disease (AD). However, neither the pathophysiological significance of dysregulated Reelin cleavage, nor the involved proteases and their modulators are known. Here we identified the serine protease tissue plasminogen activator (tPA) and two matrix metalloproteinases, ADAMTS-4 and ADAMTS-5, as Reelin cleaving enzymes. Moreover, we assessed the influence of several endogenous protease inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), α-2-Macroglobulin, and multiple serpins, as well as matrix metalloproteinase 9 (MMP-9) on Reelin cleavage, and described their complex interplay in the regulation of this process. Finally, we could demonstrate that in the murine hippocampus, the expression levels and localization of Reelin proteases largely overlap with that of Reelin. While this pattern remained stable during normal aging, changes in their protein levels coincided with accelerated Reelin aggregation in a mouse model of AD. PMID:23082219

  11. Hugoniot and opacity measurements of polystyrene and carbon up to 80 TPa from radiography of converging shocks at the National Ignition Facility

    NASA Astrophysics Data System (ADS)

    Kritcher, A. L.; Doeppner, T.; Swift, D. C.; Bachmann, B.; Kraus, D.; Hawreliak, J.; Gaffney, J.; Collins, G.; Glenzer, S.; Chapman, D.; Rothman, S. D.; Rose, S.; Falcone, R. W.

    2015-06-01

    Converging shocks were induced with hohlraum-driven x-ray radiation on spherical samples of poly alpha-methyl styrene and diamond. The time-history of density profiles through the sample was measured by x-ray radiography using a laser-heated backlighter and a streak camera, viewing a diameter across the sample through slots in the hohlraum wall. Profile-matching in radius and time was used to increase the accuracy of density inferred from the transmission. The shock temperature reached several hundred eV, causing ionization which significantly reduced the opacity to the 9 kV x-ray energy. The opacity change at the shock was inferred from the change in apparent mass inside a radiographic marker layer. The speed and compression of the shock were measured from the density profiles. The shock pressure increased with convergence, so a range of Hugoniot states was obtained from each experiment. Shock states were measured between 10 and 80 TPa. Presently at Washington State University.

  12. EFFECT OF PHORBOL ESTERS ON CLONAL CULTURES OF HUMAN, HAMSTER, AND RAT RESPIRATORY EPITHELIAL CELLS

    EPA Science Inventory

    The effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth of epithelial cells from rat, hamster, and human respiratory tract has been measured by monitoring colony formation in culture. TPA and its active derivatives stimulated colony formation of ...

  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. Selective inhibition of JAK2/STAT1 signaling and iNOS expression mediates the anti-inflammatory effects of coniferyl aldehyde.

    PubMed

    Akram, Muhammad; Kim, Kyeong-A; Kim, Eun-Sun; Shin, Young-Jun; Noh, Dabi; Kim, Eunji; Kim, Jeong-Hyeon; Majid, Arshad; Chang, Sun-Young; Kim, Jin-Ki; Bae, Ok-Nam

    2016-08-25

    Urgent needs still exist for selective control of excessive inflammation. Despite the therapeutic potential of natural compounds against inflammation-associated chronic conditions, lack of specific molecular targets renders these bioactive compounds difficult for further development. Here we examined the bioactivity of coniferyl aldehyde (CA), a natural phenolic compound found in several dietary substances and medicinal plants, elucidating its efficacy both in vivo and in vitro with underlying molecular mechanisms. IFN-γ/TNF-α-stimulated human keratinocytes and lipopolysaccharide (LPS)-stimulated murine macrophages were used to examine the effect of CA in vitro and to elucidate the underlying mechanisms. In vivo models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and carrageenan (CRG)-induced paw edema were employed to investigate the topical and systemic anti-inflammatory effects of CA, respectively. CA significantly reduced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated macrophages. While nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) pathways, the representative cellular pathways for iNOS induction, were not affected by CA, phosphorylation of Janus kinase 2 (JAK2) and signal Transducers and Activators of Transcription 1 (STAT1) and subsequent nuclear translocation of p-STAT1 were significantly decreased by CA. The effect of CA on JAK2-STAT1-iNOS axis was also observed in human keratinocytes stimulated with IFN-γ/TNF-α. Topical application of CA to mice produced significant protection against TPA-induced ear edema along with suppressed epidermal hyperproliferation and leucocyte infiltration. Systemic administration of CA significantly reduced CRG-induced paw edema in rats, where CRG-induced iNOS expression and STAT1 phosphorylation were decreased by CA. In summary, CA has significant anti-inflammatory properties both in vitro and in vivo, mediated by

  15. 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

  16. A New Chemiluminescent Method for Evaluation of the Functional Activity of Neutrophils in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Proskurnina, E V; Sozarukova, M M; Polimova, A M; Prudnikova, M A; Ametov, A S; Vladimirov, Yu A

    2016-06-01

    Functional activity of neutrophils was evaluated by the chemiluminescent method with successive double stimulation by soluble stimuli with different mechanisms of action: phorbol-12-myristate-13-acetate (PMA) and phormyl-methionyl-leucyl-phenilalanine (fMLP). The study was carried out in 26 patients receiving oral sugar-reducing therapy. In addition to the functional activity of neutrophils, the levels of TBA reactive products, inflammation markers, blood clotting values, and biochemical parameters were measured. The results showed mainly reduction of the granulocytic component of the immune system in the patients. PMID:27388632

  17. Melatonin protects mast cells against cytotoxicity mediated by chemical stimuli PMACI: possible clinical use.

    PubMed

    Maldonado, M D; Garcia-Moreno, H; Calvo, J R

    2013-09-15

    Melatonin has documented cytoprotective effects on a wide variety of immune cells. The mechanism of action on mast cells (RBL-2H3) still remains in the dark. We found that melatonin significantly attenuated phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-induced cytotoxicity in a concentration and time-dependent manner. It appears that the effect of melatonin on mast cells is two-fold: dependent (MT1 and MT2) and independent membrane receptors. In conclusion, melatonin treatment reduced the cytotoxicity, mediated by PMACI, and could provide a useful therapeutic option in processes where an excessive activation of mast cells occurs. PMID:23870536

  18. Cyanine fluorophores for cellular protection against ROS in stimulated macrophages and two-photon ROS detection.

    PubMed

    Chan, M S; Xu, D; Guo, L; Tam, D Y; Liu, L S; Chen, Y; Wong, M S; Lo, P K

    2015-07-14

    We report the first example of a novel two-photon active, biocompatible, and macrophage cell-membrane permeable carbazole-based cyanine fluorophore for the detection of three biologically important ROS, namely, ˙OH, O2(-) and OCl(-) in solution. This versatile probe shows cellular protection not only in stimulated macrophages from phorbol-12-myristate-13-acetate-induced morphological changes but also lipopolysaccharide-induced cytotoxicity by quenching with the O2(-) and OCl(-) production, respectively. Such protection could be visualized by a distinct change in the fluorescence intensity of the probe. PMID:26059852

  19. Effects of protein kinase C activation on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule.

    PubMed Central

    Hays, S R; Baum, M; Kokko, J P

    1987-01-01

    Several hormones induce phosphatidylinositol turnover in cell membranes and thus activate protein kinase C. Activation of protein kinase C can, in turn, have effects on epithelial transport. These experiments were designed to investigate the effects of two activators of protein kinase C, phorbol 12-myristate,13-acetate (PMA) and L-alpha-1,2-dioctanoylglycerol (L-alpha-1,2-DOG), and two inactive analogues, 4 alpha-phorbol and 4-O-methyl phorbol 12-myristate,13-acetate, on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule. Utilizing in vitro microperfusion techniques, we found that activation of protein kinase C with either PMA or L-alpha-1,2-DOG significantly inhibited net sodium absorption, net potassium secretion and transepithelial voltage in a dose-dependent manner. There was no effect on net chloride or total CO2 transport. In contrast, the inactive phorbol analogues did not alter either sodium or potassium transport. These studies demonstrate that in the rabbit cortical collecting tubule sodium and potassium transport can be inhibited by compounds known to activate proteins kinase C. Thus, hormones that induce phosphatidylinositol turnover in the rabbit cortical collecting tubule may lead to inhibition of sodium transport by activation of protein kinase C. PMID:3680514

  20. Do β-Cells Generate Peroxynitrite in Response to Cytokine Treatment?*

    PubMed Central

    Broniowska, Katarzyna A.; Mathews, Clayton E.; Corbett, John A.

    2013-01-01

    The purpose of this study was to determine the reactive species that is responsible for cytokine-mediated β-cell death. Inhibitors of inducible nitric oxide synthase prevent this death, and addition of exogenous nitric oxide using donors induces β-cell death. The reaction of nitric oxide with superoxide results in the generation of peroxynitrite, and this powerful oxidant has been suggested to be the mediator of β-cell death in response to cytokine treatment. Recently, coumarin-7-boronate has been developed as a probe for the selective detection of peroxynitrite. Using this reagent, we show that addition of the NADPH oxidase activator phorbol 12-myristate 13-acetate to nitric oxide-producing macrophages results in peroxynitrite generation. Using a similar approach, we demonstrate that cytokines fail to stimulate peroxynitrite generation by rat islets and insulinoma cells, either with or without phorbol 12-myristate 13-acetate treatment. When forced to produce superoxide using redox cyclers, this generation is associated with protection from nitric oxide toxicity. These findings indicate that: (i) nitric oxide is the likely mediator of the toxic effects of cytokines, (ii) β-cells do not produce peroxynitrite in response to cytokines, and (iii) when forced to produce superoxide, the scavenging of nitric oxide by superoxide is associated with protection of β-cells from nitric oxide-mediated toxicity. PMID:24194521

  1. Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis.

    PubMed Central

    Wilhelm, S M; Eisen, A Z; Teter, M; Clark, S D; Kronberger, A; Goldberg, G

    1986-01-01

    Human skin fibroblasts secrete collagenase as two proenzyme forms (57 and 52 kDa). The minor (57-kDa) proenzyme form is the result of a partial posttranslational modification of the major (52-kDa) proenzyme through the addition of N-linked complex oligosaccharides. Human endothelial cells as well as fibroblasts from human colon, cornea, gingiva, and lung also secrete collagenase in two forms indistinguishable from those of the skin fibroblast enzyme. In vitro tissue culture studies have shown that the level of constitutive synthesis of this fibroblast-type interstitial collagenase is tissue specific, varies widely, and correlates with the steady-state level of a single collagenase-specific mRNA of 2.5 kilobases. The tumor promoter, phorbol 12-myristate 13-acetate, apparently blocks the control of collagenase synthesis resulting in a similarly high level of collagenase expression (approximately equal to 3-7 micrograms of collagenase per 10(6) cells per 24 hr) in all examined cells. The constitutive level of synthesis of a 28-kDa collagenase inhibitor does not correlate with that of the enzyme. Phorbol 12-myristate 13-acetate stimulates the production of this inhibitor that in turn modulates the activity of collagenase in the conditioned media. As a result, the apparent activity of the enzyme present in the medium does not accurately reflect the rate of its synthesis and secretion. Images PMID:3012533

  2. Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.

    PubMed

    Tee, Andrew R; Anjum, Rana; Blenis, John

    2003-09-26

    The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo. PMID:12867426

  3. Periplogenin induces necroptotic cell death through oxidative stress in HaCaT cells and ameliorates skin lesions in the TPA- and IMQ-induced psoriasis-like mouse models.

    PubMed

    Zhang, Wen-Jing; Song, Zhen-Bo; Bao, Yong-Li; Li, Wen-Liang; Yang, Xiao-Guang; Wang, Qi; Yu, Chun-Lei; Sun, Lu-Guo; Huang, Yan-Xin; Li, Yu-Xin

    2016-04-01

    Psoriasis is a multifactorial skin disease that inconveniences many patients. Considering the side effects and drug resistance of the current therapy, it is urgent to discover more effective and safer anti-psoriatic drugs. In the present study, we screened over 250 traditional Chinese medicine compounds for their ability to inhibit the cell viability of cultured human HaCaT keratinocytes, a psoriasis-relevant in vitro model, and found that periplogenin was highly effective. Mechanistic studies revealed that apoptosis and autophagy were not induced by periplogenin in HaCaT cells. However, periplogenin caused PI to permeate into cells, increased lactate LDH release and rapidly increased the number of necrotic cells. Additionally, the typical characteristics of necrosis were observed in the periplogenin-treated HaCaT cells. Notably, the necroptosis inhibitor Nec-1 and NSA were able to rescue the cells from necrotic cell death, supporting that necroptosis was involved in periplogenin-induced cell death. Furthermore, the ROS levels were elevated in the periplogenin-treated cells, NAC (an antioxidant) and Nec-1 could inhibit the ROS levels, and NAC could attenuate necroptotic cell death, indicating that the periplogenin-induced necroptotic cell death was mediated by oxidative stress. More importantly, in the murine models of TPA-induced epidermal hyperplasia and IMQ-induced skin inflammation, topical administration of periplogenin ameliorated skin lesions and inflammation. In sum, our results indicate, for the first time, that periplogenin is a naturally occurring compound with potent anti-psoriatic effects in vitro and in vivo, making it a promising candidate for future drug research. PMID:26850986

  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. COLONY FORMATION ENHANCEMENT OF RAT TRACHEAL AND NASAL EPITHELIAL CELLS BY POLYACETATE, INDOLE ALKALOID, AND PHORBOL ESTER TUMOR PROMOTERS

    EPA Science Inventory

    The phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), teleocidin, and two polyacetate tumor promoters (aplysiatoxin and debromoaplysiatoxin) have been tested for their effect on colony forming efficiency (CFE) of rat tracheal and nasal turbinate epithelial cells. In rat t...

  7. Antitumor-promoting activity of scopadulcic acid B, isolated from the medicinal plant Scoparia dulcis L.

    PubMed

    Nishino, H; Hayashi, T; Arisawa, M; Satomi, Y; Iwashima, A

    1993-01-01

    Scopadulcic acid B (SDB), a tetracyclic diterpenoid isolated from a medicinal plant, Scoparia dulcis L., inhibited the effects of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro and in vivo; SDB inhibited TPA-enhanced phospholipid synthesis in cultured cells, and also suppressed the promoting effect of TPA on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene. The potency of SDB proved to be stronger than that of other natural antitumor-promoting terpenoids, such as glycyrrhetinic acid. PMID:8451033

  8. Synergistic activation by serotonin and GTP analogue and inhibition by phorbol ester of cyclic Ca2+ rises in hamster eggs.

    PubMed Central

    Miyazaki, S; Katayama, Y; Swann, K

    1990-01-01

    1. Synergistic activation of a GTP-binding protein (G protein) by external serotonin (5-hydroxytryptamine, 5-HT) and internally applied guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) in hamster eggs was demonstrated by the facilitation of repetitive increases in cytoplasmic Ca2+ as measured by their associated hyperpolarizing responses (HRs) and by aequorin luminescence. 2. Rapid application of 70 nM-5-HT caused a single HR of 10-12 s duration and with a delay of 80 s. The critical concentration of 5-HT to cause an HR was 50 nM. 3. With 10 microM-5-HT four to six HRs were often elicited with a delay to the first HR of 8-30 s. HRs disappeared after prolonged or repeated application of 5-HT, indicating an apparent desensitization. 4. 5-HT-induced HRs were completely inhibited by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (TPA) (100 nM). Conversely, the PKC inhibitor sphingosine (2 microM) enhanced the series of HRs by shortening the delay to the first HR (3-9 s) and by causing more HRs. 5. Ionophoretic injection of GTP gamma S into the egg usually produced a large HR with a delay of 120-240 s followed by a series of much smaller HRs. When 5-HT was applied within 1 min of injection of GTP gamma S. 70 nM-5-HT induced a number of large HRs and even 1 nM-5-HT could induce HR(s). In contrast, when 5-HT was applied after the size of GTP gamma S-induced HRs had declined, as much as 10 microM-5-HT could only elicit a single large HR. Thus, GTP gamma S apparently caused a sensitization and then a desensitization of the action of 5-HT. 6. GTP gamma S-induced Ca2+ transients were facilitated when injected in the presence of 5-HT concentrations as low as 0.1 nM. The time delay to the first HR was 65 s in 0.1 nM-5-HT or 4 s in 100 nM-5-HT whereas it was 170 s without 5-HT (mean values). The magnitude as well as frequency of HRs succeeding the first HR was enhanced by 5-HT at concentrations above 0.01 nM. 7. TPA (100 nM) blocked the GTP gamma S-plus-5

  9. Studies on the mechanism of skin tumor promotion: evidence for several stages in promotion. [Mice

    SciTech Connect

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

    1980-06-01

    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. The results suggest that although mezerein by itself is a weak promotor and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen.

  10. 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. PMID:22729280

  11. [Inhibition of neutrophil adhesion by pectic galacturonans].

    PubMed

    Popov, S V; Ovodova, R G; Popova, G Iu; Nikitina, I R; Ovodov, Iu S

    2007-01-01

    The inhibition of the adhesion of neutrophils to fibronectin by the fragments of the main galacturonan chain of the following pectins was demonstrated: comaruman from the marsh cinquefoil Comarum polustre, bergenan from the Siberian tea Bergenia crassifolia, lemnan from the duckweed Lemna minor, zosteran from the seagrass Zostera marina, and citrus pectin. The parent pectins, except for comaruman, did not affect the cell adhesion. Galacturonans prepared from the starting pectins by acidic hydrolysis were shown to reduce the neutrophil adhesion stimulated by phorbol 12-myristate 13-acetate (1.625 microM) and dithiothreitol (0.5 mM) at a concentration of 50-200 microg/ml. The presence of carbohydrate chains with molecular masses higher than 300, from 100 to 300, and from 50 to 100 kDa in the galacturonan fractions was proved by membrane ultrafiltration. PMID:17375675

  12. Decrease in free-radical production with age in rat peritoneal macrophages.

    PubMed Central

    Alvarez, E; Conde, M; Machado, A; Sobrino, F; Santa Maria, C

    1995-01-01

    The respiratory-burst reaction has been studied in rat peritoneal macrophages of different ages (3, 12 and 24 months) using phorbol 12-myristate 13-acetate (PMA) to stimulate NADPH oxidase. Production of O2-. and H2O2 decreased with age (about 50 and 75% respectively); however, no difference in NADPH oxidase activity was found. NO. production was also reduced with age (40%). Furthermore, a progressive and significant decrease in the pentose phosphate flux was detected as a function of age in control and PMA-stimulated macrophages. The NADPH/NADP+ ratio decreased with age in control and PMA-stimulated macrophages. Glucose uptake was lower in middle-aged (12 months) and old (24 months) animals but no differences were found between these groups. PMID:8526870

  13. 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.

  14. The in vitro biological effect of nerve growth factor is inhibited by synthetic peptides.

    PubMed Central

    Longo, F M; Vu, T K; Mobley, W C

    1990-01-01

    Nerve growth factor (NGF)1 is a neurotrophic polypeptide that acts via specific receptors to promote the survival and growth of neurons. To delineate the NGF domain(s) responsible for eliciting biological activity, we synthesized small peptides corresponding to three regions in NGF that are hydrophilic and highly conserved. Several peptides from mouse NGF region 26-40 inhibited the neurite-promoting effect of NGF on sensory neurons in vitro. Inhibition was sequence-specific and could be overcome by increasing the concentration of NGF. Moreover, peptide actions were specific for NGF-mediated events in that they failed to block the neurotrophic activity of ciliary neuronotrophic factor (CNTF) or phorbol 12-myristate 13-acetate (PMA). In spite of the inhibition of NGF activity, peptides did not affect the binding of radiolabeled NGF. These studies define one region of NGF that may be required for neurotrophic activity. Images PMID:2100197

  15. Cytoprotection against neutrophil-delivered oxidant attack by antibiotics.

    PubMed

    Ottonello, L; Dallegri, F; Dapino, P; Pastorino, G; Sacchetti, C

    1991-11-27

    In the present study we have investigated the effect of six antibiotics (penicillin G, ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin) on the neutrophil cytolytic activity by using a system constituted of phorbol-12-myristate-13-acetate-triggered neutrophils and 51Cr-labelled lymphoblastoid Daudi target cells. The results demonstrate that five of these drugs (ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin) are capable of inhibiting the neutrophil cytolytic activity by inactivating the hypochlorous acid (HOCl) generated extracellularly by the myeloperoxidase pathway and crucial to the target cell lysis. Penicillin G had no effect on neutrophil-mediated cytolysis. Thus, these data demonstrate that ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin lower the neutrophil-mediated target cell damage by a HOCl-scavenging mechanism, suggesting a possible cytoprotective role for these drugs during infections. PMID:1662510

  16. 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.

  17. External Application of Apo-9'-fucoxanthinone, Isolated from Sargassum muticum, Suppresses Inflammatory Responses in a Mouse Model of Atopic Dermatitis.

    PubMed

    Han, Sang-Chul; Kang, Na-Jin; Yoon, Weon-Jong; Kim, Sejin; Na, Min-Chull; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2016-04-01

    Allergic skin inflammation such as atopic dermatitis is characterized by skin barrier dysfunction, edema, and infiltration with various inflammatory cells. The anti-inflammatory effects of Apo-9'-fucoxanthinone, isolated from Sargassum muticum, have been described in many diseases, but the mechanism by which it modulates the immune system is poorly understood. In this study, the ability of Apo-9'-fucoxanthinone to suppress allergic reactions was investigated using a mouse model of atopic dermatitis. The Apo-9'-fucoxanthinone-treated group showed significantly decreased immunoglobulin E in serum. Also, Apo-9'-fucoxanthinone treatment resulted in a smaller lymph node size with reduced the thickness and length compared to the induction group. In addition, Apo-9'-fucoxanthinone inhibited the expression of interleukin-4, interferon-gamma and tumor necrosis factor-alpha by phorbol 12-myristate 13-acetate and ionomycin-stimulated lymphocytes. These results suggest that Apo-9'-fucoxanthinone may be a useful therapeutic strategy for treating chronic inflammatory diseases. PMID:27123161

  18. Effects of phorbol esters on adrenergic receptors of DDT MF-2 smooth muscle cells

    SciTech Connect

    Cowlen, M.; Toews, M.

    1986-03-05

    Phorbol esters have been reported to induce redistribution or internalization of several types of cell surface receptors, including beta-adrenergic receptors (BAR) in some cells. They investigated the effects of phorbol esters on adrenergic receptor distribution in DDT/sub 1/ MF-2 smooth muscle cells in suspension culture. Exposure of cells to epinephrine, an agonist for both BAR and alpha-1 adrenergic receptors (AAR), led to a shift of about half of BAR from plasma membrane to light vesicle fractions on sucrose density gradient centrifugation. This change correlates with other evidence for internalization or sequestration of BAR away from the cell surface. AAR distribution remained unaltered following agonist treatment. Pretreatment of cells with phorbol 12-myristate 13-acetate, which caused about 80% inhibition of epinephrine-stimulated turnover of inositol phospholipids, did not lead to redistribution of either BAR or AAR.

  19. Special type of morphological reorganization induced by phorbol ester: reversible partition of cell into motile and stable domains

    SciTech Connect

    Dugina, V.B.; Svitkina, T.M.; Vasiliev, J.M.; Gelfand, I.M.

    1987-06-01

    The phorbol ester phorbol 12-myristate 13-acetate (PMA) induced reversible alteration of the shape of fibroblastic cells of certain transformed lines-namely, partition of the cells into two types of domains: motile body actively extending large lamellas and stable narrow cytoplasmic processes. Dynamic observations have shown that stable processes are formed from partially retracted lamellas and from contracted tail parts of cell bodies. Immunofluorescence microscopy and electron microscopy of platinum replicas of cytoskeleton have shown that PMA-induced narrow processes are rich in microtubules and intermediate filaments but relatively poor in actin microfilaments; in contrast, lamellas and cell bodies contained numerous microfilaments. Colcemid-induced depolymerization of microtubules led to contraction of PMA-induced processes; cytochalasin B prevented this contraction. It is suggested that PMA-induced separation of cell into motile and stable parts is due to directional movement of actin structures along the microtubular framework. Similar movements may play an important role in various normal morphogenetic processes.

  20. 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.

  1. 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.

  2. Effects of maglev-spectrum magnetic field exposure on CEM T-lymphoblastoid human cell growth and differentiation

    SciTech Connect

    Groh, K.R.; Chubb, C.B.; Collart, F.R.; Huberman, E.

    1992-01-01

    Exposure to magnetic fields similar to those produced by maglev vehicles (combined ac and dc components) was studied for the ability to alter cell growth and chemically induced cellular differentiation processes in cultured human CEM Tlymphoblastoid leukemia cells. A series of continuous and intermittent magnetic field (MF) exposures for varying lengths of time were tested at intensities up to 7-fold greater than that produced by the German TR07 maglev vehicle. Phorbol 12-myristate 13-acetate or mycophenolic acid were used to induce cell differentiation. Changes in cell number, morphology, and fluorescence expression of antigenic markers of differentiation were monitored. The results indicated that maglev-spectrum magnetic field exposures up to 2 gauss had little effect on culture growth or chemically induced cellular differentiation when exposed to maglev-spectrum magnetic fields compared to chemically treated but MF-unexposed controls.

  3. 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

  4. Comparative study of histamine H4 receptor expression in human dermal fibroblasts.

    PubMed

    Ikawa, Yoshiko; Shiba, Kayoko; Ohki, Emi; Mutoh, Nanami; Suzuki, Masahiko; Sato, Hiromi; Ueno, Koichi

    2008-10-01

    The histamine H4 receptor (H4R) is the newest receptor identified of four histamine receptors. Its expression in numerous immune and inflammatory organs has been implicated in relation to immune systems and allergic diseases. In the present study, we demonstrate the expression of H4R in human dermal fibroblasts and investigate changes in its expression level when stimulated by histamine, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), dexamethasone and indomethacin. Histamine and PMA showed no effects on H4R expression. LPS and indomethacin up-regulated H4R mRNA expression, and 20 microM dexamethasone increased H4R protein levels. These results indicate a good prospective for this new receptor in the development of effective treatments of inflammatory diseases and pruritus or for the appropriate prevention of toxicities. PMID:18827451

  5. Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy

    PubMed Central

    Weber, Petra; Schickinger, Sarah; Wagner, Michael; Angres, Brigitte; Bruns, Thomas; Schneckenburger, Herbert

    2015-01-01

    Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure. PMID:25761242

  6. A region of the rat N-methyl-D-aspartate receptor 2A subunit that is sufficient for potentiation by phorbol esters.

    PubMed

    Grant, E R; Guttmann, R P; Seifert, K M; Lynch, D R

    2001-09-01

    N-methyl-D-aspartate (NMDA) receptors are modulated by protein kinase C (PKC) in vivo and in heterologous expression systems. In heterologous expression systems, PKC-mediated modulation is subunit specific with NR2A-containing receptors being potentiated by phorbol 12-myristate 13-acetate (PMA), while NR2C-containing receptors are inhibited or unaffected. In the present study we have produced chimeric receptors containing NR2A and NR2C to define the components of NR2A which are sufficient for potentiation by PMA. Amino acids 1105-1400 of NR2A placed onto the C-terminus of NR2C at amino acid 1102 was the minimum region sufficient for producing a PMA-stimulated receptor. This suggests that this region contains structural determinants for PKC-mediated potentiation of NR2A receptors. PMID:11524145

  7. Transrepression function of the glucocorticoid receptor regulates eyelid development and keratinocyte proliferation but is not sufficient to prevent skin chronic inflammation.

    PubMed

    Donet, Eva; Bosch, Pilar; Sanchis, Ana; Bayo, Pilar; Ramírez, Angel; Cascallana, José L; Bravo, Ana; Pérez, Paloma

    2008-04-01

    Glucocorticoids (GCs) play a key role in skin homeostasis and stress responses acting through the GC receptor (GR), which modulates gene expression by DNA binding-dependent (transactivation) and -independent (transrepression) mechanisms. To delineate which mechanisms underlie the beneficial and adverse effects mediated by GR in epidermis and other epithelia, we have generated transgenic mice that express a mutant GR (P493R, A494S), which is defective for transactivation but retains transrepression activity, under control of the keratin 5 promoter (K5-GR-TR mice). K5-GR-TR embryos exhibited eyelid opening at birth and corneal defects that resulted in corneal opacity in the adulthood. Transgenic embryos developed normal skin, although epidermal atrophy and focal alopecia was detected in adult mice. GR-mediated transrepression was sufficient to inhibit keratinocyte proliferation induced by acute and chronic phorbol 12-myristate 13-acetate exposure, as demonstrated by morphometric analyses, bromodeoxyuridine incorporation, and repression of keratin 6, a marker of hyperproliferative epidermis. These antiproliferative effects were mediated through negative interference of GR with MAPK/activator protein-1 and nuclear factor-kappaB activities, although these interactions occurred with different kinetics. However, phorbol 12-myristate 13-acetate-induced inflammation was only partially inhibited by GR-TR, which efficiently repressed IL-1beta and MMP-3 genes while weakly repressing IL-6 and TNF-alpha. Our data highlight the relevance of deciphering the mechanisms underlying GR actions on epithelial morphogenesis as well as for its therapeutic use to identify more restricted targets of GC administration. PMID:18174358

  8. 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

  9. Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators.

    PubMed

    Cerda, S R; Mustafi, R; Little, H; Cohen, G; Khare, S; Moore, C; Majumder, P; Bissonnette, M

    2006-05-25

    PKC-delta is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-delta slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-delta dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-delta using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-delta caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21(Waf1), a cyclin-dependent kinase (cdk) inhibitor in PKC-delta transfectants compared with empty vector (EV) transfected cells, whereas the PKC-delta specific inhibitor rottlerin (3 microM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21(Waf1) expression. Concomitantly, compared to EV control cells, PKC-delta upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-delta increased binding of cdk inhibitor p27(Kip1) to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-delta plays in cell growth and cell cycle regulation, we knocked down PKC-delta using specific siRNA oligonucleotides. PKC-delta specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-delta protein by more than 80% in Caco-2 cells. Moreover, PKC-delta knockdown enhanced cell proliferation ( approximately 1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression ( approximately 1.7-fold, P<0.05). This was a specific

  10. The effect of phorbols on metabolic cooperation between human fibroblasts

    SciTech Connect

    Mosser, D.D.; Bols, N.C.

    1982-01-01

    Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of (/sup 3/H)hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total (/sup 3/H)hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of (/sup 3/H)nucleotides and is an indication of an interference with intercellular communication.

  11. 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

  12. 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. PMID:24734106

  13. Total synthesis of plagiochin G and derivatives as potential cancer chemopreventive agents

    PubMed Central

    Li, Rui-Juan; Zhao, Yu; Tokuda, Harukuni; Yang, Xiao-Ming; Wang, Yue-Hu; Shi, Qian; Morris-Natschke, Susan L.; Lou, Hong-Xiang; Lee, Kuo-Hsiung

    2014-01-01

    A new and efficient total synthesis has been developed to obtain plagiochin G (22), a macrocyclic bisbibenzyl, and four derivatives. The key 16-membered ring containing biphenyl ether and biaryl units was closed via an intramolecular SNAr reaction. All synthesized macrocyclic bisbibenzyls inhibited Epstein-Barr virus early antigen (EBVEA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells and, thus, are potential cancer chemopreventive agents. PMID:25574060

  14. Protein kinase C is involved in regulation of Ca2+ channels in plasmalemma of Nitella syncarpa.

    PubMed

    Zherelova, O M

    1989-01-01

    Ca2+ current recordings have been made on Nitella syncarpa cells using the intracellular perfusion and the voltage-clamp technique. TPA (12-O-tetradecanoylphorbol-13-acetate), a substance capable of activating protein kinase C from plasmalemma of Nitella cells, modulates voltage-dependent Ca2+ channels. Polymixin B, inhibitor of protein kinase C, blocks the Nitella plasmalemma Ca2+ channels; the rate of channel blockage depends on the concentration and exposure time of the substance. PMID:2536617

  15. 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. PMID:19394719

  16. Neutral Sphingomyelinase 2 (nSMase2) Is a Phosphoprotein Regulated by Calcineurin (PP2B)*

    PubMed Central

    Filosto, Simone; Fry, William; Knowlton, Anne A.; Goldkorn, Tzipora

    2010-01-01

    We previously reported that exposure of human airway epithelial cells to oxidative stress increased ceramide generation via specific activation of neutral sphingomyelinase2 (nSMase2). Here we show that nSMase2 is a phosphoprotein exclusively phosphorylated at serine residues. The level of nSMase2 phosphorylation can be modulated by treatment with anisomycin or phorbol 12-myristate 13-acetate (PMA/12-O-tetradecanoylphorbol-13-acetate), suggesting that p38 mitogen-activated protein kinase (MAPK) and protein kinases Cs are upstream of nSMase2 phosphorylation. Oxidative stress enhances both the activity and phosphorylation of nSMase2. Strikingly, we show here that nSMase2 is bound directly by the phosphatase calcineurin (CaN), which acts as an on/off switch for nSMase2 phosphorylation in the presence or absence of oxidative stress. Specifically, CaN is being inhibited/degraded and therefore does not bind nSMase2 under oxidative stress, and a mutant nSMase2 that lacks the CaN binding site exhibits constitutively elevated phosphorylation and increased activity relative to wild type nSMase2. Importantly, the phosphorylation and activity of the mutant no longer responds to oxidative stress, confirming that CaN is the critical link that allows oxidative stress to modulate nSMase2 phosphorylation and function. PMID:20106976

  17. Increased endocytosis and formation of multivesicular bodies in phorbol-ester-stimulated human monoblastic U-937 cells

    SciTech Connect

    Nilsson, M. ); Nilsson, K.; Forsbeck, K. )

    1989-04-01

    The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to arrest mitotic activity and induce macrophage differentiation in the U-937 monoblastic cell line. The acute effect of TPA on ultrastructural morphology and endocytic activity of U-937 cells was studied. TPA induced within 15 minutes {alpha} marked enlargement of multivesicular bodies (MVBs), comprising both volume and number of inclusion vesicles (other organelles appeared unchanged). At this stage the MVBs frequently showed tubular cytoplasmic extensions. Inclusion vesicles accumulated in MVBs with prolonged incubation (60 minutes). Cellular uptake of {sup 125}I-HRP was increased five times the control values already after 5 minutes of TPA stimulation. The uptake increased further with prolonged incubation (60 minutes), but at a slower rate. Together these indicate a TPA-induced transfer by endocytosis of portions of the plasma membrane to the lysosomal system via MVBs. Consideration of MVBs as part of the receptor-mediated endocytic pathway suggests that this effect of TPA might involve down-regulation of cell-surface receptors. The possibility of MVBs as a proton-sequestrating compartment, responsible for the cytoplasmic alkalinization previously reported for TPA-stimulated U-937 monoblastic cells, is discussed.

  18. Stroke, tPA, and Physician Decision-Making

    MedlinePlus

    ... blood vessel become starved for oxygen. When a part of the brain is starved for oxygen, it is called ischemia ... are several arteries that bring blood to the brain, but the middle cerebral artery is a major artery that supplies ...

  19. Plasma application for detoxification of Jatropha phorbol esters

    NASA Astrophysics Data System (ADS)

    Kongmany, S.; Matsuura, H.; Furuta, M.; Okuda, S.; Imamura, K.; Maeda, Y.

    2013-06-01

    Atmospheric pressure non-thermal dielectric barrier discharge (DBD) plasma generated by helium gas at high voltage and input power of about 50 W was first applied to detoxification of Jatropha curcas phorbol esters (J. PEs) as well as standard phorbol ester (4β-12-O-tetradecanoyl phorbol-13-acetate, TPA) in water and methanol. Plasma irradiation on the solution sample was conducted for 15 min. In aqueous solution, only 16% of TPA was degraded and complete degradation of J. PEs was observed. On the contrary, complete degradation of both TPA and J. PEs in methanol was achieved by the same plasma irradiation condition. Hydroxyl radical (•OH) generated by plasma irradiation of the solution is expected as the main radical inducing the degradation of PEs.

  20. Regulation of acid phosphatase activity in human promyelocytic leukemic cells induced to differentiate in culture

    PubMed Central

    1979-01-01

    Induction of differentiation of a human promyelocytic leukemic cell line (HL60) in culture is accompanied by changes in acid phosphatase (Acpase) activity. The increase in activity is less than twofold when the leukemic cells are stimulated by dimethylsulfoxide (DMSO) to differentiate into metamyelocytes and granulocytes but is eightfold when the cells are stimulated by the tumor-promoting agent 12-0- tetradecanoylphorbol 13-acetate (TPA) to differentiate into macrophage- like cells. Five different isozymes of Acpase were separated by acrylamide gel electrophoresis. Isozyme 1, the most anodal isozyme, was found to be present in undifferentiated, DMSO-treated and TPA-treated cells; isozyme 2 was a very faint band observed both in DMSO- and TPA- treated cells, the isoenzymes 3a and 3b were present only in TPA- induced cells; and isozyme 4, the most cathodal isozyme, was present both in TPA- and DMSO-induced cells. A time sequence study on the appearance of the various forms after TPA treatment indicated that the expression of the isozymes is regulated in an uncoordinated fashion. Acpase activity has been shown by ultrastructural cytochemistry to be localized in the entire rough endoplasmic reticulum (RER) and in areas of the smooth endoplasmic reticulum (SER) located near the Golgi complex in differentiating cells but to be extremely weak, if at all detectable, in undifferentiated promyelocytes. PMID:291600

  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. Protein kinase C is involved in stimulation of arachidonic acid metabolism in Madin-Darby canine kidney (MDCK) cells

    SciTech Connect

    Parker, J.; Daniel, L.W.; Waite, M.

    1986-05-01

    The authors used 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to directly stimulate protein kinase C (PKC) in order to examine the role of PKC in transduction of biological signals that increase metabolism of arachidonic acid. Release of radioactive arachidonic acid and prostaglandins from TPA-stimulated MDCK cells is inhibited by either of two PKC inhibitors: 1-(5-isoquinolinesulfonyl)piperazine and 1-octadecyl-2-methoxy-glycero-3-phosphocholine (ALP). ALP is unable to inhibit cyclooxygenase when added into an in vitro assay for this enzyme. Furthermore, TPA induces de novo synthesis of cyclooxygenase in MDCK cells but ALP fails to prevent this effect of TPA. Thus, cyclooxygenase activity appears to be independent of PKC and TPA can still induce de novo synthesis of cyclooxygenase even in the presence of the PKC inhibitor ALP. Also, ALP has no effect on the release of arachidonic acid which occurs upon addition of the calcium ionophore A23187 to MDCK cells suggesting that there are multiple mechanisms to mobilize arachidonic acid. Their data indicate that activation of PKC by TPA leads to increased release of arachidonic acid through regulation of phospholipase(s) by PKC.

  3. Role of the Slug Transcription Factor in Chemically-Induced Skin Cancer

    PubMed Central

    von Maltzan, Kristine; Li, Yafan; Rundhaug, Joyce E.; Hudson, Laurie G.; Fischer, Susan M.; Kusewitt, Donna F.

    2016-01-01

    The Slug transcription factor plays an important role in ultraviolet radiation (UVR)-induced skin carcinogenesis, particularly in the epithelial-mesenchymal transition (EMT) occurring during tumor progression. In the present studies, we investigated the role of Slug in two-stage chemical skin carcinogenesis. Slug and the related transcription factor Snail were expressed at high levels in skin tumors induced by 7,12-dimethylbenz[α]anthracene application followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. TPA-induced transient elevation of Slug and Snail proteins in normal mouse epidermis and studies in Slug transgenic mice indicated that Slug modulates TPA-induced epidermal hyperplasia and cutaneous inflammation. Although Snail family factors have been linked to inflammation via interactions with the cyclooxygenase-2 (COX-2) pathway, a pathway that also plays an important role in skin carcinogenesis, transient TPA induction of Slug and Snail appeared unrelated to COX-2 expression. In cultured human keratinocytes, TPA induced Snail mRNA expression while suppressing Slug expression, and this differential regulation was due specifically to activation of the TPA receptor. These studies show that Slug and Snail exhibit similar patterns of expression during both UVR and chemical skin carcinogenesis, that Slug and Snail can be differentially regulated under some conditions and that in vitro findings may not recapitulate in vivo results. PMID:26848699

  4. Phorbol ester-stimulated phosphorylation of basolateral membranes from canine kidney

    SciTech Connect

    Hammerman, M.R.; Rogers, S.; Morrissey, J.J.; Gavin, J.R. III

    1986-06-01

    To determine whether protein kinase C is present in the basolateral membrane of the renal proximal tubular cell, we performed experiments to ascertain whether specific binding of (/sup 3/H)phorbol 12,13-dibutyrate could be demonstrated in basolateral membranes isolated from canine kidney. Specific binding was demonstrable that was half maximal at between 10(-7) and 10(-8) M phorbol 12,13-dibutyrate. Binding was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other tumor-promoting phorbol esters, but not by inactive phorbol esters, including 4 alpha-phorbol. Incubation of basolateral membranes with TPA and phorbol 12,13-dibutyrate, but not with 4 alpha-phorbol, in the presence of submicromolar concentrations of free calcium, enhanced phosphorylation of several proteins demonstrable in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels originating from membranes subsequently exposed to (gamma-32P)ATP for 30 s. Dephosphorylation of (/sup 32/P)phosphoproteins was observed in gels from membranes incubated with (gamma-32P)ATP over time. TPA-stimulated phosphorylation of one protein band with Mr 135,000 was quantitated and was found to increase as a function of (TPA). Half-maximal TPA-stimulated phosphorylation of this protein band occurred at slightly less than 10(-9) M TPA. Our findings are consistent with a role for protein kinase C-effected phosphorylation of basolateral membrane proteins in the mediation or modulation of hormonal actions in the proximal tubular cell.

  5. Phorbol ester tumor promoter induced the synthesis of two major cytoplasmic proteins: identity with two proteins induced under heat-shocked and glucose-starved conditions

    SciTech Connect

    Zhang, H.; Chen, K.Y.; Liu, A.Y.C.

    1987-05-01

    The regulation of specific protein synthesis by the phorbol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was evaluated using the L-8 and C-2 myoblast and the 3T3-L1 fibroblast cell cultures. TPA increased, by 2-4 fold, the synthesis rates of two cytoplasmic proteins with apparent molecular weights of 89,000 and 74,000 as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. The concentration of TPA and the time of incubation needed to elicit this induction was determined to be 10 ..mu..g/ml and 20 hrs, respectively. Increasing the concentration of TPA to 100, 200, and 500 ng/ml did not result in a greater magnitude of induction. The possibility that these two TPA-induced proteins may be identical to proteins with similar molecular weights induced under heat-shocked or glucose-starved conditions was evaluated by 1-D and 2-D gel electrophoresis and autoradiography. Results provided evidence that the TPA-induced 89,000- and 74,000-dalton proteins were identical to hsp 89 and hsp 74, 2 out of a set of 8-9 proteins induced under heat shocked conditions. Furthermore, they are identical to two of the set of glucose-regulated proteins induced under a glucose-starved condition.

  6. Human serum amyloid A genes are expressed in monocyte/macrophage cell lines.

    PubMed Central

    Urieli-Shoval, S.; Meek, R. L.; Hanson, R. H.; Eriksen, N.; Benditt, E. P.

    1994-01-01

    Serum amyloid A (apoSAA) is a family of proteins found, mainly associated with high density lipoproteins, in the blood plasma of mammals and at least one avian species, the Pekin duck. These proteins are present in small amounts under normal circumstances, but their concentration is capable of rising 100- to 1,000-fold in situations involving tissue injury or infection. Like classic acute phase proteins they are produced in the liver; however, expression of one of the apoSAA genes is known to occur in activated macrophages of mice. We examined three human macrophage precursor cell lines (THP-1, U-937, and HL-60), before and after differentiation with phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxy-vitamin D3, for apoSAA messenger (m)-RNA expression and found that: 1) induction of steady-state apoSAA mRNA by lipopolysaccharide, interleukin-1, or interleukin-6 required the presence of the synthetic glucocorticoid dexamethasone; 2) the three known active genes, apoSAA1, apoSAA2, and apoSAA4, were induced in THP-1 cells, whereas the pseudogene apoSAA3 was not; 3) differentiated and undifferentiated THP-1 cells expressed apoSAA mRNA, but U-937 cells expressed apoSAA mRNA (low levels) only after phorbol 12-myristate 13-acetate differentiation and HL-60 cells did not express apoSAA mRNA whether differentiated or not; 4) apoSAA protein was detectable immunologically at a low level in lyophilized medium from induced THP-1 cells. Our findings are compatible with the hypotheses that 1) apoSAA gene expression in human monocytes/macrophages in vivo is differentiation dependent; 2) activated macrophages provide a local source of apoSAA at sites of tissue injury or inflammation; 3) apoSAA is induced in tissue macrophages by local stimuli, under conditions that may not evoke the systemic acute phase response. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8080047

  7. Modulation of human basophil histamine release by protein kinase C inhibitors differs with secretagogue and with inhibitor.

    PubMed

    Bergstrand, H; Lundquist, B; Karabelas, K; Michelsen, P

    1992-03-01

    To assess possible involvement of protein kinase C (PKC) in human basophil degranulation, the present work compared effects of various purported PKC inhibitors on leukocyte histamine release triggered by different stimuli. The effects recorded varied with the inhibitor and the secretagogue used; moreover, with a given secretagogue, different inhibitors often displayed different activities. Thus, histamine release triggered by the PKC activator 4 beta-phorbol 12-myristate 13-acetate was blocked by K252a, staurosporine and the purported specific PKC inhibitor Ro 31-7549, and reduced by calphostin C, H-7, TMB-8 and W-7 but not affected by polymyxin B; it was augmented by 2.1 microM palmitoyl carnitine. The leukocyte response induced by another putative activator of PKC, 1,2-isopropylidene-3-decanoyl-sn-glycerol, was also enhanced by 2.1 microM palmitoyl carnitine, slightly increased by staurosporine, TMB-8 and W-7 but not affected by calphostin C, H-7, K252a or Ro 31-7549, whereas the hyperosmolar mannitol-induced response was reduced by H-7, calphostin C, TMB-8 and W-7 and slightly augmented by staurosporine. Anti-IgE-induced histamine release was blocked by staurosporine and K252a and reduced by calphostin C, sphingosine, TMB-8 and W-7 but not affected by H-7, polymyxin B or retinal. It was enhanced by Ro 31-7549. In contrast, leukocyte histamine release induced by calcium ionophore A23187 or by ionomycin was blocked by retinal, TMB-8 and W-7 and reduced by calphostin C and palmitoyl carnitine but enhanced by H-7, staurosporine and polymyxin B; K252a and Ro 31-7549 did not affect such responses. Formyl-methionyl-leucyl-phenylalanine-triggered histamine release was barely affected by any agent used. Thus, the specific PKC inhibitor Ro 31-7549 selectively blocked 4 beta-phorbol 12-myristate 13-acetate-triggered leukocyte histamine release. These results imply that examined secretagogues trigger human leukocyte histamine release through partly separate pathways

  8. Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture

    SciTech Connect

    Laszlo, A.; Radke, K.; Chin, S.; Bissell, M.J.

    1981-10-01

    We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response to TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment (Bissell, M.J., Hatie, C. and Calfin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352). Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.

  9. Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil.

    PubMed

    Pelletier, Guillaume; Padhi, Bhaja K; Hawari, Jalal; Sunahara, Geoffrey I; Poon, Raymond

    2015-06-01

    New health safety concerns may arise from the increasing production and use of Jatropha oil, a biodiesel feedstock that also contains toxic, pro-inflammatory, and co-carcinogenic phorbol esters. Based on the exceptional sensitivity of Madin-Darby canine kidney (MDCK) cells to the model phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a robust bioassay was developed to quantify the biological activity of Jatropha phorbol esters directly in oil, without sample extraction. We first verified that the characteristic response of MDCK cells to TPA was also observed following direct exposure to phorbol esters in Jatropha oil. We further confirmed that similarly to TPA, Jatropha oil's phorbol esters can activate protein kinase C (PKC). We then assessed the transcriptional response of MDCK cells to Jatropha oil exposure by measuring the expression of cyclooxygenase-2 (COX-2), a gene involved in inflammatory processes which is strongly upregulated following PKC activation. Based on the parameterization of a TPA dose-response curve, the transcriptional response of MDCK cells to Jatropha oil exposure was expressed in term of TPA toxic equivalent (TEQ), a convenient metric to report the inflammatory potential of complex mixtures. The sensitive bioassay described in this manuscript may prove useful for risk assessment, as it provides a quantitative method and a convenient metric to report the inflammatory potential of phorbol esters in Jatropha oil. This bioassay may also be adapted for the detection of bioactive phorbol esters in other matrices. PMID:25588777

  10. 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. PMID:23983093

  11. 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.

  12. 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.

  13. Modulation of the Tumor Metastatic Microenvironment and Multiple Signal Pathways by Prunella vulgaris in Human Hepatocellular Carcinoma.

    PubMed

    Su, Yu-Chieh; Lin, I-Hsin; Siao, Yu-Miao; Liu, Ching-Ju; Yeh, Chia-Chou

    2016-01-01

    Prunella vulgaris (PV) is a traditional Chinese medicine that has been used clinically for centuries in Asian countries to treat herpetic keratitis. In previous studies, PV was shown to suppress TPA-induced activation of MMP-9 and inhibit cell invasion and migration in hepatoma cell lines. However, the detailed molecular mechanism underlying these effects is still unclear. In this study, we investigated the mechanisms underlying PV-mediated inhibition of 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced cell invasion and inhibition of secreted and cytosolic MMP-9 production in human hepatoma cells (Huh-7 and HA22T). PV suppressed VEGF and MMP-9 transcription by inhibiting activator protein (AP)-1 and nuclear factor-[Formula: see text]B (NF-[Formula: see text]B) activity. PV suppressed TPA-induced AP-1 activity by inhibiting phosphorylation of the extracellular signal-related kinase (ERK), downregulating p38 signaling pathways, and suppressing TPA-induced inhibition of NF-[Formula: see text]B nuclear translocation through I[Formula: see text]B. PV suppressed TPA-induced activation of ERK/phosphatidylinositol-3-kinase/Akt upstream of NF-[Formula: see text]B and AP-1. These data suggest that PV modifies the metastatic microenvironment of hepatocellular carcinoma (HCC) by inhibiting multiple signal transduction pathways. PV thus may have the therapeutic potential to inhibit the migration and invasion of HCC and act as potential agent for systemic therapies. PMID:27222069

  14. 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. PMID:8552199

  15. 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. PMID:15158981

  16. 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.

  17. Chemically induced skin carcinogenesis in a transgenic mouse line (TG.AC) carrying a v-Ha-ras gene.

    PubMed

    Spalding, J W; Momma, J; Elwell, M R; Tennant, R W

    1993-07-01

    A transgenic mouse line (TG.AC) created in the FVB/N strain, carries a v-Ha-ras gene fused to a zeta-globin promoter gene. These trangenic mice have the properties of genetically initiated skin and have been shown to be sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA), a well-described promoter of skin papillomas in the two-stage mouse skin tumorigenesis model. It was of interest to determine whether the TG.AC mouse strain was also responsive to other known promoters. Groups of heterozygous or homozygous TG.AC mice were treated topically, 2x/week, for up to 20 weeks with benzoyl peroxide (BPO), 2-butanol peroxide (2-BUP), phenol (PH), acetic acid (AA), TPA and acetone (ACN), the vehicle control. Skin papillomas were induced in all groups treated with TPA, BPO and 2-BUP. Papillomas were observed in some treatment groups as early as 3 weeks. The relative activity of the promoters was TPA > 2-BUP > BPO > PH = AA = ACN. No papillomas were observed in any of the uninitiated FVB/N mice treated in a similar manner and which served as treatment control groups. Studies to determine the sensitivity of TG.AC mice to TPA, indicated that a total dose of 25-30 micrograms of TPA administered in 3 or 10 applications, was sufficient to induce an average incidence of 11-15 papillomas per mouse. The papilloma incidence continued to increase and was maintained up to 15 weeks after TPA treatment was terminated. The short latency period and high incidence of papilloma induction indicate that TG.AC mice have a high sensitivity to known skin promoters. The TG.AC line should prove to be a sensitive model for identifying putative tumor promoters or complete carcinogens. PMID:8330346

  18. 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

  19. Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

    PubMed Central

    Nishizawa, N; Okano, Y; Chatani, Y; Amano, F; Tanaka, E; Nomoto, H; Nozawa, Y; Kohno, M

    1990-01-01

    We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation. Images PMID:2129194

  20. Malignant conversion and metastasis of mouse skin tumors: a comparison of SENCAR and CD-1 mice

    SciTech Connect

    Hennings, H.; Spangler, E.F.; Shores, R.; Mitchell, P.; Devor, D.; Shamsuddin, A.K.M.; Elgjo, K.M.; Yuspa, S.H.

    1986-09-01

    The progression of papillomas to squamous cell carcinomas (malignant conversion) was studied in the skin of SENCAR and Charles River CD-1 mice, using a three-stage treatment protocol. After initiation with 7,12-dimethylbenz(a)anthracene (DMBA) (stage I) and limited promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) (stage II), papilloma-bearing mice were treated (stage III) with either tumor initiators, such as urethane, N-methyl-N'nitro-N nitrosoguanidine (MNNG) or 4-nitroquinoline-n-oxide (R-NQO), the promoter TPA, or solvent (acetone). Similar final carcinoma yields were found in the mice treated in stage III with TPA or acetone, although carcinomas developed earlier in the TPA-treated mice. In contrast, treatment with tumor initiators in stage III increased both the rate of appearance and the final yield of carcinomas. Similar results were obtained in both SENCAR and CD-1 mice. A papilloma stage appears to be necessary for carcinoma development since elimination of TPA treatment in stage II greatly reduced the incidence of both papillomas and carcinomas in both stocks of mice. The heterogeneity of papillomas with regard to progression to carcinomas is demonstrated by the low rate of conversion of TPA-dependent papillomas and the high rate of conversion of persistent papillomas in CD-1 mice. The carcinomas that develop using the three-stage regimen vary in metastatic potential. In CD-1 mice, the frequency of metastases to lymph nodes were similar in groups treated in stage III with MNNG, urethane, 4-NQO, TPA, or acetone, but treatment with urethane substantially increased metastases to the lung. In SENCAR mice, this effect of urethane was not observed, but lymph node and lung metastases appeared too be increased by stage III treatment with MNNG.

  1. Antioxidants inhibit the enhancement of malignant cell transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Wölfle, D; Marquardt, H

    1996-06-01

    The mechanisms of the tumor promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied using as in vitro model the enhancement ('promotion') of malignant transformation of C3H/M2 mouse fibroblasts induced by N-methyl-N'-nitro-N-nitrosoguanidine or 3-methylcholanthrene. In this assay, the promoting effect of TCDD was maximal at a very low concentration of 1.5 pM and was comparable to the effect of the reference tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.25 microg/ml). The role of reactive oxygen species in the promoting action was investigated: mannitol, a scavenger of hydroxyl radicals, or antioxidants, i.e. ascorbic acid plus alpha-tocopherol, abolished the in vitro promoting effects of TPA and TCDD. Furthermore, the involvement of protein kinase C (PKC) activation was studied: the protein kinase inhibitor H-7 markedly reduced the in vitro promoting activity of TPA but did not affect the promotion by TCDD. In accord with these results, TPA, but not TCDD, enhanced the PKC activity in C3H/M2 fibroblasts. Since the TPA-mediated activation of PKC was not affected by ascorbate plus alpha-tocopherol, it is concluded that the antioxidants interfere with tumor promotion at a step beyond PKC activation. Thus, the results suggest that the enhancement of malignant cell transformation by TPA and TCDD is dependent on a common mechanism, possibly induced by oxygen radicals, and, in addition, on further mechanisms that may involve agent-specific signalling pathways (e.g. PKC activation by TPA). PMID:8681442

  2. 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.

  3. 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

  4. 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.

  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. 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).

  7. 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.

  8. Damnacanthal inhibits the NF-κB/RIP-2/caspase-1 signal pathway by inhibiting p56lck tyrosine kinase.

    PubMed

    Kim, Min-Ho; Jeong, Hyun-Ja

    2014-10-01

    Damnacanthal is a major constituent of Morinda citrifolia L. (noni) and exhibits anti-cancer and anti-inflammatory activities. However, the effects of damnacanthal on allergic diseases have not been determined. In this study, we investigated the effect of damnacanthal on mast cell-mediated allergic inflammatory responses. Damnacanthal significantly and dose-dependently inhibited compound 48/80-induced systemic anaphylactic shock, histamine release and intracellular calcium levels. In particular, IgE-mediated passive cutaneous anaphylaxis was significantly inhibited by the oral administration of damnacanthal. In addition, we report for the first time that p56lck tyrosine kinase was expressed in phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated mast cells. Furthermore, damnacanthal inhibited the up-regulation of p56lck tyrosine kinase activity by PMACI and repressed PMACI-induced histidine decarboxylase expression and activity. Damnacanthal also inhibited PMACI-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α expressions by suppressing nuclear factor-kappa B (NF-κB) activation and suppressed the activation of caspase-1 and the expression of receptor interacting protein-2. This study shows damnacanthal inhibits the NF-κB/receptor-interacting protein-2/caspase-1 signal pathway by inhibiting p56lck tyrosine kinase and suggests that damnacanthal has potential for the treatment of mast cell-mediated allergic disorders. PMID:25139491

  9. 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.

  10. Effect of cisplatin treatment on the urinary excretion of guanidinoacetic acid, creatinine and creatine in patients with urinary tract neoplasm, and on superoxide generation in human neutrophils.

    PubMed

    Yasuda, M; Sugahara, K; Zhang, J; Shuin, T; Kodama, H

    2000-01-01

    Production of guanidinoacetic acid, a precursor of creatinine is known to be reduced by metabolic disturbance when kidney function is damaged, and thus it may be a sensitive marker of renal damage. Therefore, the urinary levels of guanidinoacetic acid, creatinine and creatine from patients with urinary tract neoplasm who received cisplatin treatment were measured by liquid chromatography-mass spectrometry. Following the administration of cisplatin, the urinary excretion of guanidinoacetic acid decreased significantly, and the low concentration was maintained for at least five days. The concentrations of creatinine and creatine gradually decreased until the third day after cisplatin administration, and slightly increased on the fifth day. As superoxide might be concerned in renal damage by cisplatin, the effect of cisplatin on superoxide generation was also investigated using human neutrophils. Cisplatin significantly enhanced phorbol 12-myristate 13-acetate-induced superoxide generation in a concentration-dependent manner, but had no effect on the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine and arachidonic acid. The superoxide generation increased by cisplatin was inhibited by staurosporine, an inhibitor of protein kinase C, but was rather enhanced by genistein, an inhibitor of protein tyrosine kinase. PMID:11383133

  11. Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14.

    PubMed

    Lahaie, Nicolas; Kralikova, Michaela; Prézeau, Laurent; Blahos, Jaroslav; Bouvier, Michel

    2016-03-25

    Mechanisms controlling the metabotropic γ-aminobutyric acid receptor (GABAB) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly down-regulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABAB(1b)subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABABin a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination. PMID:26817839

  12. Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: Involvement of a phosphatidylcholine-hydrolyzing phospholipase C

    SciTech Connect

    Nishijima, J.; Wright, T.M.; Hoffman, R.D.; Liao, F.; Symer, D.E.; Shin, H.S. )

    1989-04-04

    The authors have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using ({sup 14}C)glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.

  13. 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.

  14. Reactive oxygen species (ROS) production in human peripheral blood neutrophils exposed in vitro to static magnetic field.

    PubMed

    Poniedziałek, Barbara; Rzymski, Piotr; Karczewski, Jacek; Jaroszyk, Feliks; Wiktorowicz, Krzysztof

    2013-12-01

    The aim of this study was to determine the effect of gradient static magnetic field (SMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. Blood samples collected from healthy individuals were incubated in an inhomogeneous SMF (in a south or north pole of the field) for 15, 30 or 45 minutes. The maximum value of induction (B max) amounted to ≈ 60 mT. To determine the strength of the ROS production, dihydrorhodamine (123DHR) as fluorophore and phorbol 12-myristate 13-acetate (PMA) as respiratory burst stimulator were used. 123DHR oxidation by ROS was measured by flow cytometry. The exposure of blood samples to SMF induced statistically significant changes in ROS production in unstimulated and PMA-stimulated neutrophils. The observed effects were highly correlated with the exposure time and depended on the orientation of the field. Although intracellular mechanisms underlying such interactions are not thoroughly understood, it could be presumed that SMF affects ROS metabolic oscillations and their formation and inactivation. This study emphasizes the importance of proper adjustment of exposure time to SMF for any potential therapeutic applications. PMID:23631724

  15. Piperlonguminine downregulates endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Ku, Sae-Kwang; Kim, Jeong Ah; Bae, Jong-Sup

    2014-04-01

    Endothelial cell protein C receptor (EPCR) plays an important role in coagulation and inflammation. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α-converting enzyme (TACE). Piperlonguminine (PL), an important component of Piper longum fruits, is known to exhibit antihyperlipidemic, antiplatelet, and antimelanogenesis activities. However, little is known about the effects of PL on EPCR shedding. Here, we investigated this issue by monitoring the effects of PL on phorbol-12-myristate 13-acetate (PMA) and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. PL induced potent inhibition of PMA, and CLP induced EPCR shedding through suppression of TACE expression. And treatment with PL resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, PL might have potential as an anti-sEPCR shedding reagent against PMA- and CLP-mediated EPCR shedding. PMID:24127121

  16. Small molecule glutaminase inhibitors block glutamate release from stimulated microglia.

    PubMed

    Thomas, Ajit G; O'Driscoll, Cliona M; Bressler, Joseph; Kaufmann, Walter; Rojas, Camilo J; Slusher, Barbara S

    2014-01-01

    Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest. PMID:24269238

  17. Helicobacter pylori induces IL-1β protein through the inflammasome activation in differentiated macrophagic cells.

    PubMed

    Kameoka, Shoichiro; Kameyama, Takeshi; Hayashi, Takaya; Sato, Seiichi; Ohnishi, Naomi; Hayashi, Takeru; Murata-Kamiya, Naoko; Higashi, Hideaki; Hatakeyama, Masanori; Takaoka, Akinori

    2016-01-01

    More than 50% of people in the world are infected with Helicobacter pylori (H. pylori), which induces various gastric diseases. Especially, epidemiological studies have shown that H. pylori infection is a major risk factor for gastric cancer. It has been reported that the levels of interleukin (IL)-1β are upregulated in gastric tissues of patients with H. pylori infection. In this study, we investigated the induction mechanism of IL-1β during H. pylori infection. We found that IL-1βmRNA and protein were induced in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells after H. pylori infection. This IL-1β production was inhibited by a caspase-1 inhibitor and a ROS inhibitor. Furthermore, K(+) efflux and Ca(2+) signaling were also involved in this process. These data suggest that NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) and its complex, known as NLRP3 inflammasome, are involved in IL-1β production during H. pylori infection because it is reported that NLRP3 inflammasome is activated by ROS, K(+) efflux and/or Ca(2+) signaling. These findings may provide therapeutic strategy for the control of gastric cancer in H. pylori-infected patients. PMID:26912137

  18. 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

  19. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  20. Regulation of cholesterol esterification by protein kinase C

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-03-05

    They have recently identified acyl-CoA cholesterol acyltransferase as the key enzyme for cholesterol esterification in the central nervous system. They found that the activity of glial acyl-CoA cholesterol acyltransferase could be controlled by a phosphorylation-dephosphorylation mechanism. However, repeated attempts to identify cyclic AMP as the bioregulator for this reaction failed. Recently, they have studied the possible involvement of protein kinase C in the regulation of glial cholesterol esterification. Phorbol-12-myristate 13-acetate (PMA) can activate cellular cholesterol esterification in a complex, time-dependent manner. Phorbol analogues inactive toward protein kinase C are also ineffective in this assay. Furthermore, oleoyl-acetyl-glycerol mimics the effect of PMA, confirming the proposal that protein kinase C mediates the effect of these compounds and that the natural bioregulator is probably diacylglycerol. Receptor-mediated polyphosphatidyl-inositol cleavage often produces diacylglycerol and inositol triphosphate. The synergic effects of these two compounds are known to be necessary to elicit other biological responses. Their preliminary studies using calcium ionophore A23187 indicates that Ca/sup + +/ is not required for cellular cholesterol esterification. In sum, glial cholesterol esterification is probably regulated by a calcium-independent and protein kinase C-dependent reaction.

  1. Serotonin stimulates phospholipase A sub 2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis

    SciTech Connect

    Felder, C.C.; Ma, A.L.; Axelrod, J.; Kanterman, R.Y. )

    1990-03-01

    Serotonin (5-HT) stimulated the release of arachidonic acid in hippocampal neurons cocultured with glial cells but not in glial cultures alone. Similar results were observed for the 5-HT-stimulated release of inositol phosphates. These results suggest a neural but not glial origin of both responses. Pharmacological studies suggested that release of arachidonic acid and inositol phosphates was mediated by a type 2 5-HTT (5-HT{sub 2}) receptor. 5-HT-stimulated release of arachidonic acid was also detected in cortical neurons, which contain high levels of 5-HT{sub 2} receptors, but not striatum, spinal cord, or cerebellar granule cells, which have very low levels or are devoid of 5-HT{sub 2} receptors. The phorbol ester phorbol 12-myristate 13-acetate augmented the 5-HT-stimulated release of arachidonic acid but inhibited the 5-HT-stimulated release of inositol phosphates. 5-HT-stimulated release of arachidonic acid, but not inositol phosphates, was dependent on extracellular calcium. 5-HT stimulated the release of ({sup 3}H)lysophosphatidylcholine from ({sup 3}H)choline-labeled cells with no increase in the release of ({sup 3}H)choline or phospho({sup 3}H)choline. These data suggest that 5-HT stimulated the release of arachidonic acid in hippocampal neurons through the activation of phospholipase A{sub 2}, independent of the activation of phospholipase C.

  2. High Mobility Group Box 1 Protein Induction by Mycobacterium Bovis BCG

    PubMed Central

    Hofner, Péter; Seprényi, György; Miczák, András; Buzás, Krisztina; Gyulai, Zsófia; Medzihradszky, Katalin F.; Rouhiainen, Ari; Rauvala, Heikki; Mándi, Yvette

    2007-01-01

    High mobility group box 1 protein (HMGB1), a nuclear protein, is a critical cytokine that mediates the response to infection, injury, and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB1 from supernatants of cells, following induction with Mycobacterium bovis BCG. Densitometric analysis revealed higher concentrations of HMGB1 in cell supernatants stimulated with BCG than in the supernatants of the control, nonstimulated cells. Further quantitation of the secreted HMGB1 was performed by ELISA. The BCG strain resulted in a higher amount of secreted HMGB1 (450 ± 44 ng/mL) than that of LPS (84 ± 12 ng/mL) or Staphylococcus aureus (150 ± 14 ng/mL). BCG and Phorbol −12-myristate13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 ± 125 ng/mL). The translocation of the HMGB1 towards the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations. Conclusion: Our pilot experiments draw attention to the HMGB1 inducing ability of Mycobacterium bovis. Assesment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations. PMID:18288272

  3. Interleukin-8 stimulates the migration of human colonic epithelial cells in vitro.

    PubMed

    Wilson, A J; Byron, K; Gibson, P R

    1999-09-01

    The migration of colonic epithelial cells (restitution) is an important event in the repair of mucosal injuries. Interleukin-8 (IL-8) is a physiological initiator of the chemotactic migration of leucocytes. This study aimed to determine whether IL-8 had a similar effect on migration in an in vitro model of wounded colonic epithelium. Cell migration over 24 h was assessed in circular wounds made in confluent monolayers of the human colon cancer cell line LIM1215. This migration was stimulated in a concentration-dependent manner by IL-8, with maximal effects of approx. 1.75-fold above basal migration. The motogenic effect of IL-8 was mediated independently of effects on cell proliferation. In contrast, it was partially dependent upon gene transcription and protein synthesis and involved the activation of pertussis-toxin-sensitive G-proteins. The short-chain fatty acids, acetate, propionate, butyrate and valerate, the activator of protein kinase C (phorbol-12-myristate-13-acetate) and tumour necrosis factor-alpha (TNF-alpha) all stimulated the secretion of IL-8. However, only the motogenic effect of TNF-alpha was dependent upon IL-8. In conclusion, IL-8 stimulated cell migration in an in vitro model of colonic epithelium, whereas the motogenic effect of at least one physiologically relevant factor was dependent upon an increase in its endogenous levels. If IL-8 stimulates colonic epithelial restitution in vivo, this would have ramifications for the control of repair processes following wounding of the colonic mucosa. PMID:10464065

  4. 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. PMID:12787890

  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. PMID:24974465

  6. Concord grape juice attenuates platelet aggregation, serum cholesterol and development of atheroma in hypercholesterolemic rabbits.

    PubMed

    Shanmuganayagam, Dhanansayan; Warner, Thomas F; Krueger, Christian G; Reed, Jess D; Folts, John D

    2007-01-01

    Intake of Concord grape juice (CGJ), rich in polyphenolics, inhibits platelet aggregation (PA), a risk factor for cardiovascular disease (CVD), in normocholesterolemic animals and humans. It is unclear whether CGJ can attenuate hypercholesterolemia-enhanced PA. The effects of daily CGJ consumption on hypercholesterolemia-enhanced PA and the development of atherosclerosis were investigated. Two groups of rabbits (Control and Treated; n=10 each) were fed a hypercholesterolemic diet for 48 days. Treated group then received supplemental CGJ (225mL/day) while Control group received supplemental iso-caloric sugar water for 48 days. Collagen-, collagen+epinephrine- and phorbol-12-myristate-13-acetate-induced whole blood PA responses were measured on Days 0, 48 and 96; total serum cholesterol and blood pressure were also measured. The development of aortic atheroma was quantified at the end. Both groups showed significant increases in PA and serum cholesterol at Day 48. However, at Day 96, Treated group showed significantly lower PA and development of atheroma (30.7+/-3.9% lower (p<0.001)) than Control group; Treated group also had significantly lower total serum cholesterol and blood pressure than Control group. In conclusion, daily consumption of CGJ attenuates hypercholesterolemia-enhanced PA, blood pressure, total serum cholesterol and development of atheroma in rabbits. PMID:16780846

  7. Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo.

    PubMed

    Kamezaki, Aosa; Sato, Fuminori; Aoki, Kazuhiro; Asakawa, Kazuhide; Kawakami, Koichi; Matsuzaki, Fumio; Sehara-Fujisawa, Atsuko

    2016-01-01

    Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as 'ectodomain shedding', is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo. PMID:27364328

  8. Weight gain and inflammation regulate aromatase expression in male adipose tissue, as evidenced by reporter gene activity.

    PubMed

    Polari, L; Yatkin, E; Martínez Chacón, M G; Ahotupa, M; Smeds, A; Strauss, L; Zhang, F; Poutanen, M; Saarinen, N; Mäkelä, S I

    2015-09-01

    Obesity and white adipose tissue (WAT) inflammation are associated with enhanced aromatization in women, but little is known about the regulation of aromatase (CYP19A1) gene expression in male WAT. We investigated the impact of weight gain and WAT inflammation on the regulation of CYP19A1 in males, by utilizing the hARO-Luc aromatase reporter mouse model containing a >100-kb 5'-region of the human CYP19A1 gene. We show that hARO-Luc reporter activity is enhanced in WAT of mice with increased adiposity and inflammation. Dexamethasone and TNFα, as well as forskolin and phorbol 12-myristate 13-acetate, upregulate hARO-Luc activity, suggesting the involvement of promoters I.4 and I.3/II. Furthermore, we show that diet enriched with antioxidative plant polyphenols attenuates WAT inflammation and hARO-Luc activity in obese males. In conclusion, our data suggest that obesity-associated WAT inflammation leads to increased peripheral CYP19A1 expression in males, and that polyphenol-enriched diet may have the potential to attenuate excessive aromatization in WAT of obese men. PMID:26054748

  9. Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling

    PubMed Central

    Chandrika, Goparaju; Natesh, Kumar; Ranade, Deepak; Chugh, Ashish; Shastry, Padma

    2016-01-01

    Glioblastoma (GBM) is the most aggressive type of brain tumors in adults with survival period <1.5 years of patients. The role of mTOR pathway is documented in invasion and migration, the features associated with aggressive phenotype in human GBM. However, most of the preclinical and clinical studies with mTOR inhibitors are focused on antiproliferative and cytotoxic activity in GBM. In this study, we demonstrate that mTOR inhibitors-rapamycin (RAP), temisirolimus (TEM), torin-1 (TOR) and PP242 suppress invasion and migration induced by Tumor Necrosis Factor-α (TNFα) and tumor promoter, Phorbol 12-myristate 13-acetate (PMA) and also reduce the expression of the TNFα and IL1β suggesting their potential to regulate factors in microenvironment that support tumor progression. The mTOR inhibitors significantly decreased MMP-2 and MMP-9 mRNA, protein and activity that was enhanced by TNFα and PMA. The effect was mediated through reduction of Protein kinase C alpha (PKC-α) activity and downregulation of NFκB. TNFα- induced transcripts of NFκB targets -VEGF, pentraxin-3, cathepsin-B and paxillin, crucial in invasion were restored to basal level by these inhibitors. With limited therapeutic interventions currently available for GBM, our findings are significant and suggest that mTOR inhibitors may be explored as anti-invasive drugs for GBM treatment. PMID:26940200

  10. 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

    2015-10-01

    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. PMID:26209362

  11. Endogenously produced glycosaminoglycans affecting the release of lipoprotein lipase from macrophages and the interaction with lipoproteins.

    PubMed

    Zimmermann, R; Sartipy, P; Winkler, R; Zechner, R; Hurt-Camejo, E; Kostner, G M

    2000-04-12

    Macrophages are intimately involved in the pathogenesis of atherosclerotic diseases. A key feature of this process is their uptake of various lipoproteins and subsequent transformation to foam cells. Since lipoprotein lipase (LPL) is believed to play a role in foam cell formation, we investigated if endogenously produced proteoglycans (PGs) affect the release of this enzyme from macrophages. The human leukaemic cell line THP-1 which differentiates into macrophages by treatment with phorbol ester (phorbol 12-myristate 13-acetate) served as a model. The differentiation of THP-1 macrophages promoted the release of PGs into the cell medium which caused the detachment of LPL activity from the cell surface, and prevented LPL re-uptake and inactivation. These PGs were mainly composed of chondroitin sulfate type and exerted a heparin-like effect on LPL release. LPL is known to increase the cell association of lipoproteins by the well known bridging function. Exogenous bovine LPL at a concentration of 1 microg/ml enhanced low density lipoprotein (LDL)-binding 10-fold. Endogenously produced PGs reduced LPL-mediated binding of LDL. It is proposed that the differentiation-dependent increase in the release of PGs interferes with binding of LPL and reduces lipoprotein-binding to macrophages. PMID:10760480

  12. Expression of peptide YY by human blood leukocytes.

    PubMed

    Holler, Julia Pia Natascha; Schmitz, Jessica; Roehrig, Rainer; Wilker, Sigrid; Hecker, Andreas; Padberg, Winfried; Grau, Veronika

    2014-08-01

    Peptide YY is produced by L cells in the mucosa of the distal ileum, colon, and rectum and may have systemic and paracrine functions. We hypothesized that peptide YY is expressed by peripheral blood mononuclear cells. The purpose of the present study was to evaluate the expression of peptide YY mRNA and peptide by peripheral blood mononuclear cells and differentiated THP-1 cells after lipopolysaccharide treatment as an in vitro model of inflammation. Blood was drawn by venipuncture from 18- to 63-year-old healthy male blood donors (n=63); peptide YY mRNA expression levels were detected in peripheral blood mononuclear cells from all healthy male subjects. In 3 subjects, peripheral blood mononuclear cells were cultured for 3 and 24h and peptide YY was detected in the cell culture supernatant. In human monocytic THP-1 cells treated with phorbol-12-myristate-13-acetate to induce differentiation to macrophages, treatment with lipopolysaccharide caused down-regulation of peptide YY mRNA levels. In summary, freshly isolated peripheral blood mononuclear cells from healthy humans expressed peptide YY. In vitro data suggested that peptide YY expression is down-regulated by differentiation of monocytes to macrophages and proinflammatory stimuli. PMID:24969624

  13. Culture supernatants of different colon cancer cell lines induce specific phenotype switching and functional alteration of THP-1 cells.

    PubMed

    Wu, Tsung-Han; Li, Ying-Ying; Wu, Tai-Ling; Chang, John W-C; Chou, Wen-Chi; Hsieh, Ling-Ling; Chen, Jim-Ray; Yeh, Kun-Yun

    2014-07-01

    We developed an in vitro model to evaluate the effect of products secreted from different colorectal cancer (CRC) cell lines on specific phenotypic switching and functional alterations in THP-1 cells. We co-cultured the human monocytic cell line, THP-1, or phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells, (THP-1p), with supernatants from either the HT-29 (Dukes' B), HCT-15 (Dukes' C), or Colo205 (Dukes' D) cell lines, and assessed the cells for macrophage differentiation. The surface marker and cytokine profiles suggested that secreted CRC factors differentiated THP-1 cells into a "mixed" M1/M2 phenotype, although HT-29 and Colo205 supernatants induced THP-1p cells into predominantly M1-like macrophages and M2-like macrophages, respectively. Further, all three CRC supernatants enhanced the phagocytic capacity and migration of THP-1 and THP-1p cells, altering their phenotype to a more M2-kind. Therefore, different CRC cell lines induced specific phenotype switching and functional polarization of THP-1 cells. PMID:24960291

  14. Comparative evaluation of in vitro human macrophage models for mycobacterial infection study.

    PubMed

    Mendoza-Coronel, E; Castañón-Arreola, M

    2016-08-01

    Macrophages are phagocytic cells that play a key role maintaining the homeostasis of many tissues. Their function is essential for controlling and eradicating infecting mycobacteria. Human monocytic cell lines such as THP-1 and U937 have provided interesting insights into how mycobacteria subvert the host cell response. However, immortalized cell lines could bring some disadvantages. Here we compare the response of THP-1 and U937 cell lines with human monocyte-derived macrophages (hMDMs) to determine functional differences during infection with different mycobacterial phenotypes (virulent Mycobacterium tuberculosis H37Rv and Mycobacterium bovis, and attenuated M. bovis BCG). The findings of this study indicate that the U937 cell line displays a significantly lower phagocytic capacity than hMDMs and THP-1 macrophages, regardless of the mycobacterial strain. In all cell models, interferon-γ activation leads to up-regulation of interleukin-12 and nitrite production. However, the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of U937 and THP-1 cell lines induces a significant tumor necrosis factor-α production in resting macrophages. However, this state of activation has no effect on the control of intracellular growth of mycobacteria. Moreover, U937 cells show more discrepancies with hMDM than THP-1. This study demonstrates that THP-1 macrophages exhibit closer functional similarities to hMDMs in response to mycobacterial infection, regardless of the strain. PMID:27307103

  15. Simultaneous Real-Time Monitoring of Oxygen Consumption and Hydrogen Peroxide Production in Cells Using Our Newly Developed Chip-Type Biosensor Device.

    PubMed

    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

  16. 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

  17. 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.

  18. 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.

  19. Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo

    PubMed Central

    Kamezaki, Aosa; Sato, Fuminori; Aoki, Kazuhiro; Asakawa, Kazuhide; Kawakami, Koichi; Matsuzaki, Fumio; Sehara-Fujisawa, Atsuko

    2016-01-01

    Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as ‘ectodomain shedding’, is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo. PMID:27364328

  20. Reciprocal regulation of expression of the human adenosine 5′-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors

    PubMed Central

    Davis, Warren; Chen, Zhijian J.; Ile, Kristina E.; Tew, Kenneth D.

    2003-01-01

    The human ABCA2 transporter gene encodes a member of a large family of ATP-binding proteins that transport a variety of macromolecules across biological membranes. We have performed luciferase reporter gene assays with promoter constructs comprising the 5′-flanking region to identify cis-regulatory DNA elements and have mapped the minimal promoter region to 321 bp upstream of the translation start site. We have discovered a functional role for two GC-boxes located in the proximal promoter of the ABCA2 gene that contain overlapping sites for the EGR-1 and Sp1 transcription factors. We observed that oligonucleotides containing overlapping EGR-1/Sp1 sites bind the Sp1, Sp3 and Sp4 transcription factors. When BE(2)-M17 cells were treated with phorbol 12-myristate 13-acetate, we observed inducible expression and binding of the EGR-1 transcription factor to the two GC-boxes. Transfection of Sp1, Sp3 or Sp4 expression constructs into Drosophila S2 induced a dose-dependent increase in transcriptional activation of the ABCA2 promoter, but transfection of EGR-1 alone failed to activate transcription. When increasing amounts of EGR-1 were transfected into the BE(2)-M17 neuroblastoma cells we observed a dose-dependent decrease in expression of the ABCA2 promoter, although expression of the endogenous ABCA2 gene increased following transfection of EGR-1. PMID:12560508

  1. Up-regulation of AKAP13 and MAGT1 on cytoplasmic membrane in progressive hepatocellular carcinoma: a novel target for prognosis

    PubMed Central

    Molee, Patamaporn; Adisakwattana, Poom; Reamtong, Onrapak; Petmitr, Songsak; Sricharunrat, Thaniya; Suwandittakul, Nantana; Chaisri, Urai

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers and is associated with high mortality worldwide. The current gold standards for HCC surveillance are detection of serum α-fetoprotein (AFP) and ultrasonography; however, non-specificity of AFP and ultrasonography has frequently been reported. Therefore, alternative tools, especially novel specific tumor markers, are required. In this study, cytoplasmic membrane proteins were isolated from phorbol 12-myristate 13-acetate (PMA)-induced invasive HepG2 cells and identified using nano-scale liquid chromatographic tandem mass spectrometry (NLC-MS/MS) with comparison to non-treated controls. The results showed that two proteins, magnesium transporter protein 1 (MAGT1) and A-kinase anchor protein 13 (AKAP13), were highly expressed in PMA-treated HepG2 cells. This up-regulation was confirmed by real-time RT-PCR, western blot analysis, and immunofluorescent staining studies. Furthermore, evaluation of MAGT1 and AKAP13 expression in clinical HCC tissues by immunohistochemistry suggested that both proteins were strongly expressed in tumor tissues with significantly higher average immunoreactive scores of Remmele and Stegner (IRS) than in non-tumor tissues (P ≤ 0.005). In conclusion, the expression levels of MAGT1 and AKAP13 in HCC may be potential biomarkers for the diagnosis and prognosis of this cancer. PMID:26617690

  2. Chemical modulation of the ultra-weak photon emission from Saccharomyces cerevisiae and differentiated HL-60 cells

    NASA Astrophysics Data System (ADS)

    Červinková, Kateřina; Nerudová, Michaela; Hašek, Jiří; Cifra, Michal

    2015-01-01

    The ultra-weak photon emission (UPE) is a universal phenomenon common to all cells with active oxidative metabolism. Generally accepted mechanism of the origin of the ultra-weak photon emission considers reactions of radical or nonradical reactive oxygen species (ROS) with biomolecules such as lipids and proteins which lead to the formation of electron excited species. During the transition to the ground state the excess energy is released as a photon with a wavelength in the visible range of the electromagnetic spectrum. Since the intensity of the light is very low it is possible to be measured only by highly sensitive devices. We used Hamamatsu Photonics PMT module H7360-01 mounted into a light-tight chamber for the purposes of this work. The goal of our research is to delineate an origin of UPE from two model organisms; differentiated HL-60 cells (human promyelocytic leukemia) and yeast cells Saccharomyces cerevisiae. While the UPE from the yeast cells arises spontaneously during the growth without any external stimuli, UPE from HL-60 is induced by phorbol 12-myristate, 13-acetate (PMA). It is possible to modulate the UPE production by certain antioxidants which scavenge ROS formed during the metabolism (yeast cells) or respiratory burst (HL-60 cells). The experiments are focused on the description of effects caused by antioxidants. Several kinds of antioxidants (ascorbic acid, mannitol, glutathione) with different concentration were used and we studied the changes in the UPE intensities of and the temporal developments of the optical signal.

  3. Visfatin contributes to the differentiation of monocytes into macrophages through the differential regulation of inflammatory cytokines in THP-1 cells.

    PubMed

    Yun, Mi Ran; Seo, Jeong Mi; Park, Hyun Young

    2014-04-01

    Visfatin is a novel multifunctional adipocytokine with inflammatory properties. Although a link between visfatin and atherosclerosis has recently been suggested, its actions in the development of atherosclerosis remain unknown. Therefore, we investigated a potential role and underlying mechanism(s) of visfatin in monocytes/macrophages differentiation, a critical early step in atherogenesis, using phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cell models. The co-incubation of PMA with visfatin-induced CD36 expression with a concomitant increase in the phagocytosis of latex beads compared with PMA alone treatment. Moreover, visfatin markedly increased interleukin (IL)-1β secretion by enhancing IL-1β mRNA stability in a short-term incubation. Visfatin also significantly elevated the secretion of IL-6 as well as IL-1β in a longer incubation period, which was partially suppressed by nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, and c-Jun-N-terminal kinase (JNK) inhibitor, SP600125. Furthermore, silencing IL-1β successfully blocked IL-6 secretion, CD36 expression, and NF-κB activation in response to visfatin. Collectively, these results suggest that visfatin enhances the IL-1β-dependent induction of IL-6 and CD36 via distinct signaling pathways mediated by JNK and NF-κB, respectively, and consequently, leading to the acceleration of monocytes/macrophages differentiation. PMID:24378536

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

    PubMed Central

    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. PMID:27034964

  5. Rhizoctonia bataticola lectin (RBL) induces phenotypic and functional characteristics of macrophages in THP-1 cells and human monocytes.

    PubMed

    Pujari, Radha; Kumar, Natesh; Ballal, Suhas; Eligar, Sachin M; Anupama, S; Bhat, Ganapati; Swamy, Bale M; Inamdar, Shashikala R; Shastry, Padma

    2015-02-01

    We have previously reported that a fungal lectin, Rhizoctonia bataticola lectin (RBL), stimulates proliferation and secretion of Th1/Th2 cytokines in human peripheral blood mononuclear cells (PBMC). In the present study, we evaluated the ability of RBL to differentiate human monocytes to macrophages. RBL induced morphological changes indicative of differentiation in primary monocytes and THP-1 cells. Stimulation with RBL resulted in significant up-regulation of differentiation markers - CD54, HLA-DR, CD11b and CD11c and secretion of proinflammatory cytokines - IL-1β, TNF-α and IL-6. Functionally, RBL profoundly increased phagocytic activity in monocytes. In THP-1 cells, RBL-induced phagocytosis was higher compared to the effect induced by combination of phorbol-12-myristate-13-acetate (PMA) and lipopolysaccharide (LPS). RBL induced a significant increase in matrix metalloproteinase-9 (MMP-9) activity in comparison with a combined treatment of PMA+LPS. Mechanistic studies revealed the involvement of the NF-κB pathway in RBL-induced differentiation of monocytes. The data suggest that RBL mimics the combined action of PMA and LPS to induce morphological and functional differentiation in human monocytes and monocytic cell line - THP-1 to macrophages. Human monocytes differentiated to macrophages with RBL have the potential as an in vitro model to study macrophage biology. PMID:25555439

  6. Modulation of protein tyrosine nitration and inflammatory mediators by isoprenylhydroquinone glucoside.

    PubMed

    Olmos, Ana; Giner, Rosa-María; Recio, María-Carmen; Ríos, José-Luis; Máñez, Salvador

    2007-03-01

    The nitration of tyrosine caused by peroxynitrite and other reactive nitrogen species is clearly detrimental for some physiological processes; however, its signalling role is still open to controversy. Among the natural phenolics known for their ability to oppose free tyrosine nitration, isoprenylhydroquinone glucoside is investigated due to its unusual structure, which contains a simple hydroxybenzene alkylated by a hemiterpenoid moiety. This hydroquinone was shown to be an effective inhibitor of peroxynitrite-induced protein tyrosine nitration in 3T3 fibroblasts. When tested on bovine seroalbumin nitration, however, the potency was reduced by half and the effect was almost abolished in the presence of bicarbonate. In contrast, addition of this anion had no effect on the nitrite/hydrogen peroxide/hemin system. Isoprenylhydroquinone glucoside was also active in the microM range on intra- and extracellular protein-bound tyrosine nitration by phorbol 12-myristate 13-acetate-stimulated neutrophils. The effects on nitric oxide synthase expression, interleukin-1beta and tumor necrosis factor-alpha production by lipopolysaccharide-stimulated macrophages were quite moderate. Thus, isoprenylhydroquinone glucoside is an inhibitor of protein nitration in situ, but lacks effect on the generation of either nitric oxide or inflammatory cytokines. PMID:17161592

  7. v-jun cooperates with v-erbB to transform the thrombocytic/megakaryocytic lineage.

    PubMed Central

    Garcia, M; Samarut, J

    1993-01-01

    The transforming properties of v-jun, the viral counterpart of the transcription factor AP1, were investigated in avian hematopoietic cells. Two retroviruses, called JB and JBN, expressing both v-jun and v-erbB, were constructed using an avian erythroblastosis-based vector. We show that the cooperative action of both oncogenes allowed the virus to efficiently transform bone marrow cells. No such transformation was obtained with either oncogene alone. JB-transformed bone marrow cells expressed GATA-1, TAL-1, and histone H5, suggesting that they belong to the erythrocytic/thrombocytic lineage. (Thrombocytes are the avian homologues of mammal megakaryocytes.) Moreover, after induction with phorbol 12-myristate 13-acetate JB-transformed bone marrow cells began to differentiate and synthesized high levels of platelet glycoproteins, indicating that they were of thrombocytic origin. These results were confirmed by c-ets1 analysis since this transcription factor, specifically found in cells with megakaryocytic but not erythrocytic features, was clearly detected in these cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8105467

  8. Antiinflammatory evaluation of alcoholic extract of galls of Quercus infectoria.

    PubMed

    Kaur, Gurpreet; Hamid, Hinna; Ali, Asif; Alam, M Sarwar; Athar, Mohammad

    2004-02-01

    Galls of Quercus infectoria Olivier (Fagaceae) possess pleiotropic therapeutic activities, with particular efficacy against inflammatory diseases. The present study was undertaken to evaluate the effect of alcoholic extract of Q. infectoria galls on various in vivo and in vitro experimental models of inflammation. Oral administration of gall extract significantly inhibited carrageenan, histamine, serotonin and prostaglandin E2 (PGE2) induced paw oedemas, while topical application of gall extract inhibited phorbol-12-myristate-13-acetate (PMA) induced ear inflammation. The extract also inhibited various functions of macrophages and neutrophils relevant to the inflammatory response. In vitro exposure of rat peritoneal macrophages to gall extract ameliorated lipopolysaccharide (LPS) stimulated PGE2 and nitric oxide (NO) production and PMA stimulated superoxide (O2*-) production in a dose dependent manner. Gall extract also scavenged NO and O2*-. Probing into mechanism of NO inhibition in macrophages revealed gall extract to ameliorate the induction of inducible NO synthase (iNOS), respectively without any inhibitory effect on its catalytic activities even at higher concentrations. Gall extract also significantly inhibited formyl-Met-Leu-Phe (fMLP) stimulated degranulation in neutrophils. These results suggest that alcoholic extract of galls of Q. infectoria exerts in vivo antiinflammatory activity after oral or topical administration and also has the ability to prevent the production of some inflammatory mediators. PMID:15013194

  9. 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. PMID:21165548

  10. Unique insights into the intestinal absorption, transit, and subsequent biodistribution of polymer-derived microspheres

    PubMed Central

    Reineke, Joshua J.; Cho, Daniel Y.; Dingle, Yu-Ting; Morello, A. Peter; Jacob, Jules; Thanos, Christopher G.; Mathiowitz, Edith

    2013-01-01

    Polymeric microspheres (MSs) have received attention for their potential to improve the delivery of drugs with poor oral bioavailability. Although MSs can be absorbed into the absorptive epithelium of the small intestine, little is known about the physiologic mechanisms that are responsible for their cellular trafficking. In these experiments, nonbiodegradable polystyrene MSs (diameter range: 500 nm to 5 µm) were delivered locally to the jejunum or ileum or by oral administration to young male rats. Following administration, MSs were taken up rapidly (≤5 min) by the small intestine and were detected by transmission electron microscopy and confocal laser scanning microscopy. Gel permeation chromatography confirmed that polymer was present in all tissue samples, including the brain. These results confirm that MSs (diameter range: 500 nm to 5 µm) were absorbed by the small intestine and distributed throughout the rat. After delivering MSs to the jejunum or ileum, high concentrations of polystyrene were detected in the liver, kidneys, and lungs. The pharmacologic inhibitors chlorpromazine, phorbol 12-myristate 13-acetate, and cytochalasin D caused a reduction in the total number of MSs absorbed in the jejunum and ileum, demonstrating that nonphagocytic processes (including endocytosis) direct the uptake of MSs in the small intestine. These results challenge the convention that phagocytic cells such as the microfold cells solely facilitate MS absorption in the small intestine. PMID:23922388

  11. Characterization and regulation of adenosine transport in T84 intestinal epithelial cells.

    PubMed

    Mun, E C; Tally, K J; Matthews, J B

    1998-02-01

    Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl- secretion. Previous data suggest that A2b receptor-mediated Cl- secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na(+)-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5'-(N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl- secretory responses and hence diarrheal disorders. PMID:9486178

  12. Lipoic acid suppression of neutrophil respiratory burst: effect of NADPH.

    PubMed

    O'Neill, Heidi C; Rancourt, Raymond C; White, Carl W

    2008-02-01

    Lipoic acid (LA) and its reduced product dihydrolipoic acid (DHLA) are potent antioxidants with capacity to scavenge reactive oxygen species (ROS) and recycle endogenous antioxidants. LA may increase cellular glutathione (GSH), an antioxidant lacking in the lung's epithelial lining fluid in lung disorders such as idiopathic pulmonary fibrosis (IPF). Neutrophils (PMN) are key innate responders and are pivotal in clearing bacterial infection, therefore it is crucial to understand the impact LA may have on their function. Circulating neutrophils were isolated from healthy volunteers and pretreated with LA or diluent. Cells were subsequently activated with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) to induce ROS production. SOD-inhibitable reduction of acetylated cytochrome c demonstrated the PMA-dependent respiratory burst was suppressed by LA. Oxygen consumption also was diminished when PMA-stimulated cells were pretreated with LA. PMN respiratory burst was partially restored by addition of NADPH but not other pyridine nucleotides. LA did not inhibit glucose-6-phosphate dehydrogenase activity of PMN. These data together suggest that the reduction of LA to DHLA using cellular NADPH may limit the capacity of the PMN NADPH oxidase to produce superoxide. Further studies will be required to determine if LA can diminish excessive superoxide produced by PMN and/or alveolar macrophages in IPF or relevant disease models in vivo. PMID:18158760

  13. Suppression of COX-2, IL-1β and TNF-α expression and leukocyte infiltration in inflamed skin by bioactive compounds from Rosmarinus officinalis L.

    PubMed

    Mengoni, Eleonora S; Vichera, Gabriel; Rigano, Luciano A; Rodriguez-Puebla, Marcelo L; Galliano, Silvia R; Cafferata, Eduardo E; Pivetta, Omar H; Moreno, Sivia; Vojnov, Adrián A

    2011-04-01

    In the present study, we evaluated the effects of extracts and purified compounds from fresh leaves of Rosmarinus officinalis L. Pretreatment with the major anti-inflammatory compounds, carnosic acid (CA) and carnosol (CS), inhibited phorbol 12-myristate 13-acetate (PMA)-induced ear inflammation in mice with an EC(50) of 10.20 μg/cm(2) and 10.70 μg/cm(2), respectively. To further understand the anti-inflammatory mechanism of these compounds, we analyzed the in vivo expression of several inflammation-associated genes in mouse skin by reverse transcriptase-polymerase chain reaction (RT-PCR). Our data showed that CA and CS reduced the expression of IL-1β and TNF-α but had less effect on fibronectin and ICAM-1 expression. Interestingly, both compounds selectively inhibited COX-2 but not COX-1. Histopathological analysis of hematoxylin and eosin (H&E)-stained tissue revealed a marked reduction in leukocyte infiltration and epidermal ulceration of PMA-treated ears when ears were pretreated with ethanolic extracts or pure CA. In vitro, we showed that ethanolic extract, carnosic acid and carnosol significantly inhibited the overproduction of nitric oxide (NO) in a dose-dependent manner in the RAW 264.7 murine macrophage cell line. For the first time in vivo, we showed that CA and CS differentially regulate the expression of inflammation-associated genes, thus demonstrating the pharmacological basis for the anti-inflammatory properties reported for CA and CS. PMID:21129455

  14. Interleukin-10 antisense oligodeoxynucleotide suppresses IL-10 expression and effects on proinflammatory cytokine responses to porcine reproductive and respiratory syndrome virus.

    PubMed

    Charerntantanakul, Wasin; Kasinrerk, Watchara

    2010-08-01

    Upregulation of interleukin-10 (IL-10) expression has been suggested to be the mechanism by which the porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the innate and adaptive immune response in infected pigs. In this study we evaluated the potential of phosphorothioate-modified IL-10 antisense oligodeoxynucleotide specific to the translation initiation region of porcine IL-10 mRNA (IL-10AS) in enhancing proinflammatory cytokine responses to PRRSV. Naïve peripheral blood mononuclear cells from eight PRRSV-seronegative pigs were transfected with IL-10AS in vitro prior to PRRSV inoculation and phorbol 12-myristate 13-acetate plus ionomycin or concanavalin A stimulation. The effects of IL-10AS on mRNA expression of IL-10, interferon-gamma (IFN-gamma), IFN-alpha, tumor necrosis factor-alpha (TNF-alpha), IL-2, and IL-4 were tested by real-time PCR. The percentages of IFN-gamma-producing T-cell subsets were determined by flow cytometry. Compared to the controls, the levels of IL-10 and IL-2 mRNA were significantly reduced, while those of IFN-gamma mRNA were increased, and TNF-alpha, IFN-alpha, and IL-4 mRNA were unchanged. An increase in the percentage of the IFN-gamma+ population was also observed in lymphocytes and CD8beta+ T cells. Our results suggest that IL-10AS has the potential to enhance proinflammatory cytokine responses to PRRSV infection. PMID:20712487

  15. NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone-1 under anaerobic conditions.

    PubMed Central

    Murakami, M; Nakamura, M; Minakami, S

    1986-01-01

    The stimulation-specific NADPH-dependent reduction of ubiquinone-1 (Q-1) in guinea-pig macrophages was studied. The activity was due neither to any modified product of the phagocytosis-specific NADPH oxidase nor to non-specific diaphorases of the cells, since the activity was measured in sonicated or detergent-disrupted cells by subtracting the activity in the resting cells from that in cells activated by phorbol 12-myristate 13-acetate. The activity was not mediated by superoxide anions, since strict anaerobic conditions were employed. The anaerobic reduction of Q-1 was NADPH-specific, like superoxide formation under aerobic conditions, and its maximal velocity was also essentially the same as that of superoxide formation. The oxidase does not directly reduce Q-1 under aerobic conditions [Nakamura, Murakami, Umei & Minakami (1985) FEBS Lett. 186, 215-218], and the electron transfer from NADPH to cytochrome c by the oxidase under aerobic conditions was not enhanced by the addition of Q-1. The observations indicate that the phagocytosis-specific NADPH oxidase reduces Q-1 and that oxygen competes with the reduction of Q-1. Q-1 seems to accept electrons not from the intermediary electron carriers of the oxidase but from the terminal oxygen-reducing site of the enzyme. PMID:3026322

  16. Ripe fruit of Rubus coreanus inhibits mast cell-mediated allergic inflammation.

    PubMed

    Kim, Hui-Hun; Choi, Phil Hyung; Yoo, Jin-Su; Jeon, Hoon; Chae, Byeong-Suk; Park, Jeong-Suk; Kim, Sang-Hyun; Shin, Tae-Yong

    2012-02-01

    In this study, we investigated the effect of a water extract of the ripe fruits of Rubus coreanus Miq. (Rosaceae) (RFRC) on mast cell-mediated allergic inflammation and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases such as anaphylaxis, rhinitis, asthma and atopic dermatitis. RFRC dose-dependently inhibited compound 48/80-induced systemic anaphylaxis and serum histamine release in mice. RFRC reduced the immunoglobulin E (IgE)-mediated local allergic reaction, passive cutaneous anaphylaxis. RFRC attenuated histamine release from rat peritoneal mast cells and human mast cells by the reduction of intracellular calcium. RFRC decreased the phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187 (PMACI)-stimulated expression and secretion of pro-inflammatory cytokines in human mast cells. The inhibitory effect of RFRC on cytokine production was nuclear factor (NF)-κB- and mitogen-activated protein kinase (MAPK)-dependent. In addition, RFRC suppressed the activation of caspase-1. Our findings provide evidence that RFRC inhibits mast cell-derived allergic inflammatory reactions, and for the involvement of calcium, NF-κB, MAPKs and caspase-1 in these effects. Furthermore, in vivo and in vitro anti-allergic inflammatory effects of RFRC provide affirmative proof of a possible therapeutic application of this agent in allergic inflammatory diseases. PMID:22075758

  17. Age-related alterations to immune parameters in Labrador retriever dogs.

    PubMed

    Blount, Daniel G; Pritchard, David I; Heaton, Paul R

    2005-12-15

    In order to assess age-related changes in the immune status of Labrador retriever dogs, leukocyte phenotypes, lymphocyte proliferative capacity, and serum antibody levels were measured in four cohorts of dogs, ranging from 2 to 10 years of age. Absolute numbers of white blood cells, lymphocytes, monocytes, granulocytes, and CD3+, CD4+, CD8+ and CD21+ lymphocytes significantly decreased with increasing age. Relative percentages of lymphocytes and CD4 cells were significantly decreased, and relative percentages of granulocytes and CD8 cells significantly increased, with age. The CD4:CD8 ratio showed a significant age-related decrease. Proliferative responses of T-cells to mitogens in whole-blood cultures either increased (Concanavalin A) or remained the same (phytohemagglutinin) with age when data was normalised to allow for differences in responding cell number. Similarly, normalised data of proliferative response to anti-CD3 stimulation together with phorbol 12-myristate 13-acetate showed an age-related increase. Serum levels of total IgA significantly increased with age whereas total IgG levels remained unchanged. These observations illustrate a significant change to a number of immune parameters with age. However, further work is required to determine whether the differences reported here are sufficient to cause overt or functional immune senescence in Labrador retriever dogs. PMID:16105688

  18. The role of the Fgr tyrosine kinase in the control of the adhesive properties of U937 monoblastoid cells and their derivatives.

    PubMed Central

    Faulkner, L; Patel, M; Brickell, P M; Katz, D R

    1997-01-01

    In humans, expression of the cellular proto-oncogene c-fgr is normally restricted to mature cells of the myeloid lineage, mantle zone B cells and various myeloid and B-cell lines. Previous studies of the monoblastoid cell line, U937, showed that c-fgr expression increased following differentiation, but its role in monocytes and related cells has not been defined in functional terms. We therefore investigated the role of c-fgr in U937 cells transfected with the c-fgr gene such that its expression could be manipulated independent of differentiation. Induction of the transfected c-fgr gene by cadmium ions did not affect cell proliferation, responses to phorbol 12-myristate 13-acetate (PMA), dihydroxycholecalciferol (DHCC), tumour necrosis factor-alpha (TNF-alpha) or retinoic acid, or phagocytosis of antibody-coated sheep red blood cells. However, there was increased surface expression of CD54 (intracellular adhesion molecule-1; ICAM-1) and CD102 (ICAM-2) and decreased surface expression of CD50 (ICAM-3) compared with cells that had been transfected with plasmid only and treated in the same way. These findings suggest that the product of the c-fgr gene may be important in control of relative adhesive properties of mature monocytic cells. Images Figure 1 PMID:9497494

  19. 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. PMID:23709216

  20. Annexin A1 translocates to nucleus and promotes the expression of pro-inflammatory cytokines in a PKC-dependent manner after OGD/R.

    PubMed

    Zhao, Baoming; Wang, Jing; Liu, Lu; Li, Xing; Liu, Shuangxi; Xia, Qian; Shi, Jing

    2016-01-01

    Annexin A1 (ANXA1) is a protein known to have multiple roles in the regulation of inflammatory responses. In this study, we find that after oxygen glucose deprivation/reoxygenation (ODG/R) injury, activated PKC phosphorylated ANXA1 at the serine 27 residue (p27S-ANXA1), and promoted the translocation of p27S-ANXA1 to the nucleus of BV-2 microglial cells. This in turn induced BV-2 microglial cells to produce large amounts of pro-inflammatory cytokines. The phenomenon could be mimicked by either transfecting a mutant form of ANXA1 with its serine 27 residue converted to aspartic acid, S27D, or by using the PKC agonist, phorbol 12-myristate 13-acetate (PMA) in these microglial cells. In contrast, transfecting cells with an ANXA1 S27A mutant (serine 27 converted to alanine) or treating the cells with the PKC antagonist, GF103209X (GF) reversed this effet. Our study demonstrates that ANXA1 can be phosphorylated by PKC and is subsequently translocated to the nucleus of BV-2 microglial cells after OGD/R, resulting in the induction of pro-inflammatory cytokines. PMID:27426034

  1. Persistent induction of cyclooxygenase in p60 sup v-src -transformed 3T3 fibroblasts

    SciTech Connect

    Han, Jiawen; Sadowski, H.; Young, D.A.; Macara, I.G. )

    1990-05-01

    A BALB/c 3T3 cell line infected with the temperature-sensitive Rous sarcoma virus strain LA90 has been used to investigate early, p60{sup v-src}-dependent changes in gene expression (protein synthesis). Giant two-dimensional electrophoresis, which can resolve >3,000 polypeptides from ({sup 35}S)methionine-labeled cell lysates, was used to detect the induction of a p72-74 (72-74 kDa) doublet (pI 7.5) after activation of p60{sup v-src} at 35{degree}C. Antiserum against cyclooxygenase (prostaglandin synthase or prostaglandin endoperoxide synthase) specifically immunoprecipitated the p72-74 doublet. The p72-74 doublet was also induced by platelet-derived growth factor and by phorbol 12-myristate 13-acetate and was elevated in an NIH 3T3 cell line transformed by wild-type src. Activation of p60{sup v-src} caused a persistant increase in p72-74, whereas the effect of the growth factor was transient. These dissimilar kinetics of induction were paralleled by changes in cyclooxygenase activity. Although induction of this enzyme may not be directly involved in transformation, the data support the view that oncogenic transformation may result, not from expression of transformation-specific genes, but from persistent changes in the expression of genes normally induced only transiently during passage from the G{sub 0} stage of the cell cycle.

  2. Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translational phases

    SciTech Connect

    Raz, A.; Wyche, A.; Needleman, P. )

    1989-03-01

    The authors have recently shown that the synthesis of cyclooxygenase in human dermal fibroblasts is markedly stimulated by the cytokine interleukin 1 (IL-1). They now show that the temporal sequence of the induced synthesis of PG synthase can be separated into an early transcriptional (i.e., actinomycin D inhibitable) phase and a subsequent translational (cycloheximide but not actinomycin D inhibitable) phase and that IL-1 exerts its effect during the transcriptional phase. Phorbol 12-myristate 13-acetate also stimulates synthesis of PG synthase and, together with IL-1, produces a synergistic stimulatory effect. Inhibitors of protein kinase C activation abolished the stimulatory effect of IL-1, suggesting that protein kinase C activation is a critical event in the signal-transduction sequence of the IL-1-induced increase of PG synthase synthesis. The antiinflammatory glucocorticosteroids dexamethasone and triamcinolone, but not progesterone or testosterone, were potent inhibitors of PG synthase synthesis when added during the translational phase of the synthesis sequence. The glucocorticosteroid effect was blocked by RNA and protein synthesis inhibitors. This report suggests that glucocorticosteroids exert their effect via a newly synthesized protein, causing a profound translational control of PG synthase synthesis. This novel mechanism of suppression of arachidonate metabolism is distinct from any influence of steroids on phospholipase A{sub 2} activity.

  3. 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. PMID:26756719

  4. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.

    PubMed

    Najmeh, Sara; Cools-Lartigue, Jonathan; Giannias, Betty; Spicer, Jonathan; Ferri, Lorenzo E

    2015-01-01

    Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil's antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions. In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves. A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained. We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs. PMID:25938591

  5. The CD11c antigen couples concanavalin A binding to generation of superoxide anion in human phagocytes.

    PubMed Central

    Lacal, P M; Balsinde, J; Cabañas, C; Bernabeu, C; Sánchez-Madrid, F; Mollinedo, F

    1990-01-01

    We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils. Images Fig. 2. Fig. 3. PMID:1973035

  6. Wnt1 Participates in Inflammation Induced by Lipopolysaccharide Through Upregulating Scavenger Receptor A and NF-kB.

    PubMed

    Zhao, Wenting; Sun, Zewei; Wang, Shuai; Li, Zhenwei; Zheng, Liangrong

    2015-08-01

    The study investigated the role of wnt1 in the inflammatory response initiated by lipolysaccharide (LPS), and analyzed the association between wnt1, NF-KB, and inflammatory factors. THP-1 cells were activated with phorbol-12-myristate-13-acetate (PMA) and treated with LPS to induce inflammation. THP-1 cells were transfected with wnt1siRNA and overexpression plasmid to explore the relationship among wnt1, SRA, and NF-KB. Inhibitor of β-catenin and siRNA of FZD1were used to investigate the signaling events involved in SRA activation induced by wnt1. Levels of NF-kB protein and inflammatory cytokines were assessed followingwnt1 siRNA and LPS treatment. PMA activation and LPS treatment of THP-1 cells increased wnt1 protein levels. Wnt1 promoted SRA expression through activation of canonical wnt pathway. Wnt1 increased NF-kB protein levels and enhanced the secretion of IL-6, TNF-α, and iNOS through binding to SRA. These findings suggest that wnt1 increased SRA and NF-kB protein levels and participated in the inflammatory response. PMID:25749569

  7. Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.

    PubMed Central

    Heck, L. W.; Blackburn, W. D.; Irwin, M. H.; Abrahamson, D. R.

    1990-01-01

    To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2356859

  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. PMID:27120199

  9. Protein kinase C regulates mood-related behaviors and adult hippocampal cell proliferation in rats.

    PubMed

    Abrial, Erika; Etievant, Adeline; Bétry, Cécile; Scarna, Hélène; Lucas, Guillaume; Haddjeri, Nasser; Lambás-Señas, Laura

    2013-06-01

    The neurobiological mechanisms underlying the pathophysiology and therapeutics of bipolar disorder are still unknown. In recent years, protein kinase C (PKC) has emerged as a potential key player in mania. To further investigate the role of this signaling system in mood regulation, we examined the effects of PKC modulators in behavioral tests modeling several facets of bipolar disorder and in adult hippocampal cell proliferation in rats. Our results showed that a single injection of the PKC inhibitors tamoxifen (80 mg/kg, i.p.) and chelerythrine (3 mg/kg, s.c.) attenuated amphetamine-induced hyperlocomotion and decreased risk-taking behavior, supporting the efficacy of PKC blockade in acute mania. Moreover, chronic exposure to tamoxifen (10 mg/kg/day, i.p., for 14 days) or chelerythrine (0.3 mg/kg/day, s.c., for 14 days) caused depressive-like behavior in the forced swim test, and resulted in a reduction of cell proliferation in the dentate gyrus of the hippocampus. Finally, we showed that, contrary to the PKC inhibitors, the PKC activator phorbol 12-myristate 13-acetate (PMA) enhanced risk-taking behavior and induced an antidepressant-like effect. Taken together, these findings support the involvement of PKC in regulating opposite facets of bipolar disorder, and emphasize a major role for PKC in this disease. PMID:23228462

  10. ANKRD1 acts as a transcriptional repressor of MMP13 via the AP-1 site.

    PubMed

    Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E; Davidson, Jeffrey M

    2014-04-01

    The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1(-/-) (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1(fl/fl) (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

  11. Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils.

    PubMed

    Peak, Ian R; Chen, Adrienne; Jen, Freda E-C; Jennings, Courtney; Schulz, Benjamin L; Saunders, Nigel J; Khan, Arshad; Seifert, H Steven; Jennings, Michael P

    2016-08-01

    The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells. PMID:26562068

  12. Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

    SciTech Connect

    Matsumoto, Yohsuke; Motoki, Takahiro; Kubota, Satoshi; Takigawa, Masaharu; Tsubouchi, Hirohito; Gohda, Eiichi

    2008-02-01

    Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E{sub 2} without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.

  13. 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. PMID:19409890

  14. The antiallergic mechanisms of Citrus sunki and bamboo salt (K-ALL) in an allergic rhinitis model.

    PubMed

    Oh, Hyun-A; Kim, Myong-Jo; Shin, Tae-Yong; Kim, Hyung-Min; Jeong, Hyun-Ja

    2014-01-01

    The antiallergic effects of traditional medicines have long been studied. Traditional Korean medicine, Citrus sunki and bamboo salt, has been used for the treatment of allergic diseases in Korea. K-ALL, composed of Citrus sunki and bamboo salt, is a newly prepared prescription for allergic patients. To develop the new antiallergic agent, we examined the effects of K-ALL through in vivo and in vitro models. K-ALL and naringin (an active compound of K-ALL) significantly inhibited histamine release from rat peritoneal mast cells. This inhibitory effect of K-ALL on histamine release was higher than effects from other known histamine inhibitors such as bamboo salt, Citrus sunki or disodium cromoglycate. K-ALL significantly inhibited systemic anaphylactic shock induced by the compound 48/80 and passive cutaneous anaphylaxis induced by the IgE. K-ALL also inhibited production and mRNA expression of inflammatory cytokines induced by phorbol 12-myristate 13-acetate and the calcium ionophore A23187 on HMC-1 cells (a human mast cell line). In the ovalbumin-induced allergic rhinitis animal model, rub scores, histamine, IgE, inflammatory cytokines and inflammatory cell counts were all reduced by the oral or nasal administration of K-ALL (pre and posttreatment). These results indicate the great potential of K-ALL as an active immune modulator for the treatment of mast cell-mediated allergic diseases. PMID:24131540

  15. 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.

  16. Hepatocyte injury by activated neutrophils in vitro is mediated by proteases.

    PubMed Central

    Harbrecht, B G; Billiar, T R; Curran, R D; Stadler, J; Simmons, R L

    1993-01-01

    OBJECTIVE: This study determined the mechanism used by neutrophils (PMNs) to induce hepatocellular injury. SUMMARY BACKGROUND DATA: Neutrophils have been shown to be potent mediators of cell and tissue injury and have been hypothesized to contribute to the hepatic injury that occurs after trauma and infection. Oxygen radical scavengers protect the liver in vivo from inflammatory injury and it has been suggested that PMNs are the source of these toxic oxygen radicals. The specific mechanism used by PMNs to produce hepatocellular damage, however, has not been determined. METHODS: Neutrophils were cultured in vitro with hepatocytes (HCs) and stimulated with phorbol 12-myristate 13-acetate (PMA) to induce HC injury in the presence of oxygen radical scavengers and protease inhibitors. RESULTS: PMA induced a PMN-mediated HC injury that was dependent on the number of PMNs present and the concentration of PMA. Protease inhibitors reduced the extent of HC injury, while oxygen radical scavengers had no effect. Hydrogen peroxide, directly applied, was able to injure HCs, but only at concentrations greater than those that could be produced by PMA-stimulated PMNs. CONCLUSIONS: PMNs are cytotoxic to cultured HCs, predominantly due to the release of proteolytic enzymes, while HCs appear relatively resistant to oxidative injury. Involvement of neutrophil toxic oxygen radicals in hepatic damage in vivo may require impairment of HC antioxidant defenses or may involve injury to nonparenchymal liver cells with secondary effects on HCs. PMID:8342991

  17. 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.

  18. Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation.

    PubMed

    Ostafin, Magdalena; Pruchniak, Michal Przemyslaw; Ciepiela, Olga; Reznick, Abraham Zeev; Demkow, Urszula

    2016-09-15

    A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample. PMID:27179553

  19. The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity

    PubMed Central

    Ampofo, Emmanuel; Später, Thomas; Müller, Isabelle; Eichler, Hermann; Menger, Michael D.; Laschke, Matthias W.

    2015-01-01

    Background: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis. Methods: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time. Results: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time. Conclusion: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound. PMID:26569265

  20. Diminished Macrophage Apoptosis and Reactive Oxygen Species Generation after Phorbol Ester Stimulation in Crohn's Disease

    PubMed Central

    Palmer, Christine D.; Rahman, Farooq Z.; Sewell, Gavin W.; Ahmed, Afshan; Ashcroft, Margaret; Bloom, Stuart L.; Segal, Anthony W.; Smith, Andrew M.

    2009-01-01

    Background Crohn's Disease (CD) is a chronic relapsing disorder characterized by granulomatous inflammation of the gastrointestinal tract. Although its pathogenesis is complex, we have recently shown that CD patients have a systemic defect in macrophage function, which results in the defective clearance of bacteria from inflammatory sites. Methodology/Principal Findings Here we have identified a number of additional macrophage defects in CD following diacylglycerol (DAG) homolog phorbol-12-myristate-13-acetate (PMA) activation. We provide evidence for decreased DNA fragmentation, reduced mitochondrial membrane depolarization, impaired reactive oxygen species production, diminished cytochrome c release and increased IL-6 production compared to healthy subjects after PMA exposure. The observed macrophage defects in CD were stimulus-specific, as normal responses were observed following p53 activation and endoplasmic reticulum stress. Conclusion These findings add to a growing body of evidence highlighting disordered macrophage function in CD and, given their pivotal role in orchestrating inflammatory responses, defective apoptosis could potentially contribute to the pathogenesis of CD. PMID:19907654

  1. Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling.

    PubMed

    Chandrika, Goparaju; Natesh, Kumar; Ranade, Deepak; Chugh, Ashish; Shastry, Padma

    2016-01-01

    Glioblastoma (GBM) is the most aggressive type of brain tumors in adults with survival period <1.5 years of patients. The role of mTOR pathway is documented in invasion and migration, the features associated with aggressive phenotype in human GBM. However, most of the preclinical and clinical studies with mTOR inhibitors are focused on antiproliferative and cytotoxic activity in GBM. In this study, we demonstrate that mTOR inhibitors-rapamycin (RAP), temisirolimus (TEM), torin-1 (TOR) and PP242 suppress invasion and migration induced by Tumor Necrosis Factor-α (TNFα) and tumor promoter, Phorbol 12-myristate 13-acetate (PMA) and also reduce the expression of the TNFα and IL1β suggesting their potential to regulate factors in microenvironment that support tumor progression. The mTOR inhibitors significantly decreased MMP-2 and MMP-9 mRNA, protein and activity that was enhanced by TNFα and PMA. The effect was mediated through reduction of Protein kinase C alpha (PKC-α) activity and downregulation of NFκB. TNFα- induced transcripts of NFκB targets -VEGF, pentraxin-3, cathepsin-B and paxillin, crucial in invasion were restored to basal level by these inhibitors. With limited therapeutic interventions currently available for GBM, our findings are significant and suggest that mTOR inhibitors may be explored as anti-invasive drugs for GBM treatment. PMID:26940200

  2. Okadaic acid mimics multiple changes in early protein phosphorylation and gene expression induced by tumor necrosis factor or interleukin-1.

    PubMed

    Guy, G R; Cao, X; Chua, S P; Tan, Y H

    1992-01-25

    Okadaic acid, a phosphatase inhibitor from a marine organism, mimics tumor necrosis factor/interleukin-1 (TNF/IL-1) in inducing changes in early cellular protein phosphorylation. A total of approximately 116 proteins exhibit significant and concordant changes in phosphorylation or dephosphorylation within 15 min in human fibroblasts activated by either okadaic acid, TNF, or IL-1. The fidelity of this mimicry by okadaic acid extends to the phosphorylation of the 27 hsp complex, stathmin, eIF-4E, myosin light chain, nucleolin, epidermal growth factor receptor, and other cdc2-kinase substrates (c-abl, RB, and p53). The okadaic acid-induced pattern of protein phosphorylation is distinct from that observed in cells treated with phorbol 12-myristate 13-acetate or with ligands like epidermal growth factor, cyclic AMP agonists, bradykinin, or interferons. Like TNF, okadaic acid also induces the transcription of immediate early response genes like c-jun and Egr-1 as well as the interleukin-6 genes. The overall early effects of okadaic acid uniquely parallel those of TNF/IL-1 and not those of other cytokines or ligands. Regulation of protein phosphatase inhibition is discussed as a mechanism for TNF/IL-1 signal transduction. PMID:1370482

  3. 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.

  4. Oxidation of methionine residues in proteins of activated human neutrophils.

    PubMed Central

    Fliss, H; Weissbach, H; Brot, N

    1983-01-01

    A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins. PMID:6580633

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

    PubMed Central

    Cho, Seok-Cheol; Choi, Bu Young

    2015-01-01

    Acetylshikonin, a natural naphthoquinone derivative compound, has been used for treatment of inflammation and cancer. In the present study, we have investigated whether acetylshikonin could regulate the NF-κB signaling pathway, thereby leading to suppression of tumorigenesis. We observed that acetylshikonin significantly reduced proliferation of several cancer cell lines, including human pancreatic PANC-1 cancer cells. In addition, acetylshikonin inhibited phorbol 12-myristate 13-acetate (PMA) or tumor necrosis-α (TNF-α)-induced NF-κB reporter activity. Proteome cytokine array and real-time RT-PCR results illustrated that acetylshikonin inhibition of PMA-induced production of cytokines was mediated at the transcriptional level and it was associated with suppression of NF-κB activity and matrix metalloprotenases. Finally, we observed that an exposure of acetylshikonin significantly inhibited the anchorage-independent growth of PANC-1 cells. Together, our results indicate that acetylshikonin could serve as a promising therapeutic agent for future treatment of pancreatic cancer. PMID:26336582

  6. Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages

    PubMed Central

    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. PMID:27120199

  7. Tetrandrine suppresses pro-inflammatory mediators in PMA plus A23187-induced HMC-1 cells.

    PubMed

    Kang, Ok-Hwa; An, Hyeon-Jin; Kim, Sung-Bae; Mun, Su-Hyun; Seo, Yun-Soo; Joung, Dae-Ki; Choi, Jang-Gi; Shin, Dong-Won; Kwon, Dong-Yeul

    2014-05-01

    Tetrandrine (TET), a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, is known to possess antitumor activity in various malignant neoplasms. However, the precise mechanism of TET-mediated immune modulation remains to be clarified. One of the possible mechanisms for its protective properties is by downregulation of the inflammatory responses. In the present study, the human mast cell line (HMC-1) was used to investigate this effect. TET significantly inhibited the induction of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 by phorbol 12-myristate 13-acetate (PMA) plus A23187. Moreover, TET attenuated expression of cyclooxygenase (COX)-2. In activated HMC-1 cells, the phosphorylation of extra-signal response kinase (ERK1/2) and c-jun N-terminal Kinase (JNK1/2), but not p38 mitogen-activated protein kinase, was decreased by treatment of the cells with TET. TET inhibited PMA plus A23187-induced nuclear factor (NF)-κB activation, IκB degradation and phosphorylation. Furthermore, TET suppressed the expression of TNF-α, IL-8, IL-6 and COX-2 through suppression of the ERK1/2, JNK1/2, IκBα degradation and phosphorylation, and NF-κB activation. These results indicated that TET exerted a regulatory effect on inflammatory reactions mediated by mast cells. PMID:24589569

  8. Scanning Electrochemical Microscopy Imaging during Respiratory Burst in Human Cell

    PubMed Central

    Kikuchi, Hiroyuki; Prasad, Ankush; Matsuoka, Ryo; Aoyagi, Shigeo; Matsue, Tomokazu; Kasai, Shigenobu

    2016-01-01

    Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials. PMID:26903876

  9. 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

  10. 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.

  11. Contractions Activate Hormone-Sensitive Lipase in Rat Muscle by Protein Kinase C and Mitogen-Activated Protein Kinase

    PubMed Central

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia; Ploug, Thorkil; Galbo, Henrik

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50 % by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway. PMID:12794177

  12. Identification of novel oxidized levuglandin D2 in marine red alga and mouse tissue[S

    PubMed Central

    Kanai, Yoshikazu; Hiroki, Sadahiko; Koshino, Hiroyuki; Konoki, Keiichi; Cho, Yuko; Cayme, Mirriam; Fukuyo, Yasuo; Yotsu-Yamashita, Mari

    2011-01-01

    In animals, the product of cyclooxygenase reacting with arachidonic acid, prostaglandin(PG)H2, can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form levuglandin(LG)E2 and LGD2. These LGs and their isomers are highly reactive γ-ketoaldehydes that form covalent adducts with proteins, DNA, and phosphatidylethanolamine in cells. Here, we isolated a novel oxidized LGD2 (ox-LGD2) from the red alga Gracilaria edulis and determined its planar structure. Additionally, ox-LGD2 was identified in some tissues of mice and in the lysate of phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells incubated with arachidonic acid using LC-MS/MS. These results suggest that ox-LGD2 is a common oxidized metabolite of LGD2. In the planar structure of ox-LGD2, H8 and H12 of LGD2 were dehydrogenated and the C9 aldehyde was oxidized to a carboxylic acid, which formed a lactone ring with the hydrated ketone at C11. These structural differences imply that ox-LGD2 is less reactive with amines than LGs. Therefore, ox-LGD2 might be considered a detoxification metabolite of LGD2. PMID:21893678

  13. Inhibition of ATP release from Erythrocytes: A role for EPACs and PKC

    PubMed Central

    Adderley, Shaquria P.; Sridharan, Meera; Bowles, Elizabeth A.; Stephenson, Alan H.; Sprague, Randy S.; Ellsworth, Mary L.

    2010-01-01

    Objective Here we demonstrate that, in human erythrocytes, increases in cAMP that are not localized to a specific receptor-mediated signaling pathway for ATP release can activate effector proteins resulting in inhibition of ATP release. Specifically we sought to establish that exchange proteins activated by cAMP (EPACs) inhibit ATP release via activation of protein kinase C (PKC). Methods ATP release stimulated by iloprost (ILO), or isoproterenol (ISO), was determined in the absence and presence of selective phosphodiesterase inhibitors and/or the EPAC activator, 8CPT2OMecAMP (8CPT). To determine whether EPACs inhibit ATP release via activation of PKC, erythrocytes were incubated with phorbol 12-myristate 13-acetate (PMA) prior to either forskolin or ILO in the absence and presence of a PKC inhibitor, calphostin C (CALC). Results Selective inhibition of PDEs in one pathway inhibited ATP release in response to activation of the other cAMP-dependent pathway. 8CPT and PMA inhibited both ILO- and ISO-induced ATP release. Inhibition of ATP release with 8CPT was rescued by CALC. Conclusion These results support the hypothesis that cAMP not localized to a specific signaling pathway can activate EPACs which inhibit ATP release via activation of PKC and suggest a novel role for EPACs in erythrocytes. PMID:21166931

  14. Stimulation of low-density lipoprotein uptake in HepG2 cells by epidermal growth factor via a tyrosine kinase-dependent, but protein kinase C-independent, mechanism.

    PubMed Central

    Graham, A; Russell, L J

    1994-01-01

    Epidermal growth factor (EGF), a potent mitogenic polypeptide, stimulated the uptake and degradation of [3H]sucrose-labelled low-density lipoprotein (LDL) by HepG2 cells. The increase in LDL uptake was prevented by the presence of the tyrosine kinase inhibitor genistein. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) also stimulated the uptake of [3H]LDL by HepG2 cells. When EGF and PMA were added together, PMA increased the response to EGF in an additive manner. The protein kinase C inhibitor Ro-31-8220 prevented the increase in LDL uptake caused by PMA, but did not affect EGF stimulation of LDL uptake. Similarly, down-regulation of protein kinase C activity by chronic treatment with PMA also did not affect the EGF stimulation of LDL uptake. These results suggest that the EGF stimulation of LDL uptake and degradation by HepG2 cells is mediated by a tyrosine kinase-dependent, but protein kinase C-independent, mechanism. PMID:8141769

  15. Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production.

    PubMed

    Bal-Price, Anna; Matthias, Anita; Brown, Guy C

    2002-01-01

    Cultured rat microglia produced extracellular superoxide at a rate of 814 +/- 52 pmol/min/million cells when stimulated with phorbol 12-myristate 13-acetate (PMA) as measured by extracellular cytochrome c reduction. This superoxide production resulted in a rapid rate of superoxide dismutase-sensitive nitric oxide (NO) breakdown (155 +/- 30 pmol of NO/min/million cells) when NO was added to PMA stimulated microglia. Lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-activated microglia produce NO at the rate of 145 +/- 42 pmol/min/million cells and activated astrocytes at the rate of 51 +/- 9 pmol/min/million cells as estimated by NO electrode. Both types of cells maintained a steady-state level of 0.5-0.7 microm NO, only in the presence of L-arginine. Addition of PMA to activated microglia (but not activated astrocytes) caused the rapid and complete disappearance of all extracellular NO (but was restored in the presence of superoxide dismutase) followed by the production of peroxynitrite (as measured by urate-sensitive oxidation of dihydrorhodamine). Co-incubation of activated microglia with cerebellar granule neurones resulted in NO inhibition of neuronal respiration, but this was rapidly removed by PMA-induced breakdown of the NO. Thus, microglial NADPH oxidase can regulate the bioavailability of NO and the production of peroxynitrite. PMID:11796745

  16. 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.

  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 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

  18. Annexin A1 translocates to nucleus and promotes the expression of pro-inflammatory cytokines in a PKC-dependent manner after OGD/R

    PubMed Central

    Zhao, Baoming; Wang, Jing; Liu, Lu; Li, Xing; Liu, Shuangxi; Xia, Qian; Shi, Jing

    2016-01-01

    Annexin A1 (ANXA1) is a protein known to have multiple roles in the regulation of inflammatory responses. In this study, we find that after oxygen glucose deprivation/reoxygenation (ODG/R) injury, activated PKC phosphorylated ANXA1 at the serine 27 residue (p27S-ANXA1), and promoted the translocation of p27S-ANXA1 to the nucleus of BV-2 microglial cells. This in turn induced BV-2 microglial cells to produce large amounts of pro-inflammatory cytokines. The phenomenon could be mimicked by either transfecting a mutant form of ANXA1 with its serine 27 residue converted to aspartic acid, S27D, or by using the PKC agonist, phorbol 12-myristate 13-acetate (PMA) in these microglial cells. In contrast, transfecting cells with an ANXA1 S27A mutant (serine 27 converted to alanine) or treating the cells with the PKC antagonist, GF103209X (GF) reversed this effet. Our study demonstrates that ANXA1 can be phosphorylated by PKC and is subsequently translocated to the nucleus of BV-2 microglial cells after OGD/R, resulting in the induction of pro-inflammatory cytokines. PMID:27426034

  19. Biological and Nonbiological Antioxidant Activity of Some Essential Oils.

    PubMed

    Pérez-Rosés, Renato; Risco, Ester; Vila, Roser; Peñalver, Pedro; Cañigueral, Salvador

    2016-06-15

    Fifteen essential oils, four essential oil fractions, and three pure compounds (thymol, carvacrol, and eugenol), characterized by gas chromatography and gas chromatography-mass spectrometry, were investigated for biological and nonbiological antioxidant activity. Clove oil and eugenol showed strong DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical scavenging activity (IC50 = 13.2 μg/mL and 11.7 μg/mL, respectively) and powerfully inhibited reactive oxygen species (ROS) production in human neutrophils stimulated by PMA (phorbol 12-myristate 13-acetate) (IC50 = 7.5 μg/mL and 1.6 μg/mL) or H2O2 (IC50 = 22.6 μg/mL and 27.1 μg/mL). Nutmeg, ginger, and palmarosa oils were also highly active on this test. Essential oils from clove and ginger, as well as eugenol, carvacrol, and bornyl acetate inhibited NO (nitric oxide) production (IC50 < 50.0 μg/mL). The oils of clove, red thyme, and Spanish oregano, together with eugenol, thymol, and carvacrol showed the highest myeloperoxidase inhibitory activity. Isomers carvacrol and thymol displayed a disparate behavior in some tests. All in all, clove oil and eugenol offered the best antioxidant profile. PMID:27214068

  20. 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

  1. RbAp48, a novel inhibitory factor that regulates the transcription of human immunodeficiency virus type 1.

    PubMed

    Wang, Juan; Yang, Jin; Yang, Zongxing; Lu, Xiangyun; Jin, Changzhong; Cheng, Linfang; Wu, Nanping

    2016-07-01

    Retinoblastoma binding protein 4 (RbAp48) is a histone chaperone which has been suggested to play a role in gene silencing. However, the role of RbAp48 in human immunodeficiency virus type 1 (HIV-1) infection and gene replication has not been determined to date, to the best of our knowledge. For this purpose, we demonstrated in the present study that RbAp48 expression was upregulated by HIV-1 infection, whereas the knockdown of RbAp48 promoted HIV infection and the production of virus particles. The ectopic expression of RbAp48 inhibited HIV-1 expression, and this inhibition correlated with a marked decrease in the expression of HIV-1 genomic RNA and various RNA transcripts. Further experiments to determine the mechanism responsible for the inhibitory effects of RbAp48 revealed that the ectopic expression of RbAp48 repressed HIV-1 long terminal repeat (LTR)-mediated basal transcription as well as TNF-α- and phorbol 12-myristate 13-acetate (PMA)‑activated transcription. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis revealed that RbAp48 binds to the HIV-1 LTR in vitro. Taken together, these findings demonstrate that, as a transcriptional cofactor, RbAp48 is likely to act as a potent antiretroviral defense. PMID:27222146

  2. Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes.

    PubMed Central

    Grunberger, G; Zick, Y; Taylor, S I; Gorden, P

    1984-01-01

    Solubilized lectin-purified extracts from human monocyte-like cells (U-937) and freshly isolated human mononuclear cells preincubated in the presence of phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of synthetic tyrosine-containing polymers and of casein. Tyrosine phosphorylation was confirmed by phospho amino acid analysis. PMA stimulated phosphorylation of exogenous substrates in a time- and concentration-dependent manner. This phosphorylation reaction did not require addition of phospholipid, diolein, or calcium. Biologically inactive phorbol compounds did not stimulate phosphorylation in this system. In addition, PMA enhanced phosphorylation of a Mr approximately equal to 140,000 protein as well as several other endogenous proteins in the U-937 extracts. PMA treatment stimulated predominantly phosphorylation on tyrosine residues of the Mr 140,000 protein. Tyrosine phosphorylation, typical of growth-promoting peptides such as insulin or epidermal growth factor, is believed to play a role in regulating normal and disordered cellular growth and proliferation. The demonstration of PMA-stimulated tyrosine phosphorylation might provide a clue to the mechanism of cellular differentiation and proliferation induced by the tumor promoter. Images PMID:6201862

  3. On-line HPLC-electrospray ionization mass spectrometry: a pharmacological tool for identifying and studying the stability of Gd3+ complexes used as magnetic resonance imaging contrast agents.

    PubMed

    Behra-Miellet, J; Briand, G; Kouach, M; Gressier, B; Cazin, M; Cazin, J C

    1998-01-01

    The identification of MRI contrast agents (CAg) as gadolinium complexes often used at very low concentrations in Pharmacology was carried out by ESI-MS or HPLC-ESI-MS. Firstly, Omniscan, Dotarem and Magnevist were tested. In these compounds, the Gd3+ ion must be solidly chelated by linear or macrocyclic ligands because of the severe toxicity of the free Gd3+. Spectra were obtained at low voltage, preserving the non-covalent binding integrity of the complexes, and at various higher voltages showing the progressive destruction of the complexes. Secondly, a direct reaction of these drugs with the oxidative human neutrophil production, induced in vitro by Phorbol 12-myristate 13-acetate enhancing the respiratory burst, was investigated. This was done to mimic what happens in the case of inflammatory diseases, or infection, or when people are likely to develop anaphylactoid reactions, as the i.v. injection of CAg causes contact between the complexes and neutrophils in the blood. Analysis by HPLC-ESI-MS coupling did not show any direct reaction between Gd complexes and the chemical compounds in the neutrophil oxidative metabolism, even if uncertainty remains as regards meglumine salt. HPLC-ESI-MS is a good way of visualizing characteristic, Gd isotopic distribution and of following its associations in biological samples. PMID:9470970

  4. EMMPRIN Is Secreted by Human Uterine Epithelial Cells in Microvesicles and Stimulates Metalloproteinase Production by Human Uterine Fibroblast Cells

    PubMed Central

    Dayger, C. A.; Mehrotra, P.; Belton, R. J.; Nowak, R. A.

    2012-01-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C. PMID:22729071

  5. Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans.

    PubMed

    Pejler, Gunnar; Winberg, Jan-Olof; Vuong, Tram T; Henningsson, Frida; Uhlin-Hansen, Lars; Kimata, Koji; Kolset, Svein O

    2003-10-01

    The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9. PMID:14511379

  6. 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. PMID:26874672

  7. Regulation of Receptor for Advanced Glycation End Products (RAGE) Ectodomain Shedding and Its Role in Cell Function.

    PubMed

    Braley, Alex; Kwak, Taekyoung; Jules, Joel; Harja, Evis; Landgraf, Ralf; Hudson, Barry I

    2016-06-01

    The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that can undergo proteolysis at the cell surface to release a soluble ectodomain. Here we observed that ectodomain shedding of RAGE is critical for its role in regulating signaling and cellular function. Ectodomain shedding of both human and mouse RAGE was dependent on ADAM10 activity and induced with chemical activators of shedding (ionomycin, phorbol 12-myristate 13-acetate, and 4-aminophenylmercuric acetate) and endogenous stimuli (serum and RAGE ligands). Ectopic expression of the splice variant of RAGE (RAGE splice variant 4), which is resistant to ectodomain shedding, inhibited RAGE ligand dependent cell signaling, actin cytoskeleton reorganization, cell spreading, and cell migration. We found that blockade of RAGE ligand signaling with soluble RAGE or inhibitors of MAPK or PI3K blocked RAGE-dependent cell migration but did not affect RAGE splice variant 4 cell migration. We finally demonstrated that RAGE function is dependent on secretase activity as ADAM10 and γ-secretase inhibitors blocked RAGE ligand-mediated cell migration. Together, our data suggest that proteolysis of RAGE is critical to mediate signaling and cell function and may therefore emerge as a novel therapeutic target for RAGE-dependent disease states. PMID:27022018

  8. Eomesodermin promotes interferon-γ expression and binds to multiple conserved noncoding sequences across the Ifng locus in mouse thymoma cell lines.

    PubMed

    Fukuoka, Natsuki; Harada, Misuzu; Nishida, Ai; Ito, Yuko; Shiota, Hideki; Kataoka, Takao

    2016-02-01

    The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-γ (IFN-γ). However, in contrast to T-bet, the role of Eomes in the expression of IFN-γ remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-γ in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners. PMID:26749212

  9. Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription.

    PubMed

    Marek-Bukowiec, K; Aguado, E; Miazek, A

    2016-07-01

    Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently upregulated upon T-cell receptor (TCR) engagement, but molecular mechanisms conveying TCR signaling to enhanced LAT transcription are not fully understood. Here we found that a Jurkat subline J.CaM2, initially characterized as LAT deficient, conditionally re-expressed LAT upon the treatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). We took advantage of the above observation for studying cis-elements and trans-acting factors contributing to the activation-induced expression of LAT. We identified a LAT gene region spanning nucleotide position -14 to +357 relative to the ATG start codon as containing novel cis-regulatory elements that were able to promote PMA-induced reporter transcription in the absence of the core LAT promoter. Interestingly, a point mutation in LAT intron 1, identified in J.CaM2 cells, downmodulated LAT promoter activity by 50%. Mithramycin A, a selective Sp1 DNA-binding inhibitor, abolished LAT expression upon PMA treatment as did calcium ionophore ionomycin (Iono) and valproic acid (VPA), widely used as an anti-epileptic drug. Our data introduce J.CaM2 cells as a model for dissecting drivers and blockers of activation induced expression of LAT. PMID:27278128

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

    PubMed Central

    Hou, Shuai; Shen, Ping-Ping; Zhao, Ming-Ming; Liu, Xiu-Ping; Xie, Hong-Yan; Deng, Fang; Feng, Jia-Chun

    2016-01-01

    We observed mitochondrial connexin43 (mtCx43) expression under cerebral ischemia-reperfusion (I/R) injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO). Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. MtCx43, p-mtCx43, protein kinase C (PKC), and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX) and diazoxide (DZX) groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD) groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA) reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists. PMID:27164087

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

    PubMed Central

    Miñana, M D; Felipo, V; Grisolía, S

    1990-01-01

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 microM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 microM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased approximately 7-fold after 48 hr with 500 microM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed. Images PMID:1693437

  12. In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.

    PubMed Central

    Borghini, I; Geering, K; Gjinovci, A; Wollheim, C B; Pralong, W F

    1994-01-01

    The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves. Images PMID:8016140

  13. Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei.

    PubMed

    Homa, Joanna; Zorska, Anna; Wesolowski, Dawid; Chadzinska, Magdalena

    2013-04-01

    Due to the specific habitat conditions in which they live, earthworms are constantly exposed to pathogens. Consequently, they have evolved various immuno-defense mechanisms, including cellular (coelomocytes) and humoral responses, which may help to eliminate deleterious micro-organisms but also repair and/or protect host cells and tissues. Similar to mammalian phagocytes, coelomocytes can kill ingested pathogens with reactive oxygen species (ROS) and nitric oxide. In the present work, we studied the effects of the dermal exposure of Eisenia andrei earthworms to different immuno-stimulants: phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) or concanavalin A (ConA). After 3 days of treatment with all immuno-stimulants, decreased numbers and changed composition of the coelomocytes were observed. The immuno-stimulants also induced numerous changes in bactericidal activity, including ROS production. Furthermore, all stimulants increased cell proliferation while only LPS-treatment significantly elevated apoptosis of coelomocytes. These results demonstrate that in vivo treatment of earthworms with immuno-stimulants induces various changes in their coelomocyte response. PMID:23014884

  14. Glaucine inhibits breast cancer cell migration and invasion by inhibiting MMP-9 gene expression through the suppression of NF-κB activation.

    PubMed

    Kang, Hyereen; Jang, Sung-Wuk; Pak, Jhang Ho; Shim, Sungbo

    2015-05-01

    Matrix metalloproteinase-9 (MMP-9) plays a central role in the invasion and metastasis of various types of cancer cells. Here, we demonstrate that glaucine, an alkaloid isolated from the plant Corydalis turtschaninovii tuber (Papaveraceae), can inhibit the migration and invasion of human breast cancer cells. We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner. Results from reporter gene and electrophoretic mobility shift assays revealed that glaucine inhibits MMP-9 expression by suppressing activation of the nuclear transcription factor nuclear factor-κB (NF-κB). Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB. Finally, we also found that glaucine inhibits invasion and MMP-9 expression in the highly metastatic MDA-MB-231 breast cancer cell line. Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness. PMID:25670016

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

    SciTech Connect

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

    1988-02-01

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

  16. Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells

    SciTech Connect

    Cannistra, S.A.; Groshek, P.; Griffin, J.D. ); Garlick, R.; Miller, J. )

    1990-01-01

    Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors. By using radiolabeled GM-CSF with high specific activity, the authors have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, they also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.

  17. 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.

  18. 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.

  19. The Na+/H+ exchange inhibitor HOE642 prevents stress-induced epithelial barrier dysfunction.

    PubMed

    Nowak, Peter; Blaheta, Roman; Schuller, Alina; Cinatl, Jindrich; Wimmer-Greinecker, Gerhard; Moritz, Anton; Scholz, Martin

    2004-08-01

    Recently, evidence has been obtained that the Na+/H+ exchange (NHE) inhibitor HOE642 may stabilize endothelial and epithelial barrier function in vivo. However, the underlying mechanisms are not known. Therefore, we studied the influence of HOE642 on the barrier function of the epithelial cell line CaCo2. The phorbolester phorbol 12-myristate 13-acetate (PMA) was used to induce hyperpermeability of the epithelial layer which was indirectly determined by measuring the transepithelial electrical resistance (TER). Confocal laser scan microscopy (LSM) served to analyze the intracellular localization of adherens and tight junction molecules. In five independent experiments we found that HOE642 increased TER in non-treated CaCo2 cells (control: 350 +/- 28 Omega/cm2; HOE642: 444 +/- 53 Omega/cm2) and prevented PMA-induced barrier dysfunction (PMA: 33 +/- 12 Omega/cm2; PMA plus HOE642: 496 +/- 47 Omega/cm2). LSM showed that HOE642 prevented the PMA-induced disassociation of the zonula adherens molecule beta-catenin from the cell membrane and the decreased expression of the zonula occludens molecule ZO-1. From our data we conclude that HOE642 may prevent stress-induced epithelial dysfunction by stabilization of cell membrane-associated junction molecules. PMID:15254761

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

    PubMed

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

    2012-12-01

    This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. PMID:22407864

  1. Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells

    SciTech Connect

    Didolkar, A.K.; Sundaram, K.

    1987-07-27

    Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to insositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA) could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA augmented hCG induced T secretion, while at higher concentrations they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5,8,11,14 Eicosatetrayenoic acid, a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. The authors conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclo-oxygenase products. 28 references, 4 figures, 2 tables.

  2. 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. PMID:21344174

  3. 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

    2016-01-01

    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

  4. Effects of Modified Simiao Decoction on IL-1β and TNFα Secretion in Monocytic THP-1 Cells with Monosodium Urate Crystals-Induced Inflammation

    PubMed Central

    Liu, Ya-Fei; Tu, Sheng-Hao; Chen, Zhe; Wang, Yu; Hu, Yong-Hong; Dong, Hui

    2014-01-01

    Simiao pill, a Chinese herbal formula containing four herbs, has been used in the treatment of gouty arthritis for many years. The aim of this study was to explore the effects of modified Simiao decoction (MSD) on IL-1β and TNFα secretion in monocytic THP-1 cells with monosodium urate (MSU) crystals-induced inflammation. The MSU crystals-induced inflammation model in THP-1 cells was successfully established by the stimulation of phorbol 12-myristate 13-acetate (PMA) and MSU crystals. Then, the MSD-derived serum or control serum extracted from rat was administered to different treatment groups. The morphology of MSU crystals and THP-1 cells was observed. IL-1β and TNFα protein expression in supernatant of THP-1 cells were determined by ELISA. Our data demonstrated that MSU crystals induced time-dependent increase of IL-1β and TNFα. Moreover, MSD significantly decreased IL-1β release in THP-1 cells with MSU crystals-induced inflammation. These results suggest that MSD is promising in the treatment of MSU crystals-induced inflammation in THP-1 cells. MSD may act as an anti-IL-1 agent in treating gout. The underlying mechanism may be related to NALP3 inflammasome which needs to be validated in future studies. PMID:24999366

  5. 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

  6. 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.

  7. Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitro.

    PubMed

    Gönczi, Mónika; Szentandrássy, Norbert; Fülöp, László; Telek, Andrea; Szigeti, Gyula P; Magyar, János; Bíró, Tamás; Nánási, Péter P; Csernoch, László

    2007-04-01

    Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of -27 +/- 4 to -51 +/- 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases. PMID:17359336

  8. Transactivation of human immunodeficiency virus type 1 long terminal repeats by cell surface tumor necrosis factor alpha.

    PubMed Central

    Tadmori, W; Mondal, D; Tadmori, I; Prakash, O

    1991-01-01

    Tumor necrosis factor alpha (TNF-alpha) is expressed in secreted and cell surface (csTNF-alpha) forms by activated monocytic and T cells. In this report, we demonstrate that csTNF-alpha may predominantly regulate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activation in the promonocytic cell line U937 and in the Epstein-Barr virus-transformed B-cell line BH1. Anti-TNF-alpha antibody suppressed both the constitutive expression of the HIV-1 LTR in BH1 cells and the expression induced by phorbol 12-myristate 13-acetate in U937 cells. This suppression was found to be mediated via csTNF-alpha. No correlation between the HIV-1 LTR activation and the secretion of TNF-alpha was evident in these cell lines. Suppression of TNF-alpha secretion by cyclosporin A or by a serine protease inhibitor did not suppress the HIV-1 LTR activation. These observations suggest a novel biological role for csTNF-alpha in the immunopathogenesis of AIDS. PMID:1942242

  9. Glucocorticosteroids and in vitro effects on chemiluminescence of isolated bovine blood granulocytes.

    PubMed

    Hoeben, D; Burvenich, C; Massart-Leën, A M

    1998-08-01

    The effects of glucocorticosteroids on respiratory burst of bovine granulocytes were studied in vitro by means of (1) chemiluminescence (luminol-dependent, phorbol 12-myristate 13-acetate (PMA)-stimulated), (2) a cell-free chemiluminescence assay, and (3) a myeloperoxidase assay. Significant effects on cellular chemiluminescence were only observed at the highest, not obtainable in vivo, concentration for all drugs except betamethasone. Prednisolone induced inhibition at therapeutic doses. Also, flumethasone and dexamethasone induced significant inhibition at lower concentrations. In the cell-free assay, all glucocorticosteroids, except betamethasone, inhibited chemiluminescence at high concentrations. None of the glucocorticosteroids tested affected myeloperoxidase activity. The results indicated that the drugs do not affect NADPH-oxidase activity. The adverse effects may be due to scavenging of free oxygen radicals, or to interference with the interaction between luminol and the myeloperoxidase-H2O2-halide system. It can be concluded that most glucocorticosteroids show no adverse effects on the respiratory burst of bovine granulocytes in vitro at therapeutical concentrations. PMID:9754921

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

    SciTech Connect

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

    1987-04-01

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

  11. 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.

  12. Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density*

    PubMed Central

    Manna, Paul T.; Smith, Andrew J.; Taneja, Tarvinder K.; Howell, Gareth J.; Lippiat, Jonathan D.; Sivaprasadarao, Asipu

    2010-01-01

    Pancreatic ATP-sensitive potassium (KATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed KATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that KATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases KATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native KATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated KATP channel trafficking may play a role in the regulation of insulin secretion. PMID:20026601

  13. Bryostatins activate protein kinase C in intact human platelets

    SciTech Connect

    Smith, J.B.; Tallant, E.A.; Pettit, G.R.; Wallace, R.W.

    1986-05-01

    Bryostatins, macrocyclic lactones isolated from a marine bryozoan, have antineoplastic activity in the P388 lymphocytic leukemia system. These compounds also stimulate growth in Swiss 3T3 cells, induce secretion in leukocytes, inhibit phorbol dibutyrate binding to a high affinity receptor, and activate the C-kinase in vitro. In human platelets, phorbol esters induce aggregation and activate protein kinase C, resulting in phosphorylation of a 47K protein and the 20K myosin light chain. The authors now show that bryostatin 7 (B-7) triggers platelet aggregation to the same rate and extent as phorbol 12-myristate 13-acetate (PMA). B-7 also causes the in vivo activation of the C-kinase, resulting in phosphorylation of both the 47K and the 20K proteins; the time courses and dose-responses of these B-7-induced phosphorylations were similar to those found with PMA. In addition, B-7 increases the level of /sup 32/P-incorporation into the platelet polyphosphoinositides, which also occurs in response to PMA. Bryostatin 3 (B-3), which has been shown to be much less potent than B-7 in mimicking other PMA effects, was much less effective than PMA or B-7 in inducing platelet aggregation and in stimulating /sup 32/P-incorporation into both proteins and the phosphoinositides. These results demonstrate that, intact human platelets, bryostatins mimic the phorbol esters tumor promoters and directly activate protein kinase C.

  14. 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.

  15. Promoted megakaryocytic differentiation of K562 cells through oxidative stress caused by near ultraviolet irradiation.

    PubMed

    Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Nomura, Naoki; Taya, Masahito

    2014-12-01

    Reactive oxygen species (ROS) have been proven to be important activators for various cellular activities, including cell differentiation. Several reports showed the necessity of ROS during cell differentiation of the megakaryocytic (MK) lineage. In this study, we employed near ultraviolet (near-UV) irradiation to generate endogenous oxidative stress in an MK differentiation process of K562 cells with phorbol 12-myristate 13-acetate (PMA) induction. A significant increase in the intracellular ROS level was detected on day 1 after near-UV irradiation. In the initial stage of differentiation, a shifted fraction of G1 and G2 phase cells was obtained using near-UV irradiation, giving an increased percentage of G2 phase cells (up from 31.1 to 68.7%). The near-UV irradiation-induced upregulation of the p21 gene, which is a cell cycle inhibitor, suggested that the G2 phase cells were prevented from undergoing cell division. It was found that the percentage of high ploidy (8N and 16N) cells was enhanced significantly at the later stage of the K562 cell culture with near-UV irradiation. Moreover, time-lapse analysis showed that near-UV irradiation encouraged the expression of CD41, a specific surface marker of megakaryocytes. This is the first report that the elevated oxidative stress through the near-UV irradiation promoted the MK differentiation of PMA-induced K562 cells. PMID:25338769

  16. Studies on transcriptional regulation of the mucosal T-cell integrin αEβ7 (CD103)

    PubMed Central

    Robinson, Paul W; Green, Sally J; Carter, Christine; Coadwell, John; Kilshaw, Peter J

    2001-01-01

    Integrin αEβ7 is expressed almost exclusively by mucosal T cells and mucosal dendritic antigen-presenting cells (APCs) and is thought to be induced locally by transforming growth factor-β (TGF-β). In mice, mRNA for the αE subunit was found to be abundant in mucosal T cells but absent from other tissues. Exposure of a T-cell line to TGF-β strongly up-regulated αE mRNA levels within 30 min, and nuclear run-on experiments established that regulation occurred at the level of transcription. The organization of the human αE gene and a very closely linked novel gene, ELG, was determined. The αE promoter was tested in T cells and fibroblasts and functioned equally well in both cell types and did not confer TGF-β responsiveness. Regions of the promoter providing enhancer activity and phorbol 12-myristate 13-acetate (PMA) responsiveness were identified by deletion studies. DNAse 1 hypersensitivity analysis of 36 kb of the αE gene revealed one hypersensitive site, found only in αE+ cells, located near the transcription start points. These results show that, unlike the situation with other integrins, lineage specificity and cytokine responsiveness of αE transcription are not conferred by the proximal promoter. Specificity may depend on distant control elements that have not yet been identified. PMID:11412301

  17. Nimbidin suppresses functions of macrophages and neutrophils: relevance to its antiinflammatory mechanisms.

    PubMed

    Kaur, Gurpreet; Sarwar Alam, M; Athar, M

    2004-05-01

    Nimbidin is a mixture of tetranortriterpenes and is the major active principle of the seed oil of Azadirachta indica A. Juss (Meliaceae) possessing potent antiinflammatory and antiarthritic activities. The present study revealed that nimbidin significantly inhibited some of the functions of macrophages and neutrophils relevant to the inflammatory response following both in vivo and in vitro exposure. Oral administration of 5-25 mg/kg nimbidin to rats for 3 consecutive days significantly inhibited the migration of macrophages to their peritoneal cavities in response to inflammatory stimuli and also inhibited phagocytosis and phorbol-12-myristate-13-acetate (PMA) stimulated respiratory burst in these cells. In vitro exposure of rat peritoneal macrophages to nimbidin also inhibited phagocytosis and PMA stimulated respiratory burst in these cells. Nimbidin also inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS) stimulated macrophages following in vitro exposure, whereas interleukin 1 (IL-1) was only weakly inhibited. Probing the mechanism of NO inhibition revealed that nimbidin ameliorated the induction of inducible NO synthase (iNOS) without any inhibition in its catalytic activity. In addition, nimbidin also attenuated degranulation in neutrophils assessed in terms of release of beta-glucuronidase, myeloperoxidase and lysozyme. The results suggest that nimbidin suppresses the functions of macrophages and neutrophils relevant to inflammation. Thus nimbidin can be valuable in treating inflammation/inflammatory diseases. PMID:15174005

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

    PubMed

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

    2014-01-01

    Although a growing body of evidence suggests that plant polyphenols can modulate human immune responses, their simultaneous action on monocyte and neutrophil oxidative burst is currently poorly understood. Based on the hypothesis that various polyphenols contained in plant extracts might affect the oxidative burst of phagocytes, we evaluated the effects of ethanolic O. paradoxa extract polyphenols on monocyte and neutrophil oxidative burst in vitro activated by different stimuli, including opsonized bacteria E. coli, phorbol 12-myristate 13-acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (fMLP). Samples were analyzed by the dihydrorhodamine flow cytometry assay. Our results showed that the extract repressed significantly and dose-dependently reactive oxygen species production in both cell types stimulated with E. coli and PMA (P < 0.05) and its inhibitory efficiency was stimulus- and cell-type-dependent. Interestingly, there was significant stimulatory effect of the extract on bursting phagocytes induced by fMLP (P < 0.05). Additionally, several flavonoids and phenolic compounds as well as penta-galloyl-β-(D)-glucose (PGG), the representative of hydrolyzable tannins, were identified in the 60% extract by high-performance liquid chromatography (HPLC) coupled to electrospray ionization in negative ion mode. In summary, the ethanolic O. paradoxa extract, rich in flavonoids and phenolic compounds, exhibits dual stimulus-dependent effect on the respiratory burst in human leukocytes; hence, it might affect immune responses in humans. PMID:25298860

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

    PubMed

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

    2011-05-25

    There is a great interest in the potential health benefits of biologically active phenolic compounds in cocoa (Theobroma cacao) and dark chocolate. We investigated the anti-inflammatory potential of clovamide (a N-phenylpropenoyl-L-amino acid amide present in cocoa beans) and two phenolic extracts from unroasted and roasted cocoa beans, by evaluating superoxide anion (O(2)(-)) production, cytokine release, and NF-κB activation in human monocytes stimulated by phorbol 12-myristate 13-acetate (PMA). The effects of rosmarinic acid are shown for comparison. Clovamide and rosmarinic acid inhibited PMA-induced O(2)(-) production and cytokine release (with a bell-shaped curve and maximal inhibition at 10-100 nM), as well as PMA-induced NF-κB activation; the two cocoa extracts were less effective. In all tests, clovamide was the most potent compound and also enhanced peroxisome proliferator-activated receptor-γ (PPARγ) activity, which may exert anti-inflammatory effects. These findings indicate clovamide as a possible bioactive compound with anti-inflammatory activity in human cells. PMID:21486087

  20. Repressed PKCδ activation in glycodelin-expressing cells mediates resistance to phorbol ester and TGFβ.

    PubMed

    Hautala, Laura C; Koistinen, Riitta; Koistinen, Hannu

    2016-10-01

    Glycodelin is a glycoprotein mainly expressed in well-differentiated epithelial cells in reproductive tissues. In normal secretory endometrium, the expression of glycodelin is abundant and regulated by progesterone. In hormone-related cancers glycodelin expression is associated with well-differentiated tumors. We have previously found that glycodelin drives epithelial differentiation of HEC-1B endometrial adenocarcinoma cells, resulting in reduced tumor growth in a preclinical mouse model. Here we show that glycodelin-transfected HEC-1B cells have repressed protein kinase C delta (PKCδ) activation, likely due to downregulation of PDK1, and are resistant to phenotypic change and enhanced migration induced by phorbol 12-myristate 13-acetate (PMA). In control cells, which do not express glycodelin, the effects of PMA were abolished by using PKCδ and PDK1 inhibitors, and knockdown of PKCδ, MEK1 and 2, or ERK1 and 2 by siRNAs. Similarly, transforming growth factor β (TGFβ)-induced phenotypic change was only seen in control cells, not in glycodelin-producing cells, and it was mediated by PKCδ. Taken together, these results strongly suggest that PKCδ, via MAPK pathway, is involved in the glycodelin-driven cell differentiation rendering the cells resistant to stimulation by PMA and TGFβ. PMID:27373413

  1. On cell signalling mechanism of Mycobacterium leprae soluble antigen (MLSA) in Jurkat T cells.

    PubMed

    Joshi, Beenu; Khedouci, Sihem; Dagur, Pradeep Kumar; Hichami, Aziz; Sengupta, Utpal; Khan, Naim Akhtar

    2006-07-01

    We investigated the role of Mycobaterium leprae soluble antigen (MLSA) in the modulation of calcium signalling, phosphorylation of mitogen-activated protein (MAP) kinases and IL-2 mRNA expression in human Jurkat T cells. We observed that MLSA induced an increase in free intracellular calcium concentrations, [Ca2+]i, via opening CRAC (Ca2+-release activated- Ca2+) channels. Furthermore, MLSA failed to potentiate both thapsigargin- and anti-CD3 antibodies-induced capacitative calcium influx in Jurkat T cells. We observed that MLSA failed to affect the degree of phosphorylation of two MAP kinases, i.e., ERK1/ERK2, stimulated by anti-CD3 antibodies alone or phorbol 12-myristate 13-acetate (PMA) alone. In order to mimic co-stimulation of T cells, we stimulated them by both PMA and anti-CD3 antibodies. MLSA significantly curtailed the phosphorylation of ERK1/ERK2, stimulated by both PMA and anti-CD3 antibodies in Jurkat T cells. Also MLSA was found to reduce the transcription of IL-2 gene in PMA plus anti-CD3 antibodies-activated Jurkat T cells. Our finding demonstrates that Ca2+ influx via CRAC channels, inhibition of ERK1/ERK2 phosphorylation and IL-2 gene transcription may be implicated in immunosuppressive effects of MLSA antigen. PMID:16583135

  2. The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells

    PubMed Central

    Cartlidge, Robert A; Knebel, Axel; Peggie, Mark; Alexandrov, Andrei; Phizicky, Eric M; Cohen, Philip

    2005-01-01

    A substrate for protein kinase B (PKB)α in HeLa cell extracts was identified as methyltransferase-like protein-1 (METTL1), the orthologue of trm8, which catalyses the 7-methylguanosine modification of tRNA in Saccharomyces cerevisiae. PKB and ribosomal S6 kinase (RSK) both phosphorylated METTL1 at Ser27 in vitro. Ser27 became phosphorylated when HEK293 cells were stimulated with insulin-like growth factor-1 (IGF-1) and this was prevented by inhibition of phosphatidyinositol 3-kinase. The IGF-1-induced Ser27 phosphorylation did not occur in 3-phosphoinositide-dependent protein kinase-1 (PDK1)-deficient embryonic stem cells, but occurred normally in PDK1[L155E] cells, indicating that the effect of IGF-1 is mediated by PKB. METTL1 also became phosphorylated at Ser27 in response to phorbol-12-myristate 13-acetate and this was prevented by PD 184352 or pharmacological inhibition of RSK. Phosphorylation of METTL1 by PKB or RSK inactivated METTL1 in vitro, as did mutation of Ser27 to Asp or Glu. Expression of METTL1[S27D] or METTL1[S27E] did not rescue the growth phenotype of yeast lacking trm8. In contrast, expression of METTL1 or METTL1[S27A] partially rescued growth. These results demonstrate that METTL1 is inactivated by PKB and RSK in cells, and the potential implications of this finding are discussed. PMID:15861136

  3. l-Cystathionine Inhibits the Mitochondria-Mediated Macrophage Apoptosis Induced by Oxidized Low Density Lipoprotein

    PubMed Central

    Zhu, Mingzhu; Du, Junbao; Chen, Siyao; Liu, Angie Dong; Holmberg, Lukas; Chen, Yonghong; Zhang, Chunyu; Tang, Chaoshu; Jin, Hongfang

    2014-01-01

    This study was designed to investigate the regulatory role of l-cystathionine in human macrophage apoptosis induced by oxidized low density lipoprotein (ox-LDL) and its possible mechanisms. THP-1 cells were induced with phorbol 12-myristate 13-acetate (PMA) and differentiated into macrophages. Macrophages were incubated with ox-LDL after pretreatment with l-cystathionine. Superoxide anion, apoptosis, mitochondrial membrane potential, and mitochondrial permeability transition pore (MPTP) opening were examined. Caspase-9 activities and expression of cleaved caspase-3 were measured. The results showed that compared with control group, ox-LDL treatment significantly promoted superoxide anion generation, release of cytochrome c (cytc) from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and cell apoptosis, in addition to reduced mitochondrial membrane potential as well as increased MPTP opening. However, 0.3 and 1.0 mmol/L l-cystathionine significantly reduced superoxide anion generation, increased mitochondrial membrane potential, and markedly decreased MPTP opening in ox-LDL + l-cystathionine macrophages. Moreover, compared to ox-LDL treated-cells, release of cytc from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and apoptosis levels in l-cystathionine pretreated cells were profoundly attenuated. Taken together, our results suggested that l-cystathionine could antagonize mitochondria-mediated human macrophage apoptosis induced by ox-LDL via inhibition of cytc release and caspase activation. PMID:25514411

  4. 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

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

    PubMed Central

    Shabrish, Snehal; Gupta, Maya; Madkaikar, Manisha

    2016-01-01

    Natural killer (NK) cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a) is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC) or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA) and calcium ionophore (Ca2+-ionophore) instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p < 0.0001). It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL) with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells. PMID:27413758

  6. 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

  7. Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

    PubMed Central

    Ghadessy, Roxana S; Kelly, Eamonn

    2002-01-01

    The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma×rat glioma hybrid cells. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5′-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A2 adenosine receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA). In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of Gs-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells. PMID:11959806

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

    PubMed

    Habtemariam, S

    1998-05-01

    Treatment of human endothelial cells with cytokines such as tumour necrosis factor-alpha (TNF) or E. coli lipopolysaccharide (LPS) induces the expression of several adhesion molecules and enhances leukocyte adhesion to endothelial cell surface. Interfering with this leukocyte adhesion or adhesion molecules upregulation is an important therapeutic target for the treatment of bacterial sepsis and various inflammatory diseases. In the course of screening marketed European anti-inflammatory herbal drugs for TNF antagonistic activity, a crude ethanolic extract of corn silk (stigma of Zea mays) exhibited significant activity. The extract at concentrations of 9-250 micrograms/ml effectively inhibited the TNF- and LPS-induced adhesiveness of EAhy 926 endothelial cells to monocytic U937 cells. Similar concentration ranges of corn silk extract did also block the TNF and LPS but not the phorbol 12-myristate 13-acetate-induced ICAM-1 expression on EAhy 926 endothelial cell surface. The extract did not alter the production of TNF by LPS-activated macrophages and failed to inhibit the cytotoxic activity of TNF. It is concluded that corn silk possesses important therapeutic potential for TNF- and LPS-mediated leukocyte adhesion and trafficking. PMID:9619111

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

    PubMed Central

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

    2016-01-01

    Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at P<0.05. Results: The results showed that the ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

  10. P-selectin promotes neutrophil extracellular trap formation in mice.

    PubMed

    Etulain, Julia; Martinod, Kimberly; Wong, Siu Ling; Cifuni, Stephen M; Schattner, Mirta; Wagner, Denisa D

    2015-07-01

    Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin(-/-) mice. Moreover, NETosis was also promoted by P-selectin-immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectin(ΔCT/ΔCT) mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis) were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectin(ΔCT/ΔCT) neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases. PMID:25979951

  11. 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. PMID:27034964

  12. 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. PMID:27095915

  13. Effects of the antioxidants Trolox, Tiron and Tempol on neutrophil extracellular trap formation.

    PubMed

    Vorobjeva, Nina V; Pinegin, Boris V

    2016-02-01

    Neutrophils can entrap and kill pathogens by releasing of neutrophil extracellular traps (NETs), in addition to their routine functions such as phagocytosis and degranulation. NETs consist of a DNA backbone supplemented by multiple bactericidal proteins from the nucleus, the cytoplasm and the granules. Neutrophils release NETs after their activation by a number of physiological and pharmacological stimuli. In addition to the antimicrobial function, NETs are involved in the pathogenesis of various autoimmune and inflammatory diseases. Since NET formation predominantly depends on the generation of reactive oxygen species (ROS), all substances that are capable of scavenging ROS or inhibiting the enzymes responsible for their synthesis should prevent ROS-associated NET release. The aim of this study was to test substances with an antioxidant activity, such as Trolox, Tiron, and Tempol, for their capacity to inhibit NET formation by primary human neutrophils in vitro. We revealed for the first time an inhibitory effect of Trolox on ROS-dependent NET release. We also established a suppressive effect of Tempol on NET formation that manifested itself in a wide range of concentrations. In this study, no inhibitory influence of Tiron on NET release was revealed. All tested substances exerted a significant dose-dependent antioxidative effect on ROS generation induced by phorbol 12-myristate 13-acetate (PMA). We suggest that the antioxidants Trolox and Tempol should be recommended for treating autoimmune and inflammatory diseases that implicate ROS-dependent NET release. PMID:26371849

  14. Dedifferentiation of human epidermal melanocytes into melanoblasts in vitro.

    PubMed

    Zhao, Zhiguo; Jin, Cheng; Ding, Keyun; Ge, Xiaopeng; Dai, Lllan

    2012-07-01

    Melanoblasts (MB) are also called melanocyte (MC) precursor cells. In recent years, people have successfully cultivated human and mouse MB. Previous studies have shown that EDN3 induces cultivated bird MC to re-differentiate into double potential progenitor cells of MB. However, no study has reported whether in vitro cultivated human MC can be dedifferentiated. Our research on MC that were purified and cultivated in vitro found that adding 10 nm endothelin 1 (EDN1) (ET-1) to the MC medium without phorbol 12-myristate 13-acetate (PMA) induced a few MC to dedifferentiate and become a new type of cell. This new cell type was separated, purified, cloned and identified using multiple approaches. The results show that 88.7%, 8.69% and 2.5% of this new cell type were cells in the G(0) -G(1) , G(2) -M and S stages, respectively. The new cell type did not exhibit an apparent apoptotic peak, and its apoptotic rate was 0.09%. Stage I melanosomes were observed in the cytoplasm and were negative for the DOPA reaction. The cell surface antigen expression was positive for tyrosinase-related protein 2, negative or positive for c-kit and negative for S-100 and HMB45, showing that these cells were dedifferentiated MB of MC. Our findings provided evidence for atavism of mature human MC under certain conditions. PMID:22540983

  15. Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L.

    PubMed

    Popov, Sergey V; Golovchenko, Victoria V; Ovodova, Raisa G; Smirnov, Vasily V; Khramova, Daria S; Popova, Galina Yu; Ovodov, Yury S

    2006-06-29

    Lemnan LM, apiogalacturonanic pectin of duckweed Lemna minor L. was tested for adjuvant properties following oral administration with protein antigen. Male Swiss mice were orally immunized thrice with weekly intervals with free OVA or OVA with lemnan (LM). Lemnan LM was shown to increase delayed type hypersensitivity (DTH) and serum anti OVA IgG responses. LM was established to increase levels of both serum IgG1 and IgG2a subclasses, intestinal IgA and failed to elevate levels of serum IgE. Lemnan was found to increase the adhesion of macrophages and to enhance the generation of oxygen radicals by macrophages in response to phorbol 12-myristate 13-acetate. Serum OVA levels were four-fold higher in mice immunized with the mixture of OVA and LM in comparison with those in mice immunized with OVA only. Thus, substantial systemic and local mucosal immune responses were attained by oral immunization with the mixture of OVA and lemnan. Lemnan appeared to elicit adjuvant activity via induction of both Th1- and Th2-type responses. The immunopotentiating effect of lemnan may result from enhanced antigen ingestion and stimulation of macrophage activity. PMID:16725237

  16. Protein kinase C translocation in human blood platelets

    SciTech Connect

    Wang, Hoauyan; Friedman, E. )

    1990-01-01

    Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 {mu}M concentrations did not induce redistribution of platelet PKC. Serotonin and the specific 5-HT{sub 2} receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI) but not the 5-HT{sub 1A} or 5-HT{sub 1B} agonists, ({plus minus}) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT{sub 2} receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT{sub 2} receptors.

  17. Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C*

    PubMed Central

    Mamenko, Mykola; Zaika, Oleg L.; Boukelmoune, Nabila; Berrout, Jonathan; O'Neil, Roger G.; Pochynyuk, Oleh

    2013-01-01

    We have recently documented that the Ca2+-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca2+ responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca2+]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 [Ca2+]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 [Ca2+]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 [Ca2+]i responses and greatly increased basal [Ca2+]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. PMID:23709216

  18. 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.

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

    SciTech Connect

    Kazlauskas, A.; DiColeto, P.E.

    1986-05-01

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

  20. Continuous presence of phorbol ester is required for its IL-1 beta mRNA stabilizing effect.

    PubMed

    Siljander, P; Hurme, M

    1993-01-01

    The protein kinase C (PKC) activating phorbol esters are known to prevent the decay of mRNA of several cytokines and proto-oncogenes. To examine whether the phorbol ester signal is continuously required for this stabilizing effect, THP-1 monocytic cells were stimulated either with phorbol 12,13-dibutyrate (PDBu), which can be removed from the cells by washings, or with the more hydrophobic phorbol 12-myristate 13-acetate (PMA). Both of these stimuli induced high levels of interleukin-1 beta (IL-1 beta) mRNA. When the cells were washed at the peak of the IL-1 beta mRNA expression, this mRNA decayed rapidly in the PDBu stimulated cells while in PMA stimulated cells the mRNA levels were not affected. Moreover, this mRNA degradation induced by the removal of PDBu could be inhibited by readdition of the phorbol ester. This restabilization could be prevented by pharmacologic inhibitors of PKC, but not by inhibiting protein or RNA synthesis. Thus these data suggest that the phorbol ester must be continuously present to exert its mRNA stabilizing effect and that its effect is PKC-mediated but does not require active protein or RNA synthesis. PMID:8416817

  1. Increased internal Ca2+ mediates neural induction in the amphibian embryo.

    PubMed Central

    Moreau, M; Leclerc, C; Gualandris-Parisot, L; Duprat, A M

    1994-01-01

    The molecular mechanism of neural induction is still unknown and the identity of the natural inducer remains elusive. It has been suggested that both the protein kinase C and cAMP signal transduction pathways may be involved in mediating its action. Here we provide evidence that Ca2+ is implicated in the process of transduction of the neuralizing signal. We find that an increase in intracellular Ca2+ concentration [Ca2+]i occurs during neural induction provoked in vitro by the lectin Con A in Pleurodeles waltl embryo. We demonstrate that specific L-type Ca2+ channel agonists also trigger neural induction. Conversely, noninducing lectins do not raise [Ca2+]i. Ryanodine and caffeine trigger neural induction. An increase in [Ca2+]i was also observed after treatment with the phorbol 12-myristate 13-acetate, which has been reported to be inductive. The [Ca2+]i increase triggered by phorbol ester and Con A was abolished by staurosporine and by L-type Ca2+ channel antagonists. Our findings demonstrate that the [Ca2+]i increase occurs via L-type Ca2+ channels. We suggest an amplification of this increase by a Ca(2+)-induced Ca2+ release mechanism which involves intracellular ryanodine-sensitive stores. We propose that Ca(2+)-dependent processes controlled by protein kinase C are implicated in the regulation of gene expression in response to neural induction. Images Fig. 1 PMID:7809092

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

    PubMed Central

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

    2014-01-01

    Although a growing body of evidence suggests that plant polyphenols can modulate human immune responses, their simultaneous action on monocyte and neutrophil oxidative burst is currently poorly understood. Based on the hypothesis that various polyphenols contained in plant extracts might affect the oxidative burst of phagocytes, we evaluated the effects of ethanolic O. paradoxa extract polyphenols on monocyte and neutrophil oxidative burst in vitro activated by different stimuli, including opsonized bacteria E. coli, phorbol 12-myristate 13-acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (fMLP). Samples were analyzed by the dihydrorhodamine flow cytometry assay. Our results showed that the extract repressed significantly and dose-dependently reactive oxygen species production in both cell types stimulated with E. coli and PMA (P < 0.05) and its inhibitory efficiency was stimulus- and cell-type-dependent. Interestingly, there was significant stimulatory effect of the extract on bursting phagocytes induced by fMLP (P < 0.05). Additionally, several flavonoids and phenolic compounds as well as penta-galloyl-β-(D)-glucose (PGG), the representative of hydrolyzable tannins, were identified in the 60% extract by high-performance liquid chromatography (HPLC) coupled to electrospray ionization in negative ion mode. In summary, the ethanolic O. paradoxa extract, rich in flavonoids and phenolic compounds, exhibits dual stimulus-dependent effect on the respiratory burst in human leukocytes; hence, it might affect immune responses in humans. PMID:25298860

  3. Lundep, a Sand Fly Salivary Endonuclease Increases Leishmania Parasite Survival in Neutrophils and Inhibits XIIa Contact Activation in Human Plasma

    PubMed Central

    Chagas, Andrezza C.; Oliveira, Fabiano; Debrabant, Alain; Valenzuela, Jesus G.; Ribeiro, José M. C.; Calvo, Eric

    2014-01-01

    Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET) were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep) from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep) shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate)- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized) Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation. PMID:24516388

  4. The antipsoriatic drug, anthralin, inhibits protein kinase C--implications for its mechanism of action.

    PubMed

    Hegemann, L; Fruchtmann, R; van Rooijen, L A; Müller-Peddinghaus, R; Mahrle, G

    1992-01-01

    In psoriatic patients, anthralin is known to attenuate lesional inflammation, but often generates perilesional dermatitis. This phenomenon is well reflected by the contrasting action of anthralin on human leukocytes. The release of reactive oxygen species (ROS) is inhibited by anthralin in phorbol ester-activated leukocytes, whereas anthralin directly induces this cellular response in unstimulated cells. In order to elaborate further the underlying mechanisms, we compared the kinetics of anthralin and different well-characterized stimuli, including the phorbol ester, phorbol-12-myristate-13-acetate, in this test system. Compared with standard stimuli, anthralin only marginally induced the release of ROS from human leukocytes and displayed different kinetics. Protein kinase C (PKC), the major cellular phorbol ester receptor, is considered to be involved in the regulation of this cellular response. Furthermore, its involvement in the pathophysiology of psoriasis has been suggested. Therefore, we also investigated the effects of anthralin on purified PKC. Anthralin was found to inhibit the enzyme activity in a dose-dependent manner but not to display any stimulatory effects. The present results provide first evidence that the therapeutic activity of anthralin, at least in part, might be mediated by inhibition of PKC. PMID:1503504

  5. Phosphatidylinositol kinase is activated in membranes derived from cells treated with epidermal growth factor.

    PubMed Central

    Walker, D H; Pike, L J

    1987-01-01

    The ability of epidermal growth factor (EGF) to stimulate phosphatidylinositol (PtdIns) kinase activity in A431 cells was examined. The incorporation of 32P from [gamma-32P]ATP into PtdIns by A431 membranes was increased in membranes prepared from cells that had been pretreated with EGF. Demonstration of a stimulation of the PtdIns kinase activity by EGF required the use of subconfluent cultures and was dependent on the inclusion of protease inhibitors in the buffers used to prepare the membranes. Stimulation of the PtdIns kinase activity was rapid. The activation peaked 2 min after the addition of EGF and declined slowly thereafter. Half-maximal stimulation of the PtdIns kinase occurred at 7 nM EGF. Kinetic analyses of the reaction indicated that treatment of the cells with EGF resulted in a decrease in the Km for PtdIns with no change in the Vmax. The kinetic parameters for the utilization of ATP were unchanged in the EGF-treated membranes compared to the control membranes. Pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the ability of EGF to stimulate PtdIns kinase activity. These findings demonstrate that a PtdIns kinase activity in A431 cells is regulated by EGF and provide a good system for examining the mechanism by which EGF stimulates the activity of this intracellular enzyme. PMID:2823265

  6. 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.

  7. 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.

  8. 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.

  9. Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

    SciTech Connect

    Emoto, T.; Kasai, K.; Hiraiwa, M.; Shimoda, S.

    1988-01-01

    In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E/sub 1/ or E/sub 2/ (PGE/sub 1/ and PGE/sub 2/) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10/sup -9/ M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.

  10. Serotonin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis.

    PubMed Central

    Felder, C C; Kanterman, R Y; Ma, A L; Axelrod, J

    1990-01-01

    Serotonin (5-HT) stimulated the release of arachidonic acid in hippocampal neurons cocultured with glial cells but not in glial cultures alone. Similar results were observed for the 5-HT-stimulated release of inositol phosphates. These results suggest a neural but not glial origin of both responses. Pharmacological studies suggested that release of arachidonic acid and inositol phosphates was mediated by a type 2 5-HT (5-HT2) receptor. 5-HT-stimulated release of arachidonic acid was also detected in cortical neurons, which contain high levels of 5-HT2 receptors, but not striatum, spinal cord, or cerebellar granule cells, which have very low levels or are devoid of 5-HT2 receptors. The phorbol ester phorbol 12-myristate 13-acetate augmented the 5-HT-stimulated release of arachidonic acid but inhibited the 5-HT-stimulated release of inositol phosphates. 5-HT-stimulated release of arachidonic acid, but not inositol phosphates, was dependent on extracellular calcium. 5-HT stimulated the release of [3H]lysophosphatidylcholine from [3H]choline-labeled cells with no increase in the release of [3H]choline or phospho[3H]choline. These data suggest that 5-HT stimulated the release of arachidonic acid in hippocampal neurons through the activation of phospholipase A2, independent of the activation of phospholipase C. PMID:2315313

  11. Different roles of protein kinase C-beta and -delta in arachidonic acid cascade, superoxide formation and phosphoinositide hydrolysis.

    PubMed Central

    Duyster, J; Schwende, H; Fitzke, E; Hidaka, H; Dieter, P

    1993-01-01

    In contrast with protein kinase C (PKC)-beta, PKC-delta is exclusively detectable in the membrane fraction of liver macrophages. After long-term treatment with phorbol 12-myristate 13-acetate (PMA) PKC-beta is depleted faster (within 3 h) than PKC-delta (> 7h). Simultaneously, pretreatment with PMA for 3 h inhibits the PMA- and zymosan-induced generation of superoxide and the PMA-induced formation of prostaglandin (PG) E2, whereas a preincubation of more than 7 h is required to affect the zymosan-induced release of PGE2 and inositol phosphates. These results support an involvement of PKC-beta in the PMA-induced activation of the arachidonic acid cascade and in superoxide formation and imply an involvement of PKC-delta in zymosan-induced phosphoinositide hydrolysis and PGE2 formation. Two phorbol ester derivates, sapintoxin A (SAPA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), which have been previously reported to activate preferentially PLC-beta but not PKC-delta in vitro [Ryves, Evans, Olivier, Parker and Evans (1992) FEBS Lett. 288, 5-9], induce the formation of PGE2 and superoxide, down-regulate PKC-delta and potentiate inositol phosphate formation in parallel SAPA, but not DOPPA, down-regulates PKC-beta and inhibits the PMA-induced formation of eicosanoids and superoxide. Images Figure 1 Figure 2 Figure 5 PMID:8389125

  12. Proteasome inhibitors induce peroxisome proliferator-activated receptor transactivation through RXR accumulation and a protein kinase C-dependent pathway

    SciTech Connect

    Tsao, W.-C.; Wu, H.-M.; Chi, K.-H.; Chang, Y.-H.; Lin, W.-W. . E-mail: wwl@ha.mc.ntu.edu.tw

    2005-03-10

    Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of nuclear hormone receptors, forms a heterodimeric DNA binding complex with retinoid X receptor (RXR) and serves as a transcriptional regulator of gene expression. In this study, using luciferase assay of a reporter gene containing PPAR response element (PPRE), we found PPRE transactivity was additively induced by PPAR{gamma} activator (15dPGJ{sub 2}) and RXR activator (9-cis retinoic acid, 9-cis RA). Proteasome inhibitors MG132 and MG262 also stimulate PPRE transactivity in a concentration-dependent manner, and this effect is synergistic to 15dPGJ{sub 2} and 9-cis RA. PKC activation by 12-myristate 13-acetate (PMA) and ingenol 3,20-dibenzoate (IDB) also led to an increased PPRE activation, and this action was additive to PPAR{gamma} activators and 9-cis RA, but not to proteasome inhibitors. Results indicate that the PPAR{gamma} enhancing effect of proteasome inhibitors was attributed to redox-sensitive PKC activation. Western blot analysis showed that the protein level of RXR{alpha}, but not PPAR{gamma}, RXR{beta}, or PKC isoforms, was accumulated in the presence of proteasome inhibitors. Taken together, we conclude that proteasome inhibitors can upregulate PPRE activity through RXR{alpha} accumulation and a PKC-dependent pathway. The former is due to inhibition of RXR{alpha} degradation through ubiquitin-dependent proteasome system, while the latter is mediated by reactive oxygen species (ROS) production.

  13. Phorbol esters alter alpha4 and alphad integrin usage during eosinophil adhesion to VCAM-1.

    PubMed

    Kikuchi, Matsuo; Tachimoto, Hiroshi; Nutku, Esra; Hudson, Sherry A; Bochner, Bruce S

    2003-01-01

    We examined the effect of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA) on the human eosinophil adhesion molecule phenotype and attachment to VCAM-1 via alpha4 and alphad integrins under static and flow conditions. PMA increased surface expression of alphad integrins and decreased alpha4 integrin expression. Under static conditions, eosinophils bound well to VCAM-1, primarily via alpha4beta1 integrins, with a minor alphadbeta2 integrin component. Unexpectedly, PMA-stimulated eosinophils bound equally well to VCAM-1 and albumin in a temperature- and divalent cation-dependent manner, yet adhesion was independent of beta1 and beta2 integrins. Under flow conditions, eosinophils readily attached to VCAM-1, and adhesion was inhibited by both alpha4 and alphad mAbs (95 and 50% inhibition, respectively). Many fewer PMA-stimulated eosinophils bound to VCAM-1 under flow conditions, but both alpha4 and alphad mAbs inhibited adhesion equally. Thus, PMA alters eosinophil integrin expression and the relative contributions of alpha4 and alphad integrins during attachment to VCAM-1. PMID:14668059

  14. Effects of the root of Platycodon grandiflorum on airway mucin hypersecretion in vivo and platycodin D(3) and deapi-platycodin on production and secretion of airway mucin in vitro.

    PubMed

    Ryu, Jiho; Lee, Hyun Jae; Park, Su Hyun; Kim, Jinwoong; Lee, Dongho; Lee, Sang Kook; Kim, Yeong Shik; Hong, Jang-Hee; Seok, Jeong Ho; Lee, Choong Jae

    2014-03-15

    We investigated whether aqueous extract of the root of Platycodon grandiflorum A. de Candolle (APG), platycodinD(3) and deapi-platycodin significantly affect the production and secretion of airway mucin using in vivo and in vitro experimental models. Effect of APG was checked on hypersecretion of pulmonary mucin in sulfur dioxide-induced bronchitis in rats. Confluent NCI-H292 cells were pretreated with platycodinD(3) or deapi-platycodin for 30min and then stimulated with PMA (phorbol 12-myristate 13-acetate) for 24h. The MUC5AC mucin production and secretion were measured by ELISA. The results were as follows: (1) APG stimulated the secretion of airway mucin in sulfur dioxide-induced bronchitis rat model; (2) platycodinD(3) and deapi-platycodin inhibited the production of MUC5AC mucin induced by PMA from NCI-H292 cells, respectively; (3) however, platycodinD(3) and deapi-platycodin did not inhibit but stimulated the secretion of MUC5AC mucin induced by PMA from NCI-H292 cells, respectively. This result suggests that aqueous extract of P. grandiflorum A. de Candolle and the two natural products derived from it, platycodinD(3) and deapi-platycodin, can regulate the production and secretion of airway mucin and, at least in part, explains the traditional use of aqueous extract of P. grandiflorum A. de Candolle as expectorants in diverse inflammatory pulmonary diseases. PMID:24290472

  15. Comparative study of cell cycle kinetics and induction of apoptosis or necrosis after exposure of human Mono Mac 6 cells to radiofrequency radiation.

    PubMed

    Lantow, M; Viergutz, T; Weiss, D G; Simkó, M

    2006-09-01

    The possible harmful effects of radiofrequency electromagnetic fields (RF EMFs) are controversial. We have used human Mono Mac 6 cells to investigate the influence of RF EMFs in vitro on cell cycle alterations and BrdU uptake, as well as the induction of apoptosis and necrosis in human Mono Mac 6 cells, using flow cytometry after exposure to a 1,800 MHz, 2 W/kg specific absorption rate (SAR), GSM-DTX signal for 12 h. No statistically significant differences in the induction of apoptosis or necrosis, cell cycle kinetics, or BrdU uptake were detected after RF EMF exposure compared to sham or incubator controls. However, in the positive control cells treated with gliotoxin and PMA (phorbol 12 myristate-13 acetate), a significant increase in apoptotic and necrotic cells was seen. Cell cycle analysis or BrdU incorporation for 72 h showed no differences between RF EMF- or sham-exposed cells, whereas PMA treatment induced a significant accumulation of cells in G(0)/G(1)-phase and a reduction in S-phase cells. RF EMF radiation did not induce cell cycle alterations or changes in BrdU incorporation or induce apoptosis and necrosis in Mono Mac 6 cells under the exposure conditions used. PMID:16953672

  16. 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

  17. Effects of raspberry fruit extracts and ellagic acid on respiratory burst in murine macrophages.

    PubMed

    Raudone, Lina; Bobinaite, Ramune; Janulis, Valdimaras; Viskelis, Pranas; Trumbeckaite, Sonata

    2014-06-01

    The mechanism of action of polyphenolic compounds is attributed to their antioxidant, anti-inflammatory, and anti-proliferative properties and their effects on subcellular signal transduction, cell cycle impairment and apoptosis. A raspberry (Rubus idaeus L.) fruit extract contains various antioxidant active compounds, particularly ellagic acid (EA); however the exact intracellular mechanism of their action is not fully understood. The aim of the study was to evaluate the antioxidant effect of raspberry extracts, and that of ellagic acid by assessment of the production of the reactive oxygen species (ROS) by murine macrophage J774 cells. Raspberry extracts and their active compound EA did not affect or had very minor effects on cell viability. No significant difference in the ROS generation in arachidonic acid stimulated macrophages was determined for raspberry extracts and EA whereas in the phorbol-12 myristate-13 acetate model ROS generation was significantly (p < 0.05) reduced. Our observation that raspberry pomace extracts in vitro reduce ROS production in a J774 macrophage culture suggests that raspberry extract and ellagic acid mediated antioxidant effects may be due to the regulation of NADPH oxidase activity. PMID:24699912

  18. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps

    PubMed Central

    Guimarães-Costa, Anderson B.; Nascimento, Michelle T. C.; Froment, Giselle S.; Soares, Rodrigo P. P.; Morgado, Fernanda N.; Conceição-Silva, Fátima; Saraiva, Elvira M.

    2009-01-01

    Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania–neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania–neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site. PMID:19346483

  19. Regulation of 5-oxo-ETE synthesis by nitric oxide in human polymorphonuclear leucocytes upon their interaction with zymosan and Salmonella typhimurium

    PubMed Central

    Viryasova, Galina M.; Galkina, Svetlana I.; Gaponova, Tatjana V.; Romanova, Julia M.; Sud’ina, Galina F.

    2014-01-01

    In the present study we have presented data on the regulation of LT (leukotriene) and 5-oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) syntheses in human neutrophils upon interaction with OZ (opsonized zymosan) or Salmonella typhimurium. Priming of neutrophils with PMA (phorbol 12-myristate 13-acetate) and LPS (lipopolysaccharide) elicits 5-oxo-ETE formation in neutrophils exposed to OZ, and the addition of AA (arachidonic acid) significantly increases 5-oxo-ETE synthesis. We found that NO (nitric oxide)-releasing compounds induce 5-oxo-ETE synthesis in neutrophils treated with OZ or S. typhimurium. Exposure of neutrophils to zymosan or bacteria in the presence of the NO donor DEA NONOate (1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium) considerably increased the conversion of endogenously formed 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) to 5-oxo-ETE. To our knowledge, this study is the first to demonstrate that NO is a potent regulator of 5-oxo-ETE synthesis in human polymorphonuclear leucocytes exposed to Salmonella typhimurium and zymosan. PMID:24712762

  20. Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

    2016-01-01

    Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. PMID:26494252

  1. Stimulation of expression for the adenosine A2A receptor gene by hypoxia in PC12 cells. A potential role in cell protection.

    PubMed

    Kobayashi, S; Millhorn, D E

    1999-07-16

    The purpose of this study was to examine the regulation of adenosine A2A receptor (A2AR) gene expression during hypoxia in pheochromocytoma (PC12) cells. Northern blot analysis revealed that the A2AR mRNA level was substantially increased after a 3-h exposure to hypoxia (5% O2), which reached a peak at 12 h. Immunoblot analysis showed that the A2AR protein level was also increased during hypoxia. Inhibition of de novo protein synthesis blocked A2AR induction by hypoxia. In addition, removal of extracellular free Ca2+, chelation of intracellular free Ca2+, and pretreatment with protein kinase C inhibitors prevented A2AR induction by hypoxia. Moreover, depletion of protein kinase C activity by prolonged treatment with phorbol 12-myristate 13-acetate significantly inhibited the hypoxic induction of A2AR. A2AR antagonists led to a significant enhancement of A2AR mRNA levels during hypoxia, whereas A2AR agonists caused down-regulation of A2AR expression during hypoxia. This suggests that A2AR regulates its own expression during hypoxia by feedback mechanisms. We further found that activation of A2AR enhances cell viability during hypoxia and also inhibits vascular endothelial growth factor expression in PC12 cells. Thus, increased expression of A2AR during hypoxia might protect cells against hypoxia and may act to inhibit hypoxia-induced angiogenic activity mediated by vascular endothelial growth factor. PMID:10400659

  2. Inhibitory effect of aminoethyl-chitooligosaccharides on invasion of human fibrosarcoma cells.

    PubMed

    Hong, Sugyeong; Ngo, Dai-Nghiep; Kim, Moon-Moo

    2016-07-01

    Chitooligosaccharides (COS) have been reported to show a variety of biological efficacies such as anti-bacterial activity, anti-tumor activity and immune activity. The purpose of this study is to investigate the inhibitory effect of aminoethyl-chitooligosaccharides (AE-COS) synthesized from COS that were substituted hydroxyl groups with aminoethyl group at C-6 position on cell invasion of human fibrosarcoma cells. First of all, the effect of AE-COS on cell viability was observed using MTT assay. The cytotoxicity of AE-COS was increased in a dose dependent manner. The inhibitory effects of AE-COS on the activity and expression level of MMP-2 and MMP-9 related to invasion of cancer cells were examined using gelatin zymography and western blot. It was found that AE-COS above 20μg/ml showed the inhibitory effect on the activity and expression of MMP-9. Furthermore, AE-COS at 20μg/ml reduced the expression level of p50, a part of NF-κB, compared with phorbol-12- myristate-13- acetate (PMA) group. The available data let us hypothesize that AE-COS could provide chemoprevention as an inhibitor against cell invasion associated with metastasis. PMID:27348727

  3. Role of protein phosphatase 2A in the regulation of mitogen-activated protein kinase activity in ventricular cardiomyocytes.

    PubMed

    Braconi Quintaje, S; Church, D J; Rebsamen, M; Valloton, M B; Hemmings, B A; Lang, U

    1996-04-25

    Incubation of cultured, neonatal rat ventricular cardiomyocytes with 100 nM phorbol 12-myristate-13-acetate (PMA) induced a transient suppression of PP2A activity at 5 min, an effect that was reversed after 15 min of exposure to PMA. This inactivation was correlated with a transient increase in the phosphorylation level of the catalytic subunit of PP2A (193 +/- 38% of control levels at 5 min). Simultaneously to the transient inactivation of PP2A, we observed a rapid and reversible phosphorylation of 42-kDa MAP kinase (474 +/- 65% of control levels at 5 min, and 316 +/- 44% at 15 min) in cardiomyocytes treated with PMA. This transient phosphorylation was accompanied by a transient increase in cytosolic MAP kinase activity (209 +/- 17% of control values at 5 min and 125 +/- 7% at 15 min). Okadaic acid (1 microM ) completely blocked the decrease in the phosphorylation level and activity of MAP kinase occurring after 5 min of exposure to PMA. These data demonstrate that PP2A inactivation and MAP kinase activation are very strongly correlated in cardiomyocytes, indicating that PP2A plays a negative modulatory role in the regulation of MAP kinase activity. PMID:8629997

  4. Membrane peptidases on human osteoblast-like cells in culture: hydrolysis of calcitonin and hormonal regulation of endopeptidase-24.11.

    PubMed Central

    Howell, S; Caswell, A M; Kenny, A J; Turner, A J

    1993-01-01

    Five membrane peptidase activities have been identified on cultured human osteoblast-like cells. These consisted of the four exopeptidases aminopeptidase-A, aminopeptidase-N, aminopeptidase-W and carboxypeptidase-M, and the endopeptidase, endopeptidase-24.11. The presence of endopeptidase-24.11 was confirmed immunochemically by immunofluorescent staining and by enzyme-linked immunosorbent assay. The inclusion of phosphoramidon partially inhibited the hydrolysis of human calcitonin by a membrane fraction prepared from osteoblast-like cell membranes, thus implicating endopeptidase-24.11 in its inactivation. Another metallopeptidase also contributed substantially to calcitonin hydrolysis. Purified porcine endopeptidase-24.11 (1 microgram) was shown to hydrolyse calcitonin with a half-life of 23 min, which compared to a half-life of 0.5 min for substance P under similar conditions. Sequence data revealed that the initial site of hydrolysis of calcitonin was between residues Lys18 and Phe19. The expression of endopeptidase-24.11 by cultured osteoblast-like cells was shown to be modified by various agents: expression was decreased by phorbol 12-myristate-13-acetate (160 nM for 48 h) and increased in the presence of calcitonin (1.5 nM for 48 h) and 1,25-dihydroxyvitamin D3 (0.01-1 microM for 72 h). Images Figure 1 PMID:8439284

  5. Induction of mast cell degranulation by triterpenoidal saponins obtained from Cimicifugae rhizoma.

    PubMed

    Choi, Ji-Yoon; Jeon, Su Jin; Son, Kun Ho; Park, Young In; Dong, Mi-Sook

    2016-10-01

    Cimicifugae rhizoma has been widely used as a traditional herbal medicine to treat inflammation and menopausal symptoms. In this study, we found that some of the triterpenoidal saponins purified from the ethanol extract of Cimicifugae rhizoma dramatically induced histamine release. The structure-related induction of mast cell degranulation by them and the mechanism of action were determined. β-Hexosaminidase release in HMC-1 cells was increased in a concentration-dependent manner, with maximal 6.5- and 8.5-fold increases, by 200 μg/mL 24-epi-7,8-didehydrocimigenol-3-O-xyloside (comp 1) and cimigenol 3-O-beta-d-xyloside (comp 4) compared with those treated with phorbol 12-myristate 13-acetate and A23187 (PMACI), respectively. However, β-hexosaminidase release was not changed by 7,8-dihydrocimigenol (comp 3), or 23-OAc-shengmanol-3-O-xyloside (comp 7). These triterpenoidal saponins changed neither the intracellular Ca(2+ )level nor the activation of PKC, both of which play essential roles in histamine release. However, cromolyn and ketotifen, membrane stabilizers, effectively inhibited the β-hexosaminidase release induced by comp 1 or comp 4 by 39 and 45%, respectively. Collectively, xylose on the cimigenol-related backbone among triterpene glycosides isolated from Cimicifugae rhizoma may play an important role in activating mast cells and induction of degranulation partly via membrane destabilization of mast cells. PMID:27310149

  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. PMID:25924948

  7. Inhibitory effects of rutin on the endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Ku, Sae-Kwang; Lee, In-Chul; Han, Min-Su; Bae, Jong-Sup

    2014-10-01

    Endothelial cell protein C receptor (EPCR) has important functions in regulation of coagulation and inflammation. EPCR shedding from the cell surface is mediated by tumor necrosis factor-α converting enzyme (TACE). Rutin is one of the major flavonoids from the buckwheat plant Fagopyrum tataricum. In this study, we investigated the effects of rutin on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding. We used a CLP model because this model more closely resembles human sepsis. Data showed rutin was a potent inhibitor of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding by suppression of TACE expression. Treatment with rutin resulted in a decrease of PMA-stimulated phosphorylation of p38, extracellular regulated kinases 1/2, and c-Jun N-terminal kinase. These results suggest the potential application of rutin for treatment of PMA and CLP-mediated EPCR shedding. PMID:24622777

  8. External Application of Apo-9′-fucoxanthinone, Isolated from Sargassum muticum, Suppresses Inflammatory Responses in a Mouse Model of Atopic Dermatitis

    PubMed Central

    Han, Sang-Chul; Kang, Na-Jin; Yoon, Weon-Jong; Kim, Sejin; Na, Min-Chull; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2016-01-01

    Allergic skin inflammation such as atopic dermatitis is characterized by skin barrier dysfunction, edema, and infiltration with various inflammatory cells. The anti-inflammatory effects of Apo-9′-fucoxanthinone, isolated from Sargassum muticum, have been described in many diseases, but the mechanism by which it modulates the immune system is poorly understood. In this study, the ability of Apo-9′-fucoxanthinone to suppress allergic reactions was investigated using a mouse model of atopic dermatitis. The Apo-9′-fucoxanthinone-treated group showed significantly decreased immunoglobulin E in serum. Also, Apo-9′-fucoxanthinone treatment resulted in a smaller lymph node size with reduced the thickness and length compared to the induction group. In addition, Apo-9′-fucoxanthinone inhibited the expression of interleukin-4, interferon-gamma and tumor necrosis factor-alpha by phorbol 12-myristate 13-acetate and ionomycin-stimulated lymphocytes. These results suggest that Apo-9′-fucoxanthinone may be a useful therapeutic strategy for treating chronic inflammatory diseases. PMID:27123161

  9. Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription

    PubMed Central

    Marek-Bukowiec, K; Aguado, E; Miazek, A

    2016-01-01

    Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently upregulated upon T-cell receptor (TCR) engagement, but molecular mechanisms conveying TCR signaling to enhanced LAT transcription are not fully understood. Here we found that a Jurkat subline J.CaM2, initially characterized as LAT deficient, conditionally re-expressed LAT upon the treatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). We took advantage of the above observation for studying cis-elements and trans-acting factors contributing to the activation-induced expression of LAT. We identified a LAT gene region spanning nucleotide position −14 to +357 relative to the ATG start codon as containing novel cis-regulatory elements that were able to promote PMA-induced reporter transcription in the absence of the core LAT promoter. Interestingly, a point mutation in LAT intron 1, identified in J.CaM2 cells, downmodulated LAT promoter activity by 50%. Mithramycin A, a selective Sp1 DNA-binding inhibitor, abolished LAT expression upon PMA treatment as did calcium ionophore ionomycin (Iono) and valproic acid (VPA), widely used as an anti-epileptic drug. Our data introduce J.CaM2 cells as a model for dissecting drivers and blockers of activation induced expression of LAT. PMID:27278128

  10. Phosphorylation of Adaptor Protein Containing Pleckstrin Homology Domain, Phosphotyrosine Binding Domain, and Leucine Zipper Motif 1 (APPL1) at Ser430 Mediates Endoplasmic Reticulum (ER) Stress-induced Insulin Resistance in Hepatocytes*

    PubMed Central

    Liu, Meilian; Zhou, Lijun; Wei, Li; Villarreal, Ricardo; Yang, Xin; Hu, Derong; Riojas, Ramon A.; Holmes, Bekke M.; Langlais, Paul R.; Lee, Hakjoo; Dong, Lily Q.

    2012-01-01

    APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser430 and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser430 is stimulated by phorbol 12-myristate 13-acetate, an activator of classic PKC isoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser430 in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stress-induced APPL1 phosphorylation at Ser430 as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser430 in insulin signaling, overexpression of APPL1S430D but not APPL1S430A impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr308. Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser430. PMID:22685300

  11. Phorbol esters modulate the shape of cultured canine vascular smooth muscle cells

    SciTech Connect

    Di Salvo, J.; Kolquist, K.; Semenchuk, L.; Rengstorf, J. )

    1991-03-11

    Marked changes in the shape of vascular smooth muscle cells (VSMC) occur during early development, repair of the vascular wall, and formation of atherosclerotic plaques. Yet, surprisingly little is known about mechanisms which regulate the shape of VSMC. Since protein kinase C (PKC) is involved in regulation of multiple cellular functions including interactions between contractile and cytoskeletal proteins, the authors suspected it might also regulate VSMC shape. Accordingly, the authors studied the influence of a known activator of PKC, phorbol 12-myristate 13-acetate (PMA), on the shape of cultured canine carotid arterial BSMC. PMA produced time and concentration dependent changes from normal elongated shape to pronounced circular forms. Cells recovered normal shape within 24 hrs even though exposure to PMA was continued. Analogs of PMA which do not activate PKC did not alter shape, whereas phorbol 13, 14 diacetate, an analog which activates PKC, did produce changes in shape similar to those produced by PMA. Cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an inhibitor of mRNA synthesis, did not alter PMA-induced changes in morphology. In contrast, however, recovery of normal shape after prolonged exposure to PMA was blocked by either cycloheximide or actinomycin D. These results suggest activation of PKC produces changes in VSMC shape that are independent of transcription or translation, whereas recovery is dependent on both transcription and translation. The results also suggest PKC may modulate in vivo changes in VSMC shape occurring during different pathophysiological states.

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

    PubMed

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

    2000-06-30

    Nasopharyngeal carcinoma (NPC) is one of the high population malignant tumors among Chinese in southern China and southeast Asia. Epstein-Barr virus (EBV) is a human B lymphotropic herpes virus which is known to be closely associated with NPC. EBV DNA polymerase is a key enzyme during EBV replication and is measured by its radioactivity. The addition of phorbol 12-myristate 13-acetate to Raji cell cultures led to a large increase in EBV DNA polymerase, which was purified by sequential DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. Four tannins were isolated from the active fractions of Eugenia uniflora L., which were tested for the inhibition of EBV DNA polymerase. The results showed the 50% inhibitory concentration (IC(50)) values of gallocatechin, oenothein B, eugeniflorins D(1) and D(2) were 26.5 62.3, 3.0 and 3.5 microM, respectively. Furthermore, when compared with the positive control (phosphonoacetic acid), an inhibitor of EBV replication, the IC(50) value was 16.4 microM. In view of the results, eugeniflorins D(1) and D(2) are the potency principles in the inhibition of EBV DNA polymerase from E. uniflora. PMID:10806300

  13. 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.

  14. Luteolin inhibited the gene expression, production and secretion of MUC5AC mucin via regulation of nuclear factor kappa B signaling pathway in human airway epithelial cells.

    PubMed

    Lee, Hyun Jae; Seo, Hyo-Seok; Ryu, Jiho; Yoon, Yong Pill; Park, Su Hyun; Lee, Choong Jae

    2015-04-01

    Luteolin, a flavonoidal compound derived from Lonicera japonica Thunb. and Chrysanthemum indicum L., has been reported to show anti-inflammatory, anti-oxidative and anti-carcinogenic effects. In this study, we investigated whether luteolin significantly affects the secretion, production and gene expression of airway mucin. Confluent NCI-H292 cells were pretreated with luteolin for 30 min and then stimulated with EGF (epidermal growth factor) or PMA (phorbol 12-myristate 13-acetate) for 24 h or the indicated periods. The MUC5AC mucin gene expression was measured by RT-PCR. Production and secretion of MUC5AC mucin protein were measured by ELISA. To elucidate the action mechanism of luteolin, effect of luteolin on PMA-induced NF-κB signaling pathway was investigated by western blot analysis. The results were as follows: (1) Luteolin inhibited the secretion of MUC5AC mucin protein induced by EGF or PMA; (2) Luteolin inhibited the production of MUC5AC mucin protein and the expression of MUC5AC mucin gene induced by EGF or PMA; (3) Luteolin inhibited PMA-induced phosphorylation and degradation of inhibitory kappa Bα (IκBα); (4) Luteolin inhibited PMA-induced phosphorylation and nuclear translocation of nuclear factor kappa B (NF-κB) p65. This result suggests that luteolin can regulate the secretion, production and gene expression of mucin by acting on airway epithelial cells via regulation of NF-kB signaling pathway. PMID:25285988

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

    PubMed

    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

  16. Delphinidin suppresses PMA-induced MMP-9 expression by blocking the NF-κB activation through MAPK signaling pathways in MCF-7 human breast carcinoma cells.

    PubMed

    Im, Nam-Kyung; Jang, Won Jun; Jeong, Chul-Ho; Jeong, Gil-Saeng

    2014-08-01

    Matrix metalloproteinase-9 (MMP-9) plays an important role in the invasion and metastasis of cancer cells. The synthesis and secretion of MMP-9 can be stimulated by a variety of stimuli, including cytokines and phorbol 12-myristate 13-acetate (PMA), during various pathological processes, such as tumor invasion, atherosclerosis, inflammation, and rheumatoid arthritis, whereas MMP-2 is usually expressed constitutively. Delphinidin, an anthocyanidin present in pigmented fruits and vegetables, possesses potent antioxidant, anti-inflammatory, and antiangiogenic properties. In this study, we investigated the antiproliferative and antiinvasive effects of delphinidin on PMA-induced MMP-9 expression in MCF-7 human breast carcinoma cells using zymography, western blotting, reverse transcription-polymerase chain reaction, and Matrigel invasion assay. Delphinidin significantly suppressed PMA-induced MMP-9 protein expression in MCF-7 human breast carcinoma cells, and it also inhibited the MMP-9 gene transcriptional activity by blocking the activation of NFkappaB (NF-κB) through MAPK signaling pathways. Moreover, the Matrigel invasion assay showed that delphinidin reduces PMA-induced cancer cell invasion. These results suggest that delphinidin is a potential antimetastatic agent that suppresses PMA-induced cancer cell invasion through the specific inhibition of NF-κB-dependent MMP-9 gene expression. PMID:25000305

  17. A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype

    PubMed Central

    Toufaily, Chirine; Charfi, Cyndia; Annabi, Bayader; Annabi, Borhane

    2014-01-01

    Caveolae are specialized cell membrane invaginations known to regulate several cancer cell functions and oncogenic signaling pathways. Among other caveolar proteins, they are characterized by the presence of proteins of the cavin family. In this study, we assessed the impact of cavin-1, cavin-2, and cavin-3 on cell migration in a human HT-1080 fibrosarcoma model. We found that all cavin-1, -2 and -3 transcripts were expressed and that treatment with phorbol 12-myristate 13-acetate (PMA), which is known to prime cell migration and proliferation, specifically upregulated cavin-3 gene and protein expression. PMA also triggered matrix metalloproteinase (MMP)-9 secretion, but reduced the global cell migration index. Overexpression of recombinant forms of the three cavins demonstrated that only cavin-3 was able to reduce basal cell migration, and this anti-migratory effect was potentiated by PMA. Interestingly, cavin-3 overexpression inhibited PMA-induced MMP-9, while cavin-3 gene silencing led to an increase in MMP-9 gene expression and secretion. Furthermore, recombinant cavin-3 significantly prevented PMA-mediated dephosphorylation of AKT, a crucial regulator in MMP-9 transcription. In conclusion, our results demonstrate that cellular cavin-3 expression may repress MMP-9 transcriptional regulation in part through AKT. We suggest that the balance in cavin-3-to-MMP-9 expression regulates the extent of extracellular matrix degradation, confirming the tumor-suppressive role of cavin-3 in controlling the invasive potential of human fibrosarcoma cells. PMID:25520561

  18. Diminished production of interleukin-6 in chronic lymphocytic leukaemia (B-CLL) cells from patients at advanced stages of disease. Tampere CLL Group.

    PubMed

    Hulkkonen, J; Vilpo, J; Vilpo, L; Hurme, M

    1998-03-01

    The production of the cytokines interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) in B-CLL cells from 24 patients at different stages of chronic lymphocytic B-cell leukaemia (B-CLL) was investigated in vitro. In the majority of these cases, low spontaneous IL-6 production was measured. Mitogenic stimulation with phorbol 12-myristate 13-acetate (PMA) or PMA plus interleukin-2 (IL-2) resulted in a tremendous increase in TNF-alpha and IL-6 production in cells representing early stage (Binet A) disease. In contrast, very little, if any, production took place in cells from patients with advanced stage (Binet C) B-CLL. The results from stage B patients were intermediate. The most remarkable difference was recorded in PMA-stimulated (1 ng/ml) IL-6 production. In stimulated 72 h cultures, IL-6 concentrations were 1280 +/- 1080 pg/ml for Binet A (n = 11), 757 +/- 597 pg/ml for Binet B (n = 8) and 46.0 +/- 84.0 pg/ml for Binet C (n = 5). The differences in IL-6 production between stage C v B and stage C v A were both statistically significant (P=0.025). Similar effects, but to a lesser extent, were observed in TNF-alpha production. These results suggest that the varying capacity to produce IL-6 and TNF-alpha may play a role in B-CLL progression and in clinical manifestations of the disease. PMID:9504629

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

    SciTech Connect

    Morrison, W.J.

    1988-01-01

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

  20. Isolation of All CD44 Transcripts in Human Epidermis and Regulation of Their Expression by Various Agents

    PubMed Central

    Teye, Kwesi; Numata, Sanae; Ishii, Norito; Krol, Rafal P.; Tsuchisaka, Atsunari; Hamada, Takahiro; Koga, Hiroshi; Karashima, Tadashi; Ohata, Chika; Tsuruta, Daisuke; Saya, Hideyuki; Haftek, Marek; Hashimoto, Takashi

    2016-01-01

    CD44, a cell surface proteoglycan, is involved in many biological events. CD44 transcripts undergo complex alternative splicing, resulting in many functionally distinct isoforms. To date, however, the nature of these isoforms in human epidermis has not been adequately determined. In this study, we isolated all CD44 transcript