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Sample records for 12-myristate 13-acetate tpa

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

    SciTech Connect

    Itoh, H.; Lederis, K.

    1987-02-01

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

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

    PubMed Central

    Tada, Tomoki; Aki, Kensaku; Oboshi, Wataru; Kawazoe, Kazuyoshi; Yasui, Toshiyuki; Hosoi, Eiji

    2016-01-01

    Background The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. Objective The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. Materials and methods Human erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. Results There was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6–560 µM) and cilostazol (5–10 µM) significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8–200 µM) and sodium valproate (50–1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca2+]i. Conclusion It is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions

  7. 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.

  8. 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.

  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. A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum.

    PubMed Central

    Frost, J A; Geppert, T D; Cobb, M H; Feramisco, J R

    1994-01-01

    The role of ERK-1 and ERK-2 in wild-type (wt) Ha-Ras, phorbol 12-myristate 13-acetate (PMA), and serum-induced AP-1 activity was studied. Microinjection of ERK-specific substrate peptides inhibited the induction of AP-1 activity by all three stimuli, whereas a control peptide had no effect. By using eukaryotic expression constructs encoding wt ERK-1 and kinase-deficient mutants of ERKs 1 and 2, it was found that ERK-1 and ERK-2 activities are required for AP-1 activation stimulated by either wt Ha-Ras, PMA, or serum. Overexpression of ERK-1 augmented wt Ha-Ras stimulation of AP-1, while having no effect upon PMA or serum stimulation. Overexpression of either kinase-deficient ERK-1 or kinase-deficient ERK-2 partially inhibited AP-1 activation by wt Ha-Ras but had no effect on PMA or serum-induced activation. Coexpression of both interfering mutants abolished AP-1 induction by wt Ha-Ras, PMA, or serum. We conclude that ERKs are necessary components in the pathway leading to the activation of AP-1 stimulated by these agents. Images PMID:8170999

  11. Amphiregulin: A bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7

    SciTech Connect

    Shoyab, M.; McDonald, V.L.; Bradley, G.; Todaro, G.J. )

    1988-09-01

    A glycoprotein, termed amphiregulin (AR), inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells. It has been purified to apparent homogeneity from serum-free conditioned medium of MCF-7 human breast carcinoma cells that had been treated with phorbol 12-myristate 13-acetate. AR is a single-chain extremely hydrophilic glycoprotein containing cysteines in disulfide linkage(s) that are essential for biological activity; it is stable between pH2 and pH12 and after heating for 30 min at 56{degree}C but unstable at 100{degree}C. The apparent molecular weights of AR and N-Glycanase-treated AR are 14,000 and 15,000, respectively, as assessed by gel chromatography, and {approx}22,500 and {approx}14,000, respectively, as determined by polyacrylamide gel electrophoresis. A growth modulatory assay was performed with {sup 125}I-labeled deoxyuridine incorporation into DNA. The amino-terminal amino acid sequence of AR has been determined, and no significant sequence homology between AR and other proteins was found. The molecule thus appears to be a distinct growth regulatory protein.

  12. 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

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

    PubMed Central

    Zhu, Lei; McDavid, Sarah; Currie, Kevin P. M.

    2015-01-01

    CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. “Fast” voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of “slow” voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate “slow” inactivation of sodium channels, but little is known about if/how second messengers control “slow” inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from “slow” inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating “slow” inactivation. We postulate that the kinetics of recovery from “slow” inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe. PMID:26222492

  14. 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.

  15. 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.

  16. 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

  17. The stimulation of rat astrocytes with phorbol-12-myristate-13-acetate increases the proenkephalin mRNA: involvement of proto-oncogenes.

    PubMed

    Won, J S; Song, D K; Kim, Y H; Huh, S O; Suh, H W

    1998-03-01

    The effect of phorbol-12-myristate-13-acetate (PMA) on the regulation of proenkephalin (proENK) mRNA level, ENKCRE-2 or AP-1 DNA binding activity, and the mRNA and protein levels of proto-oncogenes (c-fos, fra-1, and c-jun) in primary cultured rat astrocytes were studied. The proENK mRNA level was elevated at 4 h after the treatment of PMA (2.5 microM) without altering the intracellular proENK protein level, and this increase was attenuated by pre-treatment with cycloheximide (CHX; 15 microM), a protein synthesis inhibitor. Both AP-1 and ENKCRE-2 DNA binding activities were markedly increased at 1-4 h by PMA treatment and these PMA-induced responses were inhibited by pre-treatment with CHX, showing that the increase of proENK mRNA level was well correlated with the AP-1 and ENKCRE-2 DNA binding activities. In contrast, although the phospho-CREBP level was also increased by PMA at 0.5-1 h, the pre-treatment with CHX further increased the PMA-induced phospho-CREBP level. In addition, PMA caused the induction of c-fos, c-jun and fra-1 mRNA level and, especially, PMA-induced increase of fra-1 mRNA level was further enhanced by CHX treatment at 4 h. Furthermore, western immunoblot assay showed that PMA caused induction of c-Fos, Fra-1, and c-Jun protein levels. PMA-induced increases of proto-oncoproteins levels were also inhibited by CHX treatment. The results suggest that newly synthesized AP-1 proteins, such as c-Fos, Fra-1, and c-Jun may play important roles in the regulation of PMA-induced proENK gene expression in cultured rat astrocytes. Phospho-CREB protein appears not to be involved in the regulation of PMA-induced proENK gene expression.

  18. Cytosolic retention of phosphorylated extracellular signal-regulated kinase and a Rho-associated kinase-mediated signal impair expression of p21(Cip1/Waf1) in phorbol 12-myristate-13- acetate-induced apoptotic cells.

    PubMed

    Lai, Jin-Mei; Wu, Sulin; Huang, Duen-Yi; Chang, Zee-Fen

    2002-11-01

    In response to treatment with phorbol-12-myristate-13-acetate (PMA), the half-population of erythromyeloblast D2 cells, a cytokine-independent variant of TF-1 cells, displayed adhesion and differentiated into a monocyte/macrophage-like morphology, while the other half-population remained in suspension and underwent apoptosis. Expression of the cell cycle inhibitor p21(Cip1/Waf1) was induced after PMA treatment in the adherent cells but not in the proapoptotic cells. We investigated the mechanism responsible for the impairment of p21(Cip1/Waf1) induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21(Cip1/Waf1) requires the activation and nuclear translocation of phosphorylated extracellular signal-regulated kinase (phospho-ERK). Although ERK was phosphorylated to comparable levels in PMA-induced proapoptotic and adherent cells, nuclear distribution of phospho-ERK was seen only in the adherent, not in the proapoptotic cells. We also found that only PMA-induced proapoptotic cells contained the phosphorylated form of myosin light chain, which is dependent on Rho-associated kinase (ROCK) activation, and that expression of a dominant-active form of ROCK suppressed activation of the p21(Cip1/Waf1) promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21(Cip1/Waf1) expression. Based on these findings, we propose that a ROCK-mediated signal is involved in interfering with the process of ERK-mediated p21(Cip1/Waf1) induction in PMA-induced proapoptotic TF-1 and D2 cells.

  19. Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase.

    PubMed Central

    Matsumoto, T; Tao, W; Sha'afi, R I

    1988-01-01

    The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their

  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. Single or multicellular origin of human T lymphocyte colonies in vitro: modification by 12-o-tetradecanoylphorbol 13-acetate (TPA).

    PubMed

    Singer, J W; Ernst, C; Whalen, C K; Steinmann, L; Fialkow, P J

    1981-04-01

    The assumption that human T lymphocyte colonies have a unicellular origin has been directly tested with peripheral blood mononuclear cells from 2 women heterozygous for the common X-linked glucose-6-phosphate dehydrogenase (G-6-PD) gene (GdB) and the variant GdA. T cells were cultured in semisolid medium in the presence of phytohemagglutinin (PHA) and T lymphocyte growth factor with or without preincubation in suspension culture with PHA (2-stage and 1-stage assays, respectively). The enzyme type of individual T cell colonies was then determined electrophoretically at the lowest colony density with adequate growth (usually less than 100 colonies/dish). In the 2-stage system, 90 of 97 tested colonies had equal amounts of A and B enzyme activities suggesting multicellular origin of the colonies. Similarly, in the single-stage system, 21 of 31 colonies had both A and B enzymes. Increasing the density of the soft agar did not influence the frequency of A/B colonies. However, when 12-O-tetradecanoylphorbol 13-acetate (TPA), a promoter of T cell colony growth shown in other systems to inhibit metabolic cooperation, was added, a striking decrease in frequency of colonies with both G-6-PD types was found. In the 2-stage culture, 0 of 9 colonies had a double-enzyme type and in the single-stage system, the frequency of A/B colonies declined to 9 of 34 (p less than 0.025). The data suggest that despite the apparent multicellular origin of T cell colonies in cultures with TPA, most colonies do originate from single cells when cultured with TPA at low colony densities. Stimulation of cell growth or inhibition of metabolic cooperation between cells by TPA are possible explanations for these differences. PMID:6970773

  2. 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

  3. 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.

  4. rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells.

    PubMed Central

    Nishiyama, T; Sasaki, T; Takaishi, K; Kato, M; Yaku, H; Araki, K; Matsuura, Y; Takai, Y

    1994-01-01

    Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings. Images PMID:8139548

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

    PubMed Central

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

    2011-01-01

    Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N′-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in “inflammation like” conditions was studied by fluorescence technique using 2′,7′-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration. PMID:22254126

  6. 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.

  7. Influence of hyaluronic acid or phorbol 12-myristate 13-acetate on the migration capacity of a murine lymphoma cell line (Eb) and its metastatic variant (ESb).

    PubMed

    Kubens, B S; Nikolai, G; Zänker, K S

    1997-10-14

    The in vitro migration of two murine T cell lymphoma cell lines (Eb and ESb) was studied employing a three-dimensional collagen matrix and time-lapse video recording. In the highly metastatic cell line ESb, which had a low spontaneous locomoting activity, migration could clearly be stimulated by hyaluronic acid (HA) whereas only a small increase was found after incubation with phorbol myristate acetate (PMA). The observed stimulation could be attributed to an increase in recruitment of locomoting cells and not to changes in migration parameters of motile individual cells such as percentage of time locomoting, velocity or distance migrated. Incubation of the low metastatic cell line Eb with HA led to a decrease in migration but blocking of CD44, the principle ligand for HA, by preincubation with an anti-CD44 mAb (KM114), followed by HA exposure increased the locomoting activity significantly. The effect was based on both an increase in recruitment as well as in all migration parameters regarding motile individual Eb cells.

  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. Tumor promoter 12-O-tetradecanoyl phorbol 13-acetate and regulatory diacylglycerols are substrates for the same carboxylesterase

    SciTech Connect

    Mentlein, R.

    1986-06-15

    Rat liver homogenate or cell fractions deacylate 12-O-tetradecanoyl phorbol 13-acetate (TPA) in vitro mainly by conversion to phorbol 13-acetate. The highest specific activity is located in the microsomal fraction. The deacylation is inhibited by bis-(4-nitrophenyl) phosphate, a selective inhibitor of nonspecific carboxylesterases. Only two of five purified esterases from rat liver endoplasmic reticulum deacylate TPA. These two esterases have formerly been characterized as acylcarnitine hydrolases and the more active one is also a potent diacylglycerol lipase. Its TPA-hydrolyzing activity is inhibited by other substrates like 1-naphthylacetate, lauroylcarnitine, or dioleoyl glycerol. The results support the view that phorbol esters act like structural analogs of diacylglycerols, not only with respect to their activating effect on protein kinase C, but also as substrates for the same lipases.

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

    PubMed

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

    1985-10-01

    The possibility that phospholipid deacylation may be a critical event in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-associated effects on mouse skin prompted us to examine in vitro the effects of TPA on arachidonic acid metabolism in neonatal mouse keratinocytes. Three-day old neonatal keratinocytes were prelabeled with [14C]arachidonic acid ([14C]AA) and [14C] stearic acid ([14C]ST) and used to characterize the lipases that were activated when these cells were treated with TPA in culture. Data from these studies demonstrate that phosphatidylcholine (PC) and phosphatidylinositol (PI) are the major phospholipids that undergo early hydrolysis to release arachidonic acid when challenged by TPA. Of particular interest was the novel observation of the hydrolysis of 14C-labeled PI in these keratinocytes, the accumulation of [14C]1,2-diacylglyceride and the lack of the [14C]diacylglyceride phosphorylation to form [14C]phosphatidic acid. This lack of [14C] phosphatidic accumulation implied that although TPA enhanced the hydrolysis of [14C]PI resulting in increased [14C]diacylglyceride it did not enhance the resynthesis of the [14C]PI via the phosphorylation of the [14C]diacylglyceride. Therefore, TPA probably is not involved in the turnover of PI in these cells but is involved in the activation of PC hydrolyzing phospholipase A2 and PI hydrolyzing phospholipase C in these keratinocytes releasing arachidonic acid which then undergoes oxygenation reactions to provide biologically active eicosanoids.

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

    SciTech Connect

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

    1985-10-01

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

  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 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

  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

    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.

  14. 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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1987-04-01

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

  18. 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.

  19. Potent inhibitory effect of silibinin from milk thistle on skin inflammation stimuli by 12-O-tetradecanoylphorbol-13-acetate.

    PubMed

    Liu, Wenfeng; Li, Yonglian; Zheng, Xi; Zhang, Kun; Du, Zhiyun

    2015-12-01

    Silibinin, a major polyphenol in milk thistle, has been reported to have multiple pharmacological activities; therefore, there is an urgent need to well understand how silibinin works on inflammation-associated skin diseases. We herein designed silibinin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated skin inflammation to test its inhibitory effects. It was demonstrated that silibinin, applied topically onto mouse ears following TPA stimulation, effectively down-regulated the expressions of TPA-induced interleukin-1β (IL-1β), interleukin-6 (IL-6), necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Further mechanistic investigations indicated that silibinin suppressed the expression of IκB kinase (IKK) by inhibiting the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, and thereby suppressing TPA-stimulated nuclear factor-κB (NF-κB) activation. Promisingly, silibinin, used for transdermal application, may be a potent naturally occurring anti-inflammatory agent for the prevention of inflammation-associated skin diseases.

  20. 1'-Acetoxychavicol acetate, a superoxide anion generation inhibitor, potently inhibits tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in ICR mouse skin.

    PubMed

    Murakami, A; Ohura, S; Nakamura, Y; Koshimizu, K; Ohigashi, H

    1996-01-01

    The anti-tumor-promoting activity of 1'-acetoxychavicol acetate (ACA) was examined in a two-stage carcinogenesis experiment in ICR mouse skin using 7,12-dimethylbenz[a]anthracene (0.19 mumol) and 12-O-tetradecanoylphorbol-13-acetate (TPA; 1.6 nmol). Topical application of ACA (160 nmol) markedly reduced the average number of tumors per mouse and the ratio of tumor-bearing mice: inhibition ratios 90% (p < 0.001) and 42% (p < 0.005), respectively. ACA even at a dose equimolar to TPA (1.6 nmol) significantly reduced the average number of tumors per mouse: inhibitory ratio 44% (p < 0.05). ACA potently inhibited TPA-induced superoxide (O2-) generation in differentiated HL-60 cells (IC50 = 4.3 microM) and suppressed the lipid hydroperoxide formation by 42% (p < 0.001) in the ethyl linoleate autoxidation test.

  1. 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

  2. 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.

  3. 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

  4. 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

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

    PubMed Central

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

    2011-01-01

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

  6. Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger.

    PubMed

    Yamamoto, T; Sakaguchi, N; Hachiya, M; Nakayama, F; Yamakawa, M; Akashi, M

    2009-04-01

    Human promonocytic cell line U937 cells can be induced to differentiate into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and CD36, with concomitant morphological changes. Moreover, TPA enhanced reactive oxygen species (ROS) generation in these cells, and phagocytic ability was also stimulated during differentiation. The antioxidant agent N-acetyl-L-cysteine inhibited the TPA-induced differentiation of U937 cells. TPA treatment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H(2)O(2)) to H(2)O and O(2). In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of superoxide into H(2)O(2) and O(2) without affecting the levels of copper-zinc superoxide dismutase or glutathione peroxidase 1, which removes H(2)O(2) using glutathione as substrate. Treatment of U937 cells with catalase inhibited the enhancement of ROS generation induced by TPA, and blocked the TPA-induced differentiation of U937 cells. Human promyelocytic cell line HL60 cells were also induced to differentiate into macrophages by TPA. However, HP100-1 cells, its variant cell line overexpressing catalase, were resistant to TPA-induced differentiation. Our results suggest that catalase inhibits monocytic differentiation by TPA; the decrease in catalase level and the accumulation of H(2)O(2) are significant events for monocyte/macrophage differentiation by TPA.

  7. 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

  8. 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.

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

    PubMed

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

    2011-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. Tumor necrosis factor-alpha-nuclear factor-kappa B-signaling enhances St2b2 expression during 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia.

    PubMed

    Matsuda, Toshihiro; Shimada, Miki; Sato, Akira; Akase, Takanori; Yoshinari, Kouichi; Nagata, Kiyoshi; Yamazoe, Yasushi

    2011-01-01

    The mouse cholesterol sulfotransferase St2b2 contributes to epidermal differentiation by biosynthesizing cholesterol sulfate (CS) from cholesterol in the epidermis. 12-O-Tetradecanoylphorbol-13-acetate (TPA) causes epidermal hyperplasia, an abnormal increase in epidermal cell numbers resulting from aberrant cell differentiation and an increase in St2b2 protein levels. The mechanisms underlying enhanced St2b2 expression and the pathophysiologic significance of the increased expression are unclear, however. To verify whether increased St2b2 levels are necessary for TPA-induced epidermal hyperplasia, the effects of St2b2-specific small hairpin RNA (St2b2-shRNA) on hyperplasia were examined in mice. St2b2-shRNA clearly suppressed TPA-induced epidermal hyperplasia and the expression of a marker of epidermal differentiation, involucrin (INV). Interestingly, treating mouse epidermal cells with tumor necrosis factor-alpha (TNFα) increased St2b2 expression. Furthermore, treatment with TNFα-siRNA or anti-TNF receptor antibodies reduced the TPA-induced enhancement of St2b2 expression. Treatment with BAY 11-7082, a specific inhibitor of nuclear factor-kappa B (NF-κB), diminished TPA-induced St2b2 expression. These results suggested that enhancement of St2b2 expression by TPA treatment occurs mainly through the TNFα-NF-κB inflammatory signaling pathway, which in turn leads to increased CS concentrations in epidermal cells and hyperplasia.

  12. Lack of a protective effect of menhaden oil on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

    PubMed

    Locniskar, M; Belury, M A; Cumberland, A G; Patrick, K E; Fischer, S M

    1990-09-01

    Fish oil has been shown to have a protective effect in some cancer models. To determine whether fish oil alters skin tumorigenesis, a study was designed using the initiation-promotion mouse skin carcinogenesis model, feeding mice during the promotion stage a constant overall amount of dietary fat (10%) in which the levels of menhaden oil (MO) varied from 0 to 8.5% or corn oil (CO) at 10%. SENCAR mice were initiated with 10 nmol dimethylbenz[a]anthracene. Two weeks later mice were divided into five groups and maintained on one of the following AIN-76 based diets consisting of: 8.5% coconut oil (CT)/1.5% CO (diet A); 1% MO/7.5% CT/1.5% CO (diet B); 4% MO/4.5% CT/1.5% CO (diet C); 8.5% MO/1.5% CO (diet D); or 10% CO (diet E). Two weeks later, promotion with twice weekly applications of 1 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) was begun and continued for 24 weeks. No statistically significant differences in kcal food consumed or body wts were observed between diet groups during the study. The final papilloma and carcinoma incidence was not different among the diet groups. However, differences were seen in the rate of papilloma appearance with the group fed diet E (10% CO) being the slowest and diet B being the most rapid. In a parallel study, ornithine decarboxylase activity, a suggested marker of promotion, was greatly elevated in the epidermis of all TPA-treated mice and the effect of diet tended to reflect the different rates of tumor formation observed among the groups. These data indicate that the diets containing fish oil were not protective in the final incidence of tumor formation and suggest that a better understanding of the complex interactions is warranted before recommendations are made to alter the human diet for cancer prevention.

  13. Lack of a protective effect of menhaden oil on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

    PubMed

    Locniskar, M; Belury, M A; Cumberland, A G; Patrick, K E; Fischer, S M

    1990-09-01

    Fish oil has been shown to have a protective effect in some cancer models. To determine whether fish oil alters skin tumorigenesis, a study was designed using the initiation-promotion mouse skin carcinogenesis model, feeding mice during the promotion stage a constant overall amount of dietary fat (10%) in which the levels of menhaden oil (MO) varied from 0 to 8.5% or corn oil (CO) at 10%. SENCAR mice were initiated with 10 nmol dimethylbenz[a]anthracene. Two weeks later mice were divided into five groups and maintained on one of the following AIN-76 based diets consisting of: 8.5% coconut oil (CT)/1.5% CO (diet A); 1% MO/7.5% CT/1.5% CO (diet B); 4% MO/4.5% CT/1.5% CO (diet C); 8.5% MO/1.5% CO (diet D); or 10% CO (diet E). Two weeks later, promotion with twice weekly applications of 1 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) was begun and continued for 24 weeks. No statistically significant differences in kcal food consumed or body wts were observed between diet groups during the study. The final papilloma and carcinoma incidence was not different among the diet groups. However, differences were seen in the rate of papilloma appearance with the group fed diet E (10% CO) being the slowest and diet B being the most rapid. In a parallel study, ornithine decarboxylase activity, a suggested marker of promotion, was greatly elevated in the epidermis of all TPA-treated mice and the effect of diet tended to reflect the different rates of tumor formation observed among the groups. These data indicate that the diets containing fish oil were not protective in the final incidence of tumor formation and suggest that a better understanding of the complex interactions is warranted before recommendations are made to alter the human diet for cancer prevention. PMID:2401054

  14. Exposure of JB-6 mouse epidermal cells to 12-O-tetradecanoyl-phorbol-13-acetate is not accompanied by a significant change in total DNA-cytosine methylation.

    PubMed

    Bondy, G P; Denhardt, D T

    1983-12-01

    The extent of methylation of the cytosine bases in DNA is believed to be a major factor influencing gene expression in eukaryotic cells. We have asked whether the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the amount of 5-methylcytosine in DNA. The amount and relative distribution of 5-methylcytosine in the DNA of two subclones of the JB-6 mouse epidermal cell line were determined respectively by high performance liquid chromatography and digestion with the restriction enzymes MspI and HpaII. Exposure to TPA for up to several cell generations had no detectable effect on the degree of DNA methylation (3.9% of the total cytosine) in the two JB-6 lines or Friend erythroleukemia cells. Reduced methylation was readily detected in DNA extracted from cells exposed to 5-azacytidine. The data suggest that tumor promotion (at least that induced by TPA) is likely not the consequence of a generalized elevation or reduction in the amount of 5-methyl-cytosine in the DNA.

  15. 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.

  16. 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).

  17. 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.

  18. Inhibition of phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate-caused inflammatory responses in SENCAR mouse skin by black tea polyphenols.

    PubMed

    Katiyar, S K; Mukhtar, H

    1997-10-01

    Over the past 10 years many studies from several laboratories defined anticarcinogenic and anti-inflammatory effects of tea, a widely consumed beverage by the human population. Much of such work has been conducted with green tea or its polyphenolic constituents. Regarding black tea, studies have shown that its water extract affords protection against tumor promotion caused by chemical carcinogens or ultraviolet B radiation in murine skin carcinogenesis models. Several studies have shown that topical application of chemical tumor promoters to murine skin results in the induction of epidermal edema, hyperplasia and ornithine decarboxylase (ODC) and cyclo-oxygenase activities, and interleukin-1 alpha (IL-1alpha) and ODC mRNA expression. In this study, we assessed whether topical application of polyphenols isolated from black tea leaves (hereafter referred to as BTP) mainly consisting of theaflavine gallates and (-)-epigallocatechin-3-gallate, inhibits phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-caused induction of these markers of inflammatory responses in murine skin. Topical application of BTP (6 mg in 0.2 ml acetone/animal) 30 min prior to TPA application on to the mouse skin resulted in significant inhibition against TPA-caused induction of epidermal edema (40%, P < 0.01), hyperplasia (57%, P < 0.005), leukocytes infiltration (50%), and induction of epidermal ODC (57%) and pro-inflammatory cytokine IL-1alpha mRNA expression (69%). Pre-application of BTP to that of TPA also resulted in significant inhibition of TPA-caused induction of epidermal ODC (23-73%, P < 0.005-0.0001), and cyclo-oxygenase, in terms of prostaglandins metabolites formation (38-65%, P < 0.01-0.0005), enzyme activities. Our data indicate that the inhibition of TPA-caused changes in these markers of inflammatory responses in murine skin by BTP may be one of the possible mechanisms of chemopreventive effects associated with black tea against tumorigenesis. The results

  19. Ascorbic acid inhibits TPA-induced HL-60 cell differentiation by decreasing cellular H₂O₂ and ERK phosphorylation.

    PubMed

    Yiang, Giou-Teng; Chen, Jen-Ni; Wu, Tsai-Kun; Wang, Hsueh-Fang; Hung, Yu-Ting; Chang, Wei-Jung; Chen, Chinshuh; Wei, Chyou-Wei; Yu, Yung-Luen

    2015-10-01

    Retinoic acid (RA), vitamin D and 12-O‑tetradecanoyl phorbol-13-acetate (TPA) can induce HL-60 cells to differentiate into granulocytes, monocytes and macrophages, respectively. Similar to RA and vitamin D, ascorbic acid also belongs to the vitamin family. High‑dose ascorbic acid (>100 µM) induces HL‑60 cell apoptosis and induces a small fraction of HL‑60 cells to express the granulocyte marker, CD66b. In addition, ascorbic acid exerts an anti‑oxidative stress function. Oxidative stress is required for HL‑60 cell differentiation following treatment with TPA, however, the effect of ascorbic acid on HL‑60 cell differentiation in combination with TPA treatment remains to be fully elucidated. The aim of the present study was to investigate the cellular effects of ascorbic acid treatment on TPA-differentiated HL-60 cells. TPA-differentiated HL-60 cells were used for this investigation, this study and the levels of cellular hydrogen peroxide (H2O2), caspase activity and ERK phosphorylation were determined following combined treatment with TPA and ascorbic acid. The results demonstrated that low‑dose ascorbic acid (5 µM) reduced the cellular levels of H2O2 and inhibited the differentiation of HL‑60 cells into macrophages following treatment with TPA. In addition, the results of the present study further demonstrated that low‑dose ascorbic acid inactivates the ERK phosphorylation pathway, which inhibited HL‑60 cell differentiation following treatment with TPA.

  20. 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.

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

    PubMed

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

    1987-10-23

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

  2. Inhibition of NF-kappaB by (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY11-7082; BAY) is associated with enhanced 12-O-tetradecanoylphorbol-13-acetate-induced growth suppression and apoptosis in human prostate cancer PC-3 cells.

    PubMed

    Zheng, Xi; Chang, Richard L; Cui, Xiao-Xing; Avila, Gina; Huang, Mou-Tuan; Liu, Yue; Kong, Ah Ng Tony; Rabson, Arnold B; Conney, Allan H

    2008-01-01

    The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) alone or in combination with an NF-kappaB inhibitor, (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), on the growth and apoptosis of human prostate cancer PC-3 cells cultured in vitro or grown in immunodeficient mice were studied. Treatment of cultured PC-3 cells with TPA (0.2-10 ng/ml) for 96 h resulted in growth inhibition and apoptosis in a concentration-dependent manner. BAY inhibited NF-kappaB activity in PC-3 cells as determined by a luciferase reporter assay and enhanced TPA-induced growth inhibition and apoptosis in cultured PC-3 cells. In animal studies, NCr immunodeficient mice were injected subcutaneously with PC-3 cells in Matrigel. Mice with well-established tumors received daily i.p. injections with TPA (100 ng/g body weight/day), BAY (4 microg/g/day), or a combination of TPA (100 ng/g/day) and BAY (4 microg/g/day) for 36 days. Tumor growth occurred in all of the vehicle-treated control mice. The percent of animals with some tumor regression after 36 days of treatment was 0% for the control group, 40% for the TPA group, 50% for the BAY group and 100% for the TPA + BAY group. Mechanistic studies indicated that treatment of the mice with TPA or TPA + BAY decreased proliferation and increased apoptosis in the tumors. Results from our studies indicate that inhibition of NF-kappaB activity is associated with enhanced TPA-induced growth inhibition and apoptosis in PC-3 cells. Inhibition of NF-kappaB activity by suitable pharmacological inhibitors may be an effective strategy for improving the therapeutic efficacy of TPA in prostate cancer.

  3. Inhibition of 12-O-tetradecanoylphorbol-13-acetate and other skin tumor-promoter-caused induction of epidermal interleukin-1 alpha mRNA and protein expression in SENCAR mice by green tea polyphenols.

    PubMed

    Katiyar, S K; Rupp, C O; Korman, N J; Agarwal, R; Mukhtar, H

    1995-09-01

    Recent studies have shown that topical application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to murine skin results in increased expression of the highly inflammatory cytokine interleukin (IL)-1 alpha in the epidermis. This has led to the suggestion that IL-1 alpha directly or indirectly mediates the inflammatory and hyperplastic responses elicited by TPA and possibly by other skin tumor promoters. In the current study, we investigated the effect of skin application of a polyphenolic fraction isolated from green tea (GTP) to SENCAR mice on skin tumor-promoter-caused induction of cutaneous edema and hyperplasia, and IL-1 alpha mRNA expression. Pretreatment of the skin with GTP 30 min before that of anthralin, benzoyl peroxide, mezerein, and TPA resulted in a significant (p < 0.05) inhibition of cutaneous edema and epidermal hyperplasia caused by each of these tumor promoters. Northern blot analysis indicated that topical application of TPA, anthralin, mezerein, or benzoyl peroxide to SENCAR mice resulted in an increased expression of epidermal IL-1 alpha mRNA. Pretreatment of the skin with GTP or individual epicatechin derivatives (ECDs) present therein, 30 min before that of TPA, resulted in a significant inhibition of enhanced expression of epidermal IL-1 alpha mRNA caused by skin application of TPA. These inhibitory effects were found to be dependent on the dose of GTP. Among four epicatechin derivatives present in GTP, (-)-epicatechin-3-gallate and (-)-epigallocatechin-3-gallate were more effective than (-)-epigallocatechin and (-)-epicatechin in affording this inhibition. Preapplication of GTP was also found to afford inhibition against anthralin-, benzoyl peroxide-, and mezerein-caused increased expression of epidermal IL-1 alpha mRNA and protein. Our study suggests that the inhibition of tumor-promoter-induced IL-1 alpha mRNA and protein expression in mouse epidermis by green tea in combination with other inhibitory effects may be

  4. Protein kinase Cepsilon is linked to 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor-alpha ectodomain shedding and the development of metastatic squamous cell carcinoma in protein kinase Cepsilon transgenic mice.

    PubMed

    Wheeler, Deric L; Ness, Kristin J; Oberley, Terry D; Verma, Ajit K

    2003-10-01

    Protein kinase Cepsilon (PKCepsilon), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately 18-fold) PKCepsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent metastatic squamous cell carcinoma (mSCC) elicited by 7,12-dimethylbenz(a)anthracene-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. We now present that PKCepsilon transgenic mice elicit elevated serum tumor necrosis factor (TNF)alpha levels during skin tumor promotion by TPA, and this increase may be linked to the development of mSCC. A single topical application of TPA (5 nmol) to the skin, as early as 2.5 h after treatment, resulted in a significant (P < 0.01) increase (2-fold) in epidermal TNFalpha and more than a 6-fold increase in ectodomain shedding of TNFalpha into the serum of PKCepsilon transgenic mice relative to their wild-type littermates. Furthermore, this TPA-stimulated TNFalpha shedding was proportional to the level of expression of PKCepsilon in the epidermis. Using the TNF-alpha converting enzyme (TACE) inhibitor, TAPI-1, TPA-stimulated TNFalpha shedding could be completely prevented in PKCepsilon transgenic mice and isolated keratinocytes. These results indicate that PKCepsilon signal transduction pathways to TPA-stimulated TNFalpha ectodomain shedding are mediated by TACE, a transmembrane metalloprotease. Using the superoxide dismutase mimetic CuDIPs and the glutathione reductase mimetic ebselen, TPA-stimulated TNFalpha shedding from PKCepsilon transgenic mice could be completely attenuated, implying the role of reactive oxygen species. Finally, i.p. injection of a TNFalpha synthesis inhibitor, pentoxifylline, during skin tumor promotion completely prevented the development of mSCC in PKCepsilon transgenic mice. Taken together, these results indicate that: (a) PKCepsilon

  5. 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.

  6. Liganded thyroid hormone receptor inhibits phorbol 12-O-tetradecanoate-13-acetate-induced enhancer activity via firefly luciferase cDNA.

    PubMed

    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

  7. ASB16165, a phosphodiesterase 7A inhibitor, reduces cutaneous TNF-alpha level and ameliorates skin edema in phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation model in mice.

    PubMed

    Kadoshima-Yamaoka, Kumiko; Goto, Megumi; Murakawa, Masao; Yoshioka, Ryosuke; Tanaka, Yoshitaka; Inoue, Hidekazu; Murafuji, Hidenobu; Kanki, Satomi; Hayashi, Yasuhiro; Nagahira, Kazuhiro; Ogata, Atsuto; Nakatsuka, Takashi; Fukuda, Yoshiaki

    2009-06-24

    Possible role of phosphodiesterase 7A (PDE7A) in skin inflammation was examined using ASB16165, a specific inhibitor for PDE7A. Epicutaneous application of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ear resulted in induction of skin edema, and topical treatment with ASB16165 inhibited the induction of skin edema in a dose-dependent manner. The TPA challenge also increased the level of TNF-alpha at the application site, and the ASB16165 treatment reduced the TNF-alpha level in the skin. In addition, ASB16165 suppressed the production of TNF-alpha by human keratinocytes stimulated in vitro with TPA and calcium ionophore. Forskolin, an activator of adenylyl cyclase, as well as dibutyryl cAMP also showed inhibitory effect on the TNF-alpha production in the cells, suggesting involvement of cAMP in TNF-alpha generation. These results demonstrate that PDE7A might regulate TNF-alpha production in keratinocytes in a cAMP-dependent fashion. As immunostaining analysis revealed that PDE7A is expressed in the epidermis and TNF-alpha is known to contribute to the TPA-induced edema, it is possible that the inhibitory effect of ASB16165 on skin edema in mouse TPA-induced dermatitis model is mediated by suppression of TNF-alpha production. This is the first report suggesting the association of PDE7A with the function of keratinocytes. ASB16165 will be useful as an agent for skin inflammation in which TNF-alpha plays a pathogenic role (e.g. psoriasis).

  8. 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

  9. 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.

  10. 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

  11. Inhibitory effect of the flowers of artichoke (Cynara cardunculus) on TPA-induced inflammation and tumor promotion in two-stage carcinogenesis in mouse skin.

    PubMed

    Yasukawa, Ken; Matsubara, Hideki; Sano, Yuri

    2010-07-01

    The methanol extract of the flowers of artichoke (Cynara cardunculus) exhibited remarkable antitumor 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 methanol extract, four triterpene alcohols and their corresponding acetates were isolated and identified. These compounds were evaluated for their inhibitory effects on TPA-induced inflammation (1 microg/ear) in mice and showed marked anti-inflammatory effects, with a 50% inhibitory dose of 0.50-0.91 micromol/ear.

  12. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase.

    PubMed

    Kanemitsu, M Y; Lau, A F

    1993-08-01

    We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat liver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.

  13. Activated Ki-Ras suppresses 12-O-tetradecanoylphorbol-13-acetate-induced activation of the c-Jun NH2-terminal kinase pathway in human colon cancer cells.

    PubMed

    Okumura, K; Shirasawa, S; Nishioka, M; Sasazuki, T

    1999-05-15

    Although the frequency of activated Ki-ras genes is high in human colorectal tumors, much less is known of activated Ki-ras-mediated signaling pathways. Using gene targeting, we examined HCT116 cells that contain the Gly-13-->Asp mutation of Ki-ras and activated Ki-ras-disrupted clones derived from HCT116. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced immediate early genes, such as c-Jun, c-Fos, and Egr-1 in activated Ki-ras-disrupted clones, whereas c-Jun induction was rare in HCT116. TPA induced both phosphorylation of stress-activated protein kinase kinase 1 (SEK1) and c-Jun NH2-terminal kinase (JNK) in the activated Ki-ras-disrupted clones but not in HCT116. On the other hand, TPA-induced mitogen-activated protein kinase kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase (ERK) activation was equally induced between HCT116 and the Ki-ras-disrupted clones. Furthermore, TPA-induced SEK1-JNK activation was observed in a DLD-1-derived activated Ki-ras-disrupted clone but not in DLD-1. The TPA-induced SEK1-JNK activation in these disrupted clones was completely inhibited by the protein kinase C (PKC) inhibitor, GF109203X (1 microM), but not by another PKC inhibitor, H7 (50 microM), whereas TPA-induced MEK1/2-ERK activation was partially and completely inhibited by GF109203X (1 microM) and H7 (50 microM), respectively. A phosphoinositol 3-kinase inhibitor, LY294002, did not inhibit the TPA-induced SEK1-JNK activation. Taken together, these results suggest that activated Ki-Ras-mediated signals are involved in the SEK1-JNK pathway through a PKC isotype that is distinct from that involved in MEK1/2-ERK activation in human colon cancer cells and independent of phosphoinositol 3-kinase activation, and the imbalance between ERK and JNK activity caused by activated Ki-Ras may play critical roles in human colorectal tumorigenesis.

  14. 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.

  15. TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells.

    PubMed

    Simboli-Campbell, M; Gagnon, A M; Franks, D J; Welsh, J

    1992-02-01

    The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.

  16. Enhancement of TPA-induced growth inhibition and apoptosis in myeloid leukemia cells by BAY 11-7082, an NF-kappaB inhibitor.

    PubMed

    Hansson, Annette; Marín, Yarí E; Suh, Junghan; Rabson, Arnold B; Chen, Suzie; Huberman, Eliezer; Chang, Richard L; Conney, Allan H; Zheng, Xi

    2005-10-01

    The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation and apoptosis in myeloid leukemia cells. In the present study, we investigated the role of the transcription factor NF-kappaB in TPA-induced growth inhibition and apoptosis in the myeloid leukemia HL-60 cell line and its TPA-resistant cell variant HL-525. Unlike the parental cell line, HL-525 cells are protein kinase C (PKC)-beta deficient and resistant to TPA-induced differentiation and apoptosis. We found that treatment of HL-60 cells with TPA resulted in a concentration-dependent growth inhibition and an increase in apoptotic cells. TPA only had a small effect on growth and apoptosis in HL-525 cells. Treatment of HL-60 cells with TPA (0.64-3.2 nM) caused a rapid activation of NF-kappaB as determined by electrophoresis mobility shift assay (EMSA) and immunocytochemistry. Although the basal level of NF-kappaB activity was low in HL-60 cells, TPA-resistant HL-525 cells had a high basal level of NF-kappaB activity. Treatment of HL-525 cells with higher concentrations of TPA (16-80 nM) resulted in a further increase in NF-kappaB activity. (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), which inhibits IkappaB alpha phosphorylation and thus decreases NF-kappaB activation, was found to decrease TPA-induced nuclear translocation of NF-kappaB. Furthermore, BAY enhanced TPA-induced growth inhibition and apoptosis in both HL-60 and HL-525 cells. Results from the present study indicate that inhibition of NF-kappaB by BAY was associated with enhanced TPA-induced growth inhibition and apoptosis in human myeloid leukemia cells. TPA in combination with pharmacological inhibitors of NF-kappaB may improve the therapeutic efficacy of TPA and overcome the resistance to TPA in some myeloid leukemia patients.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. Redox-regulation of Erk1/2-directed phosphatase by reactive oxygen species: role in signaling TPA-induced growth arrest in ML-1 cells.

    PubMed

    Traore, Kassim; Sharma, Rajni; Thimmulappa, Rajesh K; Watson, Walter H; Biswal, Shyam; Trush, Michael A

    2008-07-01

    Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.

  3. 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.

  4. Attenuation of BPDE-induced p53 accumulation by TPA is associated with a decrease in stability and phosphorylation of p53 and down-regulation of NF-κB activation: Role of p38 MAP kinase

    PubMed Central

    Mukherjee, Jagat J.; Sikka, Harish C.

    2005-01-01

    DNA damage caused by benzo[a]pyrene (BP) or other PAHs induce p53 protein as a protective measure to eliminate the possibility of mutagenic fixation of the DNA damage. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits p53 response induced by BP and other DNA-damaging agents and may cause tumor promotion. The molecular mechanism of attenuation of BP-induced p53 response by TPA is not known. We investigated the effect of TPA on p53 response in BPDE-treated mouse epidermal JB6(P+) Cl 41 cells. BPDE treatment induced p53 accumulation which was attenuated significantly by TPA. Cells treated with BPDE and TPA showed increased ratio of Mdm2 to p53 proteins in p53 immunoprecipitate and decreased p53 life span compared to BPDE-treated cells indicating p53 destabilization by TPA. TPA also inhibited BPDE-induced p53 phosphorylation at serine15. Activation of both ERKs and p38 MAPK by BPDE and attenuation of BPDE-induced p53 accumulation by U0126 or SB202190, specific inhibitor of MEK1/2 or p38 MAPK, indicate the role of ERKs and p38 MAPK in p53 accumulation. Interestingly, TPA potentiated BPDE-induced activation of ERKs whereas p38 MAPK activation was significantly inhibited by TPA, suggesting that inhibition of p38 MAPK is involved in p53 attenuation by TPA. Furthermore SB202190 treatment caused decreased p53 stability and inhibition of phosphorylation of p53 at serine 15 in BPDE-treated cells. We also observed that TPA or SB202190 attenuated BPDE-induced NF-κB activation in JB6 (Cl 41) cells harboring NF-κB reporter plasmid. To our knowledge this is the first report that TPA inhibits chemical carcinogen-induced NF-κB activation. Interference of TPA with BPDE-induced NF-κB activation implicates abrogation of p53 function which has been discussed. Overall our data suggest that abrogation of BPDE-induced p53 response and of NF-κB activation by TPA is mediated by impairment of signaling pathway involving p38 MAPK. PMID:16244358

  5. 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

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

    PubMed

    Liu, Zhaoguo; Zhu, Pingting; Tao, Yu; Shen, Cunsi; Wang, Siliang; Zhao, Lingang; Wu, Hongyan; Fan, Fangtian; Lin, Chao; Chen, Chen; Zhu, Zhijie; Wei, Zhonghong; Sun, Lihua; Liu, Yuping; Wang, Aiyun; Lu, Yin

    2015-07-01

    Epidemiologic and animal studies revealed that capsaicin (8-methyl-N-vanillyl-6-noneamide) can act as a carcinogen or cocarcinogen. However, the influence of consumption of capsaicin-containing foods or vegetables on skin cancer patients remains largely unknown. In the present study, we demonstrated that capsaicin has a cocarcinogenic effect on 9, 10-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumorigenesis. Our results showed that topical application of capsaicin on the dorsal skin of DMBA-initiated and TPA-promoted mice could significantly accelerate tumor formation and growth and induce more and larger skin tumors than the model group (DMBA + TPA). Moreover, capsaicin could promote TPA-induced skin hyperplasia and tumor proliferation. Mechanistic study found that inflammation-related factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were highly elevated by pretreatment with capsaicin, suggesting an inflammation-dependent mechanism. Furthermore, mice that were administered capsaicin exhibited significant up-regulation of phosphorylation of nuclear factor kappaB (NF-κB), Erk and p38 but had no effect on JNK. Thus, our results indicated that inflammation, Erk and P38 collectively played a crucial role in cancer-promoting effect of capsaicin on carcinogen-induced skin cancer in mice.

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

    PubMed

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

    2015-10-01

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

  8. Growth-dependent AIB and meAIB uptake in LLC-PK/sub 1/ cells: effects of differentiation inducers and of TPA

    SciTech Connect

    Amsler, K.; Shaffer, C.; Cook, J.S.

    1983-01-01

    Cultured pig kidney cells designated LLC-PK/sub 1/, previously shown to acquire Na/sup +/-dependent concentrative transport of hexoses as the cells become growth arrested, also show Na/sup +/-dependent concentrative uptake of the amino acid analogs /sup 2/-aminoisobutyric acid (AIB) and (methyl) meAIB. This A system-like transport is most active in sparse, growing cultures and becomes stepped down at confluence. The cell/medium equilibrium distribution ratio of the lipophilic cation tetraphenylphosphonium ion (TPP/sup +/) decreases in parallel fashion, suggesting that a decrease in membrane potential may be a major factor in the stepdown. Differentiation inducers (hexamethylene bisacetamide) and phosphodiesterase inhibitors (theophylline, methylisobutyl xanthine) accelerate the stepdown, but even in the presence of these compounds addition of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) results in the maintenance of a high level of AIB and meAIB uptake. In all these respects the changes in A system like amino acid transport are the reciprocal of those seen for concentrative hexose transport, although the driving force appears to be the same for both systems. The TPA analogs phorbol and 4-0-methyl TPA which are inactive in tumor promotion are inactive in this system as well. In confluent, already stepped-down cultures, addition of TPA leads to a rapid (2-6 hour) stimulation of AIB and meAIB uptake. The enhancement is sensitive to cycloheximide and actinomycin D. 30 references, 6 figures.

  9. The Consequences of edTPA

    ERIC Educational Resources Information Center

    Greenblatt, Deborah

    2016-01-01

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

  10. 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.

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

    SciTech Connect

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

    1986-03-01

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

  12. 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

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

    PubMed

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

    2013-01-01

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

  14. 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

  15. Enhancing mitochondrial respiration suppresses tumor promoter TPA-induced PKM2 expression and cell transformation in skin epidermal JB6 cells.

    PubMed

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

    2011-09-01

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

  16. Aromatic-turmerone attenuates invasion and expression of MMP-9 and COX-2 through inhibition of NF-κB activation in TPA-induced breast cancer cells.

    PubMed

    Park, Sun Young; Kim, Young Hun; Kim, YoungHee; Lee, Sang-Joon

    2012-12-01

    Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)-turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar-turmerone on expression and enzymatic activity levels of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase (MMP)-9 and cyclooxygenaase-2 (COX-2) in breast cancer cells. Our data indicated that ar-turmerone treatment significantly inhibited enzymatic activity and expression of MMP-9 and COX-2 at non-cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, MMP-2, and COX-1 did not change upon ar-turmerone treatment. We found that ar-turmerone inhibited the activation of NF-κB, whereas it did not affect AP-1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF-κB to the MMP-9 and COX-2 promoter were significantly inhibited by ar-turmerone. Our data showed that ar-turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA-induced MMP-9 and COX-2 expression. These results suggest that ar-turmerone suppressed the TPA-induced up-regulation of MMP-9 and COX-2 expression by blocking NF-κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar-turmerone significantly inhibited TPA-induced invasion, migration, and colony formation in human breast cancer cells.

  17. 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.

  18. The medical malpractice TPA: Taking it to the next level.

    PubMed

    Sicard, Lauren E; Ruzzo, Loreto

    2016-04-01

    Risk managers whose organizations self-insure their medical professional liabilities often find themselves dealing with their institution's third-party administrator (TPA), the independent entity that manages claims against the organization and its employees. By understanding better the purpose and operations of a TPA, risk managers can enhance their organizations' litigation outcomes while adding new tools to improve patient safety and quality of care. Viewing the TPA simply as an expense item to be reduced to its lowest possible level deprives the organization of a valuable resource in the form of high-quality data on the drivers of professional liability losses. This article identifies the qualities found in an exceptional medical malpractice TPA and suggests ways to create an effective partnership that will reduce the total cost of claims while supporting the risk manager's mission. PMID:27088774

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Saussurea lappa extract suppresses TPA-induced cell invasion via inhibition of NF-κB-dependent MMP-9 expression in MCF-7 breast cancer cells

    PubMed Central

    2014-01-01

    Background Saussurea lappa (SL) has been used as a traditional herbal medicine to treat abdominal pain and tenesmus, and has been suggested to possess various biological activities, including anti-tumor, anti-ulcer, anti-inflammatory, anti-viral, and cardiotonic activities. The effect of SL on breast cancer metastasis, however, is unknown. Cell migration and invasion are crucial in neoplastic metastasis. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is a major component in cancer cell invasion. Methods Cell viability was examined by MTT assay, whereas cell motility was measured by invasion assay. Western blot, Real-time PCR, and Zymography assays were used to investigate the inhibitory effects of ESL on matrix metalloproteinase-9 (MMP-9) expression level in MCF-7 cells. EMSA confirmed the inhibitory effects of ESL on DNA binding of NF- κB in MCF-7 cells. Results Cells threated with various concentrations of Saussurea lappa (ESL) for 24 h. Concentrations of 2 or 4 μM did not lead to a significant change in cell viability or morphology. Therefore, subsequent experiments utilized the optimal non-toxic concentration (2 or 4 μM) of ESL. In this study, we investigated the inhibitory effect of ethanol extract of ESL on MMP-9 expression and cell invasion in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MCF-7 cells. ESL inhibited the TPA-induced transcriptional activation of nuclear factor-kappa B (NF-κB). However, this result obtained that ESL did not block the TPA-induced phosphorylation of the kinases: p38, ERK, and JNK. Therefore, ELS-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of NF-kB pathway in MCF-7 cells. Conclusions These results indicate that ELS-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of NF-kB pathway in MCF-7 cells. Thus, ESL has potential for controlling breast cancer invasiveness in vitro. PMID:24885456

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

    PubMed

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

    1992-03-01

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

  5. 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…

  6. 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…

  7. 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…

  8. 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.

  9. 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

  10. 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.

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

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2014-07-15

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

  13. 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

  14. [The antioxidative mechanisms of tea polyphenols in inhibiting tumor promotion by TPA].

    PubMed

    Qi, L; Han, C

    1998-01-01

    In the mouse study, topical application of green tea polyphenols (GTP) significantly inhibited TPA-induced increasing of epidermal ornithine decarboxylase (ODC) and increased the activities of several antioxidant enzymes (CAT, GR and GST). In another in vitro study, when GTP was incubated with TPA and mice polymorphonuclear leukocytes (PMNs), TPA induced hydrogen peroxide formation was markedly suppressed with a dose-dependent relationship. The results suggest that the antioxidative effect of GTP may play an important role in inhibiting tumor promotion.

  15. 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…

  16. "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…

  17. Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

    SciTech Connect

    Faellman, M.; Stendahl, O.; Andersson, T. )

    1989-03-01

    Human neutrophils stimulated with a phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 minutes. In contrast, 4-{alpha}-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.

  18. Mild hypothermia markedly reduces ischemia related coronary t-PA release.

    PubMed

    van der Pals, Jesper; Götberg, Matthias; Olivecrona, Göran K; Brogren, Helen; Jern, Sverker; Erlinge, David

    2010-04-01

    In experimentally induced myocardial ischemia, mild hypothermia (33-35 degrees C) has a robust cardioprotective effect. Tissue plasminogen activator (t-PA) is a profibrinolytic enzyme that is released from the vascular endothelial cells in response to ischemia and other injurious stimuli. t-PA has also been found to have proinflammatory properties that could contribute to reperfusion injury. We postulated that hypothermia could attenuate t-PA release in the setting of myocardial ischemia. Sixteen 25-30 kg pigs were anesthetized and a temperature of 37 degrees C was established using an intravascular cooling/warming catheter. The pigs were then randomized to hypothermia (34 degrees C) or control (37 degrees C). A doppler flow wire was placed distal to a percutaneous coronary intervention balloon positioned immediately distal to the first diagonal branch of the left anterior descending artery (LAD). The LAD was then occluded for 10 min in all pigs. Coronary blood flow and t-PA was measured before, during and after ischemia/reperfusion. t-PA was measured in peripheral arterial blood and locally in the venous blood from the coronary sinus. Net t-PA release over the coronary bed was calculated by subtraction of arterial values from coronary sinus values. An estimate of differences in total t-PA release was calculated by multiplying net t-PA release with the relative increase in flow compared to baseline, measured in relative units consisting of ((ng/ml - ng/ml) x (cm/s/cm/s)). There was no observed difference in t-PA levels in peripheral arterial samples. As shown previously, net t-PA release increased during reperfusion. Hypothermia significantly inhibited the increase in t-PA release during reperfusion (peak value 9.44 +/- 4.34 ng/ml vs. 0.79 +/- 0.45 ng/ml, P = 0.02). The effect was even more prominent when an estimation of total t-PA release was performed with mean peak value in the control group 26-fold higher than in the hypothermia group (69.74 +/- 33.86 units vs

  19. Does treatment with t-PA increase the risk of developing epilepsy after stroke?

    PubMed

    Keller, Lena; Hobohm, Carsten; Zeynalova, Samira; Classen, Joseph; Baum, Petra

    2015-10-01

    Patients suffering from ischemic stroke carry an enhanced risk of developing secondary epilepsy. We sought to clarify whether thrombolytic treatment with recombinant tissue plasminogen activator (t-PA) is independently associated with post-stroke epilepsy (PSE). In this observational study, data from 302 stroke patients treated at a single academic neurological department were analyzed retrospectively. Median follow-up was 42 months (maximum 80). Variables included presence of comorbidity, stroke severity, neurological presentation, complications, infarct characteristics, and treatment with t-PA. After univariate analyses, a multivariate analysis was performed to create a model of factors that were significantly associated with PSE, including treatment with t-PA. 13.9 % of patients developed PSE during follow-up. Multivariate analysis identified 5 independent factors for PSE: low Barthel Index at discharge; hemianopia; infection acquired during the hospital stay; involvement of the temporal lobe; involvement of the perirolandic cortex. While the incidence of PSE was higher in patients treated with t-PA (20.6 vs. 10.7 %, univariate analysis; p = 0.020), the effect was lost after adjusting for several factors associated with t-PA treatment [odds ratio for PSE after treatment with t-PA 1.3 (95 % CI 0.6-2.9), p = 0.489]. This study failed to identify treatment with t-PA as an independent risk factor for PSE.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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.

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

    DOE PAGES

    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

  6. Changing contraindications for t-PA in acute stroke: review of 20 years since NINDS.

    PubMed

    Parker, Sarah; Ali, Yasmin

    2015-10-01

    When intravenous (IV) tissue-type plasminogen activator (t-PA) was originally approved by the Food and Drug Administration (FDA) for acute ischemic stroke (AIS) in 1996, there was a lengthy list of contraindications. In the 19 years since the approval of t-PA for AIS, it has been used off label and in patients with those contraindications. In February 2015, the list of contraindications for IV t-PA in AIS was revised and several of the previous contraindications were removed. As only 4 % of patients with ischemic stroke receive treatment with IV t-PA, these changes increase the number of patients eligible for treatment. Anytime there is a significant change in the indications and treatment paradigm with a medication, there can be some resistance to the adaptation of the change into physician's treating habits. We seek to review what the changes to t-PA contraindications are, how they came about, as well as the literature on the previously off-label and currently off-label use of IV t-PA for patients with AIS. PMID:26277361

  7. Isolation of inhibitors of TPA-induced mouse ear edema from Hoelen, Poria cocos.

    PubMed

    Nukaya, H; Yamashiro, H; Fukazawa, H; Ishida, H; Tsuji, K

    1996-04-01

    Triterpene carboxylic acids were isolated from the methanol extract of Hoelen, Poria cocos, and found to inhibit 12-O-tetradecanoylphorbol 13-acetate (TAP)-induced mouse ear edema. Their chemical structures were identified as 3 beta,-16 alpha-dihydroxylanosta-7,9(11),24-trien-21-oic acid, 16 alpha-hydroxydehydropachymic acid, 16 alpha-hydroxytrametenolic acid and dehydrotumulosic acid. PMID:8681415

  8. 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.

  9. 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

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

    PubMed

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

    2012-01-01

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

  11. Clinical implications of the involvement of tPA in neuronal cell death.

    PubMed

    Tsirka, S E

    1997-05-01

    Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying.

  12. 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.

  13. t-PA reduces ischemic impairment of blood-brain barrier by strengthening endothelium junction.

    PubMed

    Zhang, Zhongling; Chen, Xuhui; Li, Le; Zhang, Keling; Tian, Shuqing; Gao, Hongmei; Li, Hulun

    2013-09-01

    Cerebral ischemic stroke is one of the most prevalent diseases in senior individuals. Its therapeutical strategies include anticoagulation, thrombolysis and cell protection. Tissue-type plasminogen activator (t-PA) that interacts with thrombin for the lysis of thrombosis is widely used to treat stroke patients in early stage. The mechanism of action of t-PA is not clear. Here, we report a novel role of t-PA in protecting blood-brain barrier and its potential mechanisms. In a model of the blood-brain barrier with human umbilical vascular epithelium cells, we found that t-PA in low concentrations prevented the impairment of the blood-brain barrier as a result of oxygen and glucose deprivation. This protection was fulfilled by strengthening the junctions among vascular endothelia and by upregulating the productions of vascular endothelium growth factor and of zonula occludens-1. Therefore, t-PA may strengthen the junctions of vascular endothelia in the blood-brain barrier to improve the microenvironment of brain cells and, in turn, the outcome of stroke patients.

  14. Microglial tissue plasminogen activator (tPA) triggers neuronal apoptosis in vitro.

    PubMed

    Flavin, M P; Zhao, G; Ho, L T

    2000-02-15

    Several CNS disorders feature microglial activation. Microglia are known to have both restorative and cytotoxic capabilities. Neuronal apoptosis has been noted after an acute insult such as ischemia. Microglia may participate in this event. We previously showed that conditioned medium (CM) harvested from peritoneal macrophages or from activated microglia triggered apoptosis in rat hippocampal neurons in culture. We wished to characterize the factor responsible for triggering neuronal death. Quiescent microglia produced CM that did not disrupt hippocampal neurons. Lipopolysaccharide-activated microglia produced CM which resulted in neuronal death. This effect was blocked by plasminogen activator inhibitor-1, by tPA STOP, and by co-incubation with tPA antibody. Recombinant human tPA exaggerated the neurotoxic effects of microglial CM, while tPA alone was toxic only at very high concentrations. This in vitro system, which probably excludes any significant impact of microglial free radicals, suggests that microglial tPA may contribute significantly to hippocampal neuronal death.

  15. RACIAL DISPARITIES IN TPA TREATMENT RATE FOR STROKE: A POPULATION-BASED STUDY

    PubMed Central

    Hsia, Amie W.; Edwards, Dorothy F.; Morgenstern, Lewis B.; Wing, Jeffrey J.; Brown, Nina C.; Coles, Regina; Loftin, Sarah; Wein, Andrea; Koslosky, Sara S.; Fatima, Sabiha; Fokar, Ali; Gibbons, M. Chris; Jayam-Trouth, Annapurni; Kidwell, Chelsea S.

    2011-01-01

    Background Some prior studies have shown that racial disparities exist in intravenous tissue plasminogen activator (IV tPA) utilization for acute ischemic stroke. We sought to determine whether race was associated with tPA treatment for stroke in a predominantly black urban population. Methods Systematic chart abstraction was performed on consecutive hospitalized ischemic stroke patients from all seven acute care hospitals in the District of Columbia from Feb 1, 2008 to Jan 31, 2009. Results Of 1044 ischemic stroke patients, 74%% were black, 19% non-Hispanic white, 5% received IV tPA. Blacks were one third less likely than whites to receive IV tPA (3% vs. 10%, p<0.001). However, blacks were also less likely than whites to present within 3 hours of symptom onset (13% vs. 21%, p=0.004) and also less likely to be tPA-eligible (5% vs. 13%, p<0.001). Of those who presented within 3 hours, blacks were almost half as likely to be treated with IV tPA than whites (27% vs. 46%, p=0.023). The treatment rate for tPA-eligible patients was similar for blacks and whites (70% vs. 76%, p=0.62). Conclusions In this predominantly black urban population hospitalized for acute ischemic stroke, blacks were significantly less likely to be treated with IV tPA due to contraindications to treatment, delayed presentation, and stroke severity. Effective interventions designed to increase treatment in this population need to focus on culturally relevant education programs designed to address barriers specific to this population. PMID:21719765

  16. Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes.

    PubMed Central

    Woods, N M; Cuthbertson, K S; Cobbold, P H

    1987-01-01

    The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations. PMID:3479980

  17. X-ray diffraction of molybdenum under ramp compression to 1 TPa

    NASA Astrophysics Data System (ADS)

    Wang, Jue; Coppari, Federica; Smith, Raymond F.; Eggert, Jon H.; Lazicki, Amy E.; Fratanduono, Dayne E.; Rygg, J. Ryan; Boehly, Thomas R.; Collins, Gilbert W.; Duffy, Thomas S.

    2016-09-01

    Molybdenum (Mo) is a transition metal with a wide range of technical applications. There has long been strong interest in its high-pressure behavior, and it is often used as standard for high-pressure experiments. Combining powder x-ray diffraction and dynamic ramp compression, structural and equation of state data were collected for solid Mo to 1 TPa (10 Mbar). Diffraction results are consistent with Mo remaining in the body-centered-cubic structure into the TPa regime. Stress-density data show that Mo under ramp loading is less compressible than the room-temperature isotherm but more compressible than the single-shock Hugoniot.

  18. Involvement of tissue plasminogen activator "tPA" in ethanol-induced locomotor sensitization and conditioned-place preference.

    PubMed

    Bahi, Amine; Dreyer, Jean-Luc

    2012-01-01

    Ethanol is one of the most abused drugs in the western societies. It is well established that mesolimbic dopaminergic neurons mediate the rewarding properties of ethanol. In our previous studies we have shown that the serine protease tissue plasminogen activator (tPA) is involved in the rewarding properties of morphine and amphetamine. In the current study, we investigated the role of tPA in ethanol-induced behavioral sensitization and conditioned-place preference (CPP). Ethanol treatment dose-dependently induced tPA enzymatic activity in the nucleus accumbens (NAc). In addition, ethanol-induced increase in tPA activity was completely inhibited by pre-treatment with the dopamine D1 and D2 receptor antagonists SCH23390 and raclopride respectively. Furthermore, ethanol-induced locomotor stimulation, behavioral sensitization and conditioned-place preference were enhanced following tPA over-expression in the NAc using a lentiviral vector. In contrast, tPA knock down in the NAc with specific shRNA blocked the rewarding properties of ethanol. The defect of locomotor stimulation in shRNA-injected mice was reversed by microinjections of exogenous recombinant tPA into the nucleus accumbens. Collectively, these results indicate, for the first time, that activation of tPA is effective in enhancing the rewarding effects of ethanol. Targeting the tissue plasminogen activator system would provide new therapeutic approaches to the treatment of alcoholism.

  19. Draft Genome Sequence of Streptomyces sp. TP-A0874, a Catechoserine Producer

    PubMed Central

    Hosoyama, Akira; Ichikawa, Natsuko; Igarashi, Yasuhiro

    2016-01-01

    We report the draft genome sequence of Streptomyces sp. TP-A0874 isolated from compost. This strain produces catechoserine, a new catecholate-type inhibitor of tumor cell invasion. The genome harbors at least six gene clusters for polyketide and nonribosomal peptide biosyntheses. The biosynthetic gene cluster for catechoserines was identified by bioinformatic analysis. PMID:27795278

  20. 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…

  1. 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…

  2. 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…

  3. Is the EdTPA the Right Choice for Evaluating Teacher Readiness?

    ERIC Educational Resources Information Center

    Parkes, Kelly A.; Powell, Sean R.

    2015-01-01

    The purpose of this article is to describe and analyze the edTPA, a performance assessment created by the Stanford Center for Assessment, Learning, and Equity (SCALE) and administered by Pearson, Inc., to assess the professional readiness of student teachers. We challenge claims made in support of using this assessment, specifically within the…

  4. Male-female differences in the genetic regulation of t-PA and PAI-1 levels in a Ghanaian population.

    PubMed

    Schoenhard, J A; Asselbergs, F W; Poku, K A; Stocki, S A; Gordon, S; Vaughan, D E; Brown, N J; Moore, J H; Williams, Scott M

    2008-12-01

    Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) directly influence thrombus formation and degradation, and have been identified as risk factors for thromboembolic disease. Prior studies investigated determinants of t-PA and PAI-1 expression, but mainly in Caucasian subjects. The aim of this study was to identify the contributions of genetic and other factors to inter-individual variation in plasma levels of t-PA and PAI-1 in a large-scale population-based sample from urban West Africa. t-PA, PAI-1 and several demographic, anthropometric, and metabolic parameters were measured in 992 residents of Sunyani, the capital of the Brong-Ahafo region of Ghana. In addition, nine gene polymorphisms associated with components of the renin-angiotensin and fibrinolytic systems were determined. We found that BMI, systolic and diastolic blood pressure, total cholesterol, glucose, and triglycerides were all significant predictors of t-PA and PAI-1 in both females and males. In addition, a significant relationship was found between the PAI-1 4G/5G (rs1799768) polymorphism on PAI-1 levels in females, the TPA I/D (rs4646972) polymorphism on t-PA and PAI-1 in males, the renin (rs3730103) polymorphism on t-PA and PAI-1 in males, the ethanolamine kinase 2 (rs1917542) polymorphism on PAI-1 in males, and the renin (rs1464816) polymorphism on t-PA in females and on PAI-1 in males. This study of urban West Africans shows that t-PA and PAI-1 levels are determined by both genetic loci of the fibrinolytic and renin-angiotensin systems and other factors often associated with cardiovascular disease, and that genetic factors differ between males and females.

  5. Phorbol ester and spontaneous activity in SHR aorta

    SciTech Connect

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

    1986-03-01

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

  6. GRAPHIC REANALYSIS OF THE TWO NINDS-TPA TRIALS CONFIRMS SUBSTANTIAL TREATMENT BENEFIT

    PubMed Central

    Saver, Jeffrey L.; Gornbein, Jeffrey; Starkman, Sidney

    2010-01-01

    Background of Comment/Review Multiple statistical analyses of the two NINDS-TPA Trials have confirmed study findings of benefit of fibrinolytic therapy. A recent graphic analysis departed from best practices in the visual display of quantitative information by failing to take into account the skewed functional importance NIH Stroke Scale raw scores and by scaling change axes at up to twenty times the range achievable by individual patients. Methods Using the publicly available datasets of the 2 NINDS-TPA Trials, we generated a variety of figures appropriate to the characteristics of acute stroke trial data. Results A diverse array of figures all visually delineated substantial benefits of fibrinolytic therapy, including: bar charts of normalized gain and loss; stacked bar, bar, and matrix plots of clinically relevant ordinal ranks; a time series stacked line plot of continuous scale disability weights; and line plot, bubble chart, and person icon array graphs of joint outcome table analysis. The achievable change figure showed substantially greater improvement among TPA than placebo patients, median 66.7% (IQR 0–92.0) vs 50.0% (IQR −7.1 – 80.0), p=0.003. Conclusions On average, under 3 hour patients treated with TPA recovered two-thirds while placebo patients improved only half of the way towards fully normal. Graphical analyses of the two NINDS-TPA trials, when performed according to best practices, is a useful means of conveying details about patient response to therapy not fully delineated by summary statistics, and confirms a valuable treatment benefit of under 3 hour fibrinolytic therapy in acute stroke. PMID:20829518

  7. 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…

  8. 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

  9. Allelic imbalance of tissue-type plasminogen activator (t-PA) gene expression in human brain tissue.

    PubMed

    Tjarnlund-Wolf, A; Hultman, K; Curtis, M A; Faull, R L M; Medcalf, R L; Jern, C

    2011-06-01

    We have identified a single-nucleotide polymorphism (SNP) in the t-PA enhancer (-7351C>T), which is associated with endothelial t-PA release in vivo. In vitro studies demonstrated that this SNP is functional at the level of transcription. In the brain, t-PA has been implicated in both physiologic and pathophysiologic processes. The aim of the present study was to examine the effect of the t-PA -7351C>T SNP on t-PA gene expression in human brain tissue. Allelic mRNA expression was measured in heterozygous post-mortem brain tissues using quantitative TaqMan genotyping assay. Protein-DNA interactions were assessed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Significantly higher levels of t-PA mRNA were generated from chromosomes that harboured the wild-type -7351C allele, as compared to those generated from the mutant T allele (for the hippocampus, C to T allelic ratio of ~1.3, p=0.010, n=12; and for the cortex, C to T allelic ratio of ~1.2, p=0.017, n=12). EMSA showed reduced neuronal and astrocytic nuclear protein binding affinity to the T allele, and identified Sp1 and Sp3 as the major transcription factors that bound to the -7351 site. ChIP analyses confirmed that Sp1 recognises this site in intact cells. In conclusion, the t-PA -7351C>T SNP affects t-PA gene expression in human brain tissue. This finding might have clinical implications for neurological conditions associated with enhanced t-PA levels, such as in the acute phase of cerebral ischaemia, and also for stroke recovery.

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

    PubMed

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

    2009-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

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

    PubMed

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

    1996-04-01

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

  13. Epistatic interactions in genetic regulation of t-PA and PAI-1 levels in a Ghanaian population.

    PubMed

    Penrod, Nadia M; Poku, Kwabena A; Vaughan, Douglas E; Vaughn, Douglas E; Asselbergs, Folkert W; Brown, Nancy J; Moore, Jason H; Williams, Scott M

    2011-01-31

    The proteins, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), act in concert to balance thrombus formation and degradation, thereby modulating the development of arterial thrombosis and excessive bleeding. PAI-1 is upregulated by the renin-angiotensin system (RAS), specifically by angiotensin II, the product of angiotensin converting enzyme (ACE) cleavage of angiotensin I, which is produced by the cleavage of angiotensinogen (AGT) by renin (REN). ACE indirectly stimulates the release of t-PA which, in turn, activates the corresponding fibrinolytic system. Single polymorphisms in these pathways have been shown to significantly impact plasma levels of t-PA and PAI-1 differently in Ghanaian males and females. Here we explore the involvement of epistatic interactions between the same polymorphisms in central genes of the RAS and fibrinolytic systems on plasma t-PA and PAI-1 levels within the same population (n = 992). Statistical modeling of pairwise interactions was done using two-way ANOVA between polymorphisms in the ETNK2, RENIN, ACE, PAI-1, t-PA, and AGT genes. The most significant interactions that associated with t-PA levels were between the ETNK2 A6135G and the REN T9435C polymorphisms in females (p = 0.006) and the REN T9435C and the TPA I/D polymorphisms (p = 0.005) in males. The most significant interactions for PAI-1 levels were with REN T9435C and the TPA I/D polymorphisms (p = 0.001) in females, and the association of REN G6567T with the TPA I/D polymorphisms (p = 0.032) in males. Our results provide evidence for multiple genetic effects that may not be detected using single SNP analysis. Because t-PA and PAI-1 have been implicated in cardiovascular disease these results support the idea that the genetic architecture of cardiovascular disease is complex. Therefore, it is necessary to consider the relationship between interacting polymorphisms of pathway specific genes that predict t-PA and PAI-1 levels.

  14. Evolution of intracerebral hemorrhage after intravenous tPA: reversal of harmful effects with mast cell stabilization

    PubMed Central

    Marinkovic, Ivan; Mattila, Olli S; Strbian, Daniel; Meretoja, Atte; Shekhar, Shashank; Saksi, Jani; Abo-Ramadan, Usama; Rantanen, Ville; Lindsberg, Perttu J; Tatlisumak, Turgut

    2014-01-01

    Thrombolysis with tissue plasminogen activator (tPA) traditionally demands baseline imaging to rule out intracerebral hemorrhage (ICH), which causes delays in treatment. Preventing possible adverse effects of tPA on ICH would allow rapid on-site thrombolysis in patients with presumed acute ischemic stroke, reducing onset-to-treatment times. We examined how intravenous tPA alters ICH evolution during an extended follow-up, and how mast cell stabilization affects this process. Intracerebral hemorrhage was induced in rats by collagenase injection. Rats received either saline (n=10), tPA (n=13), tPA+low-dose cromoglycate (n=10), or tPA+high-dose cromoglycate (n=10). Magnetic resonance imaging was performed at 24, 48, and 72 hours after ICH induction, together with neurologic evaluations. During 72 hours of follow-up, tPA administration did not significantly increase hematoma volume (mean±s.d. 83.5±14.3 versus 66.7±14.7 μL; P=0.256) or hemispheric expansion (14.5±5.0 versus 11.5±5.0% P=0.457) compared with saline. However, tPA-treated animals had worse neurologic outcomes (P<0.05), and mortality (8/13 versus 3/10). Combining tPA with high-dose cromoglycate mitigated hemispheric expansion (7.4±1.7 versus 14.5±5.0% P=0.01), improved neurologic outcome (P<0.001) and decreased mortality (1/10; P<0.05) compared with tPA alone. Our results suggest tPA increases neurologic deficit in ICH, an effect that was abolished by concomitant mast cell stabilization. Further studies are needed to establish the clinical relevance of these findings. PMID:24169849

  15. t-PA acts as a cytokine to regulate lymphocyte-endothelium adhesion in experimental autoimmune encephalomyelitis.

    PubMed

    Wang, Jinghua; Zhang, Xin; Mu, Lili; Zhang, Mingqing; Gao, Zhongming; Zhang, Jia; Yao, Xiuhua; Liu, Chuanliang; Wang, Guangyou; Wang, Dandan; Kong, Qingfei; Liu, Yumei; Li, Na; Sun, Bo; Li, Hulun

    2014-01-01

    In this study, the capacity for t-PA to affect T cell-brain microvascular endothelial cell adhesion by acting as a cytokine was investigated. Following the treatment of a brain-derived endothelial cell line, bEnd.3, with various concentrations of t-PA, adhesion and transwell migration assays were performed. In the presence of t-PA, enhanced adhesion of T cells to bEnd.3 cells was observed. Using western blot analysis, an increase in ICAM-1 expression was detected for both t-PA-treated bEnd.3 cells and bEnd.3 cells treated with a non-enzymatic form of t-PA. In contrast, when LRP1 was blocked using a specific antibody, upregulation of ICAM-1 was inhibited and cAMP-PKA signaling was affected. Furthermore, using an EAE mouse model, administration of t-PA was associated with an increase in ICAM-1 expression by brain endothelial cells. Taken together, these findings suggest that t-PA can induce ICAM-1 expression in brain microvascular endothelial cells, and this may promote the development of EAE.

  16. Reciprocal actions of NCAM and tPA via a Ras-dependent MAPK activation in rat hippocampal neurons.

    PubMed

    Son, Hyeon; Seuk Kim, Jin; Mogg Kim, Jung; Lee, Sang-Hun; Lee, Yong-Sung

    2002-10-25

    In an attempt to identify the functions of neural cell adhesion molecule (NCAM) and tissue plasminogen activator (tPA) in hippocampal synaptic plasticity, we investigated the relationship between the two molecules by focusing on mitogen-activated protein kinase (MAPK), an essential enzyme in this process. NCAM clustering in cultured hippocampal neurons transiently induced MAPK within 10min. Moreover, soluble NCAM also induced a Ras-dependent MAPK activation. Conversely, MAPK activation led to an increase in the expressions of all three isoforms of NCAM. Treatment of neurons with tPA and plasminogen induced a Ras-dependent MAPK activation and tPA-plasmin degradation of NCAM was mediated in a MAPK-dependent manner. Soluble NCAM transiently inhibited tPA mRNA expression levels in a MAPK-dependent manner, while stimulation of MAPK alone induced tPA reduction in cells. These results collectively indicate that NCAM and tPA reciprocally act as important regulators in the modulation of synaptic plasticity via a Ras-MAPK-involved signaling pathway. In turn, MAPK activation may cause tPA degradation or a decrease in expression to promote synaptic plasticity.

  17. High t-PA release by neonate brain microvascular endothelial cells under glutamate exposure affects neuronal fate.

    PubMed

    Henry, Vincent Jean; Lecointre, Maryline; Laudenbach, Vincent; Ali, Carine; Macrez, Richard; Jullienne, Amandine; Berezowski, Vincent; Carmeliet, Peter; Vivien, Denis; Marret, Stéphane; Gonzalez, Bruno José; Leroux, Philippe

    2013-02-01

    Glutamate excitotoxicity is a consolidated hypothesis in neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor mediated effects. In brain microvascular endothelial cell (nBMEC) cultures from neonates, t-PA content and release upon glutamate are higher than in adult (aBMECs) cultures. Owing to the variety of t-PA substrates and receptor targets, the study was aimed at determining the putative roles of endothelial t-PA in the neonatal brain parenchyma under glutamate challenge. Basal t-PA release was 4.4 fold higher in nBMECs vs aBMECs and glutamate was 20 fold more potent to allow Evans blue vascular permeability in neonate microvessels indicating that, under noxious glutamate (50 μM) exposure, high amounts of endothelial t-PA stores may be mobilized and may access the nervous parenchyma. Culture media from nBMECS or aBMECs challenged by excitotoxic glutamate were applied to neuron cultures at DIV 11. While media from adult cells did not evoke more LDH release in neuronal cultures that under glutamate alone, media from nBMECs enhanced 2.2 fold LDH release. This effect was not observed with media from t-PA(-/-) nBMECs and was inhibited by hr-PAI-1. In Cortical slices from 10 day-old mice, hrt-PA associated with glutamate evoked neuronal necrosis in deeper (more mature) layers, an effect reversed by NMDA receptor GluN1 amino-terminal domain antibody capable of inhibiting t-PA potentiation of the receptor. In superficial layers (less mature), hrt-PA alone inhibited apoptosis, an effect reversed by the EGF receptor antagonist AG1478. Applied to immature neurons in culture (DIV5), media from nBMEC rescued 85.1% of neurons from cell death induced by serum deprivation. In cortical slices, the anti-apoptotic effect of t-PA fitted with age dependent localization of less mature neurons. These data suggest that in the immature brain, propensity of vessels to release high amounts of t-PA may not only

  18. 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.

  19. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  1. Cooperative TPA enhancement via through-space interactions in organic nanodots built from dipolar chromophores

    NASA Astrophysics Data System (ADS)

    Robin, Anne-Claire; Parthasarathy, Venkatakrishnan; Pla-Quintana, Anna; Mongin, Olivier; Terenziani, Francesca; Caminade, Anne-Marie; Majoral, Jean-Pierre; Blanchard-Desce, Mireille

    2010-08-01

    Whereas structure-properties relationships have been widely investigated at the molecular level, supramolecular structure-property relationships have been somewhat overlooked. In many cases, interchromophoric interactions are found to be detrimental (in particular in second-order NLO) and a lot of efforts have been devoted to circumvent and control these effects to achieve efficient NLO materials for electrooptics. At opposite, we have implemented a countermainstream route based on the confinement of push-pull chromophores in close proximity within organic nanodots where both their number and relative position/distance are controlled by covalent attachment onto appropriate organic scaffolds. In such multichromophoric organic superstructures (namely covalent nanoclusters), dipole-dipole interactions can be tuned by playing on the internal architecture (topology, number of chromophoric subunits, length of the covalent linkers) and on the nature and properties (polarity, polarizability) of the chromophoric subunits. Following this strategy, we present the investigation of two series of such organic nanoclusters built from push-pull chromophores where through-space interactions are shown to modify both one-photon (OPA) and two-photon absorption (TPA) of each chromophoric subunits leading to cooperative enhancement of TPA properties and improved transparency.

  2. Equation-of-state measurements for polystyrene at multi-TPa pressures in laser direct-drive experiments

    SciTech Connect

    Ozaki, N.; Ono, T.; Takamatsu, K.; Tanaka, K.A.; Nakano, M.; Kataoka, T.; Yoshida, M.; Wakabayashi, K.; Nakai, M.; Nagai, K.; Shigemori, K.; Yamanaka, T.; Kondo, K.

    2005-12-15

    Equation-of-state (EOS) measurements for polystyrene in TPa (10 Mbar) pressure regions are presented. Polystyrene Hugoniot data were obtained up to 2.7 TPa using impedance matching techniques with laser direct drive at the GEKKO/HIPER laser facility [N. Miyanaga et al., in Proceedings of the 18th International Conference on Fusion Energy (IAEA, Sorrento, Italy, 2001), IAEA-CN-77] The results were compared with theoretical models and previous experimental data and found to be in good agreement with the previous data obtained by different drive and diagnostic techniques.

  3. Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro

    PubMed Central

    Díaz, V M; Hurtado, M; Thomson, T M; Reventós, J; Paciucci, R

    2004-01-01

    Background: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. Aims: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. Methods: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. Results: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by co-immunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. Conclusion: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level. PMID:15194650

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

    PubMed

    Ulfhammer, Erik; 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.

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

    PubMed

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

    2016-01-01

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

  6. Cellulose derivatives carrying triphenylamine (TPA) moieties: synthesis and electro-optical properties.

    PubMed

    Qu, Jinqing; Liao, Wenbo; Chen, Huanqin; Masuda, Toshio

    2009-06-11

    A novel ethyl cellulose derivative [poly(1)] that carries triphenylamine moieties is synthesized with a moderate number-average molecular weight up to 78,200 in 85% yield by the reaction of 4-(diphenylamino)benzoic acid with the residual hydroxy group of ethyl cellulose. Poly(1) is soluble in common organic solvents including toluene, CHCl3, CH2Cl2, and tetrahydrofuran while insoluble in hexane, diethyl ether, and methanol. The polymer emits blue-green fluorescence with quantum yields up to 65% in CHCl3 and displays unique solvatochromism. The cyclic voltammograms of poly(1) indicate that the polymer carrying TPA moieties is electrochemically redox active. The onset temperature of weight loss of the poly(1) is about 177 degrees C according to thermogravimetric analysis in air.

  7. 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.

  8. What Does It Mean to Be Student Centered? An Institutional Case Study of edTPA Implementation

    ERIC Educational Resources Information Center

    Fayne, Harriet; Qian, Gaoyin

    2016-01-01

    This longitudinal case study investigated how one School of Education (SOE), situated in an urban, commuter, public university, responded to the New York State mandate to require the edTPA for initial teacher certification. In order to engage faculty in the work of program redesign, SOE administrators employed a covert leadership approach. Based…

  9. Vascular Risk Factors in Patients with Different Subtypes of Ischemic Stroke May Affect Their Outcome after Intravenous tPA

    PubMed Central

    Ren, Jinma; Nair, Deepak S.; Parker, Sarah; Jahnel, Jan L.; Swanson-Devlin, Teresa G.; Beck, Judith M.; Mathews, Maureen; McNeil, Clayton J.; Upadhyaya, Manas; Gao, Yuan; Dong, Qiang; Wang, David Z.

    2015-01-01

    Intravenous (IV) tissue-type plasminogen activator (tPA) is the only approved noninvasive therapy for acute ischemic stroke (AIS). However, after tPA treatment, the outcome of patients with different subtypes of stroke according to their vascular risk factors remains to be elucidated. We aim to explore the relationship between the outcome and different risk factors in patients with different subtype of acute strokes treated with IV tPA. Records of patients in this cohort were reviewed. Data collected and analysed included the demographics, vascular risk factors, baseline National Institutes of Health Stroke Scale (NIHSS) scores, 90-day modified Rankin Scores (mRS), and subtypes of stroke. By using the 90-day mRS, patients were dichotomized into favorable versus unfavorable outcome in each subtype of stroke. We identified the vascular risk factors that are likely associated with the poor outcome in each subtype. Among 570 AIS patients received IV tPA, 217 were in the large artery atherosclerosis (LAA) group, 146 in the small vessel occlusion(SVO) group, and 140 in the cardioaortic embolism(CE) group. Lower NIHSS score on admission was related to favorable outcome in patients in all subtypes. Patients with history of dyslipidemia were likely on statin treatment before their admission and hence less likely to have elevated cholesterol level on admission. Therefore, there was a possible paradoxical effect on the outcome in patients with LAA and SVO subtypes of strokes. SVO patients with history of diabetes had higher risk of unfavorable outcome. SVO patients had favorable outcome if their time from onset to treatment was short. In conclusion, the outcome of patients treated with IV tPA may be related to different vascular risk factors associated with different subtypes of stroke. PMID:26247772

  10. The gender-specific role of polymorphisms from the fibrinolytic, renin-angiotensin, and bradykinin systems in determining plasma t-PA and PAI-1 levels.

    PubMed

    Asselbergs, Folkert W; Williams, Scott M; Hebert, Patricia R; Coffey, Christopher S; Hillege, Hans L; Navis, Gerjan; Vaughan, Douglas E; van Gilst, Wiek H; Moore, Jason H

    2006-10-01

    Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thus risk for arterial thrombosis. We report here results from a genetic analysis of plasma t-PA and PAI-1 levels in a large population-based sample from the PREVEND study in Groningen, the Netherlands (n = 2,527). We measured polymorphisms from genes of the fibrinolytic system, the renin-angiotensin system (RAS), and the bradykinin system. We found that males had higher levels of natural-log transformed t-PA, and PAI-1 (P < 0.01) compared to females. When stratifying females by menopausal status, PAI-1 levels were only significantly different between pre-menopausal females and males (p < 0.001). Furthermore, we found that age, body mass index, and waist-to-hip ratio were significant predictors of t-PA and PAI-1 in both females and males, and that the regression relationships between these factors and plasma t-PA and PAI-1 were dependent on gender. In addition, we found that the PAI-1 4G/5G polymorphism was a significant predictor of PAI-1 levels in both females and males, that the angiotensin II type I receptor A1166C was a significant predictor of t-PA and PAI-1 levels in females, and that the bradykinin receptor B2 58CT polymorphism was a significant predictor of t-PA levels in females. In conclusion, this large population-based study showed that t-PA and PAI-1 levels are determined by several demographic and genetic factors involved in the fibrinolytic, RAS and bradykinin system. In addition, the results support the idea that the biology of t-PA and PAI-1 is different between females and males.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Draft Genome Sequence of Streptomyces sp. TP-A0890, a Producer of FR-900452 and A-74863a

    PubMed Central

    Ichikawa, Natsuko; Hosoyama, Akira; Fujita, Nobuyuki; Igarashi, Yasuhiro

    2015-01-01

    Here, we report the draft genome sequence of Streptomyces sp. TP-A0890, a producer of FR-900452 and A-74863a. The genome was found to contain at least eight polyketide synthase and nonribosomal peptide synthetase gene clusters. A prediction of gene functions based on the sequence similarity allowed us to assign the biosynthetic gene clusters for FR-900452 and A-74863a. PMID:26472848

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

    PubMed Central

    Hirose, Hiroshi; Hattori, Tomohiro

    2016-01-01

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

  17. Dopamine D3 receptor deletion increases tissue plasminogen activator (tPA) activity in prefrontal cortex and hippocampus.

    PubMed

    Castorina, A; D'Amico, A G; Scuderi, S; Leggio, G M; Drago, F; D'Agata, V

    2013-10-10

    Considerable evidence indicates that dopamine (DA) influences tissue plasminogen activator (tPA)-mediated proteolytic processing of the precursor of brain-derived neurotrophic factor (proBDNF) into mature BDNF (mBDNF). However, specific roles in this process for the dopamine D3 receptor (D3R) and the underlying molecular mechanisms are yet to be fully characterized. In the present study, we hypothesized that D3R deletion could influence tPA activity in the prefrontal cortex and hippocampus. Using D3R knockout (D3(-/-)) mice, we show that receptor inactivation is associated with increased tPA expression/activity both in the prefrontal cortex and, to a greater extent, in the hippocampus. Augmented tPA expression in D3(-/-) mice correlated with increased BDNF mRNA levels, plasmin/plasminogen protein ratio and the conversion of proBDNF into mBDNF, as well as enhanced tPA and mBDNF immunoreactivity, as determined by quantitative real time polymerase chain reaction (qRT-PCR), immunoblot and immunohistochemistry. In addition, when compared to wild-type controls, D3(-/-) mice exhibited increased basal activation of the canonical cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-driven Akt/cAMP-response element-binding protein (CREB) signaling cascade, as determined by the increased Akt phosphorylation both at Thr304 and Ser473 residues, of DA and cAMP-regulated protein of 32kDa (DARPP-32) at Thr34 and a phosphorylation state-dependent inhibition of glycogen synthetase kinase-3β (GSK-3β) at Ser9, a substrate of Akt whose constitutive function impairs normal CREB transcriptional activity through phosphorylation at its Ser129 residue. Accordingly, CREB phosphorylation at Ser133 was significantly increased in D3(-/-) mice, whereas the GSK-3β-dependent phosphorylation at Ser129 was diminished. Altogether, our finding reveals that mice lacking D3Rs show enhanced tPA proteolytic activity on BDNF which may involve, at least in part, a potentiated Akt/CREB signaling

  18. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  1. The reaction of [FeII(tpa)] with H2O2 in acetonitrile and acetone--distinct intermediates and yet similar catalysis.

    PubMed

    Mairata i Payeras, Antoni; Ho, Raymond Y N; Fujita, Megumi; Que, Lawrence

    2004-10-11

    The reaction of [FeII(tpa)(OTf)2] (tpa=tris(2-pyridylmethyl)amine) and its related 5-Me3-tpa complex with hydrogen peroxide affords spectroscopically distinct iron(III)-peroxo intermediates in CH3CN and acetone. The reaction in acetonitrile at -40 degrees C results in the formation of the previously reported Fe(III)-OOH intermediate, the end-on hydroperoxo coordination mode of which is established in this paper by detailed resonance Raman isotope-labeling experiments. On the other hand, the reaction in acetone below -40 degrees C leads to the observation of a different peroxo intermediate identified by resonance Raman spectroscopy to be an FeIII-OOC (CH3)2OH species; this represents the first example of an intermediate derived from the adduct of H2O2 and acetone. The peroxoacetone intermediate decays more rapidly than the corresponding FeIII-OOH species and converts to an FeIV=O species by O-O bond homolysis. This decay process is analogous to that observed for [FeIII(tpa)(OOtBu)]2+ and in fact exhibits a comparable enthalpy of activation of 54(3) kJ mol(-1). Thus, with respect to their physical properties at low temperature, the peroxoacetone intermediate resembles [FeIII(tpa)(OOtBu)]2+ more than the corresponding FeIII-OOH species. At room temperature, however, the behavior of the Fe(tpa)/H2O2 combination in acetone in catalytic hydrocarbon oxidations differs significantly from that of the Fe(tpa)/tBuOOH combination and more closely matches that of the Fe(tpa)/H2O2 combination in CH3CN. Like the latter, the Fe(tpa)/H2O2 combination in acetone catalyzes the hydroxylation of cis-1,2-dimethylcyclohexane to its tertiary alcohol with high stereoselectivity and carries out the epoxidation and cis-dihydroxylation of olefins. These results demonstrate the subtle complexity of the Fe(tpa)/H2O2 reaction surface.

  2. Epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels.

    PubMed

    Asselbergs, Folkert W; Williams, Scott M; Hebert, Patricia R; Coffey, Christopher S; Hillege, Hans L; Navis, Gerjan; Vaughan, Douglas E; van Gilst, Wiek H; Moore, Jason H

    2007-03-01

    Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thereby risk for arterial thrombosis. Activation of the renin-angiotensin system has been linked to the production of PAI-1 expression via the angiotensin II type 1 receptor (AT1R). In addition, bradykinin can induce the release of t-PA through a B2 receptor mechanism. In the present study, we aimed to investigate the epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels in a large population-based sample (n=2527). We demonstrated a strong significant interaction within genetic variations of the bradykinin B2 gene (P=0.002) and between ACE and bradykinin B2 (p=0.003) polymorphisms on t-PA levels in females. In males, polymorphisms in the bradykinin B2 and AT1R gene showed the most strong effect on t-PA levels (P=0.006). In both females and males, the bradykinin B2 gene interacted with AT1R gene on plasma PAI-1 levels (P=0.026 and P=0.039, respectively). In addition, the current study found a borderline significant interaction between PAI 4G5G and ACE I/D on plasma t-PA and PAI-1 levels. These results support the idea that the interplay between the renin-angiotensin, bradykinin, and fibrinolytic systems might play an important role in t-PA and PAI-1 biology.

  3. 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

  4. 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

  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.

  6. Synthesis, structure and properties of one novel 2D Mn-heterocyclic carboxylic acid complex [Mn(TPA)Cl(H 2O)] n

    NASA Astrophysics Data System (ADS)

    Zhu, Peng; Li, Hui-Min

    2011-04-01

    A novel 2D layer complex [Mn(TPA)Cl(H 2O)] n ( 1) has been synthesized by two methods through the reaction of MnCl 2 and TPC or TPA under hydrothermal conditions and characterized by single crystal X-ray diffraction, elemental analysis, infrared spectrometry (IR), powder X-ray diffraction (XRD) and thermogravimetric analysis (TGA), where heterocyclic carboxylic acid ligand TPA = 2-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)acetic acid, TPC = 2-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)acetonitrile. The distorted octahedral Mn(II) centers are bridged by carboxylic O atoms resulting in the formation of a 1D chain. Then the 1D chains are connected with each other through TPA ligands into a 2D (3,3)-connected topology framework. The H-bonding interactions extend the complex into a three-dimensional network, and such weak interactions further stabilized the complex. Furthermore, solid-state fluorescence spectrum of complex 1 exhibits intense broad emissions at 396 nm at room temperature, which is red-shifted by 21 nm relative to that of free ligand TPA.

  7. 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

  8. 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

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

    PubMed

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

    2016-04-19

    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

  10. 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

  11. Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer

    PubMed Central

    Witte, J H de; Sweep, C G J; Klijn, J G M; Grebenschikov, N; Peters, H A; Look, M P; Tienoven, ThH van; Heuvel, J J T M; Vries, J Bolt-De; Benraad, ThJ; Foekens, J A

    1999-01-01

    The prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100 000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient rs = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients. © 1999 Cancer Research Campaign PMID:10390010

  12. 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.

  13. 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

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

    PubMed

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

    2014-02-15

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

  15. 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…

  16. Ulinastatin decreases permeability of blood--brain barrier by inhibiting expression of MMP-9 and t-PA in postoperative aged rats.

    PubMed

    Ma, Li; Zhang, Hui; Liu, Yong-zhe; Yin, Yan-ling; Ma, Ya-qun; Zhang, Sheng-suo

    2016-01-01

    Tissue-type plasminogen activator (t-PA) and matrix metalloproteinase-9 (MMP-9) have been reported to play important roles in increased permeability of blood-brain barrier (BBB) under many pathological circumstances. We have showed that Ulinastatin, a broad-spectrum serine protease inhibitor, could alleviate inflammation in the hippocampus of aged rats following partial hepatectomy. In this study, we investigate the expression and potential roles of t-PA and MMP-9 in the protective effect of Ulinastatin. We found that partial hepatectomy increased Evans blue leakage in hippocampus at day 1 and 3 postoperatively. Furthermore, surgery decreased the protein levels of claudin-5, ZO-1, and NF-kB p65 while upregulating the mRNA and protein levels of t-PA and MMP-9 in brain capillaries. All these effects caused by surgery were partially reversed by administering Ulinastatin. Our study sheds light on the roles of t-PA and MMP-9 of BBB in post-surgical neuroinflammation and postoperative cognitive dysfunction. Besides, it could also help to understand the mechanism of Ulinastatin alleviating neuroinflammation.

  17. Anti-diphtheria antibody seroprotection rates are similar 10 years after vaccination with dTpa or DTPa using a mathematical model.

    PubMed

    Cheuvart, Brigitte; Burgess, Margaret; Zepp, Fred; Mertsola, Jussi; Wolter, Joanne; Schuerman, Lode

    2004-12-01

    The reduced antigen content diphtheria, tetanus and pertussis (dTpa) vaccine (Boostrixtrade mark) has been shown to induce a strong booster response to all the vaccine components in 4-6 year olds. However, anti-diphtheria antibody levels were observed to be lower when compared to the "full strength" paediatric DTPa vaccine. To assess the impact of this difference on long-term protection, a mathematical model was developed to predict diphtheria antibody decay over time. The model was based on a linear decrease in log-transformed antibody concentrations after the first year post-vaccination. When applied to data collected 3.5 years after vaccination of 4-6 year olds with either DTPa or dTpa, the model predicted that 10 years post-vaccination, 98.6% of subjects vaccinated with dTpa were likely to remain seroprotected against diphtheria, compared to 99.6% vaccinated with DTPa. Therefore, the difference observed in diphtheria antibody geometric mean concentrations 1 month after booster vaccination at 4-6 years with dTpa or DTPa is unlikely to be of clinical relevance 10 years later at the time of the adolescent booster.

  18. Interaction of human tissue plasminogen activator (t-PA) with pregnancy zone protein: a comparative study with t-PA-alpha2-macroglobulin interaction.

    PubMed

    Sánchez, M C; Chiabrando, G A; Guglielmone, H A; Bonacci, G R; Rabinovich, G A; Vides, M A

    1998-08-01

    Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on alpha2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of alpha-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of alpha2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.

  19. Plasma Is the Physiologic Buffer of tPA Mediated Fibrinolysis: Rationale for Plasma First Resuscitation after Life-Threatening Hemorrhage

    PubMed Central

    Moore, Hunter B; Moore, Ernest E; Gonzalez, Eduardo; Wiener, Gregory; Chapman, Michael P; Dzieciatkowska, Monika; Sauaia, Angela; Banerjee, Anirban; Hansen, Kirk C; Silliman, Christopher

    2015-01-01

    Background Pre-hospital resuscitation with crystalloid exacerbates fibrinolysis, which is associated with high mortality. We hypothesize that plasma compared to crystalloid resuscitation prevents hyperfibrinolysis in a tissue plasminogen activator (tPA) rich environment via preservation of proteins essential for regulation of fibrinolysis. Study Design Healthy individuals donated blood, which was assayed using a native (non activated) thrombelastography (TEG). Whole-blood (WB) was mixed with normal saline (NS) or platelet poor plasma (PPP) at progressive dilutions. TPA was added to promote a fibrinolytic environment. In a separate experiment PPP was run through 100 KD filter and liquid remaining on top of the filter (TFP) and below the filter (BFP) was obtained. Whole blood was diluted by 50% with TFP, BPF and NS and assayed with tPA TEG challenge. TFP and BFP were assayed for protein concentration and protein composition. Results NS and PPP dilution of WB with out tPA did not affect clot lysis at 30 minutes (LY30) (NS Spearman’s Rho 0.300 p=0.186 and PPP 0.294 p=0.288). When tPA was added NS dilution of whole blood increased LY30 in a percentage dependent manner (0.844 p<0.001) but did not significantly increase with PPP dilution (0.270 p=0.202). The difference in LY30 from WB to diluted WB with PPP (mean change −1.05 95% CI −9.42 to 7.33) was similar with TFP (1.23 95%CI −5.20 to 7.66 p=0.992). However, both BPF (37.65 95%CI 24.47 to 50.82 p=0.001) and NS (47.36 95%CI 34.3–60.45 p<0.001) showed large increases in fibrinolysis compared to PPP. Conclusions Crystalloid and plasma dilution of whole blood does not increase fibrinolysis. However NS dilution of WB, increases susceptibility to tPA mediated fibrinolysis. Plasma resuscitation, simulated by plasma dilution of whole blood, attenuates increased susceptibility to tPA mediate fibrinolysis. The benefits of plasma resuscitation are mediated through preservation of plasma proteins. PMID:25840538

  20. 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

  1. Highly effective fibrinolysis by a sequential synergistic combination of mini-dose tPA plus low-dose mutant proUK.

    PubMed

    Pannell, Ralph; Li, Shelley; Gurewich, Victor

    2015-01-01

    Results of thrombolysis by monotherapy with either tPA or proUK have not lived up to expectations. Since these natural activators are inherently complementary, this property can be utilized to a synergistic advantage; and yet, this has undergone little evaluation. ProUK is no longer available because at pharmacological concentrations it converts to UK in plasma. Therefore, a single site proUK mutant, M5, was developed to address this problem and was used in this study. Fibrinolysis was measured using preformed fluoresceinated 24 h old clots in a plasma milieu rather than by the standard automated method, because proUK/M5 is sensitive to inactivation by thrombin and activation by plasmin. The shortest 50% clot lysis time that could be achieved by tPA or M5 alone was determined: mean times were 55 and 48 minutes respectively. These bench marks were matched by 6% of the tPA monotherapy dose combined with 40% that of M5: mean lysis time 47 minutes with less associated fibrinogenolysis. Results showed that the tPA effect was limited to initiating fibrinolysis which was completed by M5 and then tcM5. Plasma C1-inhibitor inhibited fibrinogenolysis by M5, providing protection from side effects not available for proUK. In conclusion, by utilizing the complementary properties and sequential modes of action of each activator, more efficient fibrinolysis with less non-specific effects can be achieved than with traditional monotherapy. In vivo validation is needed, but in a previous clinical trial using a similar combination of tPA and proUK (5% and 50% monotherapy doses) very promising results have already been obtained. PMID:25811605

  2. 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

  3. Immunotherapy prolongs the serum CEA-TPA-CA15.3 lead time at the metastatic progression in endocrine-dependent breast cancer patients: a retrospective longitudinal study.

    PubMed

    Nicolini, A; Carpi, A; Ferrari, P; Rossi, G

    2008-05-01

    In metastatic breast cancer tumour markers' increase predicts, by a few months (lead time) disease progression. In breast cancer patients with endocrine dependent metastatic disease, we reported a prolonged clinical benefit and overall survival when first line conventional antiestrogen hormone therapy was started at the lead time and also when an immunotherapy schedule was added to the same conventional hormone treatment. Thirty-two of these last patients were considered (group a). In 27 (group b) of these 32 patients who progressed during first line salvage hormone plus immunotherapy the lead time at the progression of metastatic disease during therapy was compared with that at the onset of metastases when the same patients were without treatment and with that of a control group (group c) who did not receive immunotherapy. At disease progression, CEA-TPA-CA15.3 sensitivity was 92.5% in the group b (studied patients) and 88.5% in the group c (controls). At the progression in the group b, CEA-TPA-CA15.3 lead time (m+/-sd, months) was significantly longer than in group c (12.1+/-12.9 vs 2.4+/-4.0) (P=0.000). Besides, in group b the lead time was significantly longer at the progression than at the metastatic onset (P=0.003) while in the group c the difference was near to significance (P=0.05). The CEA-TPA-CA15.3 tumour marker panel accurately predicted metastatic disease progression and immunotherapy significantly prolonged the CEA-TPA-CA15.3 lead time. This can be used for anticipating salvage treatment in these patients.

  4. Effects of lowering the aluminium content of a dTpa vaccine on its immunogenicity and reactogenicity when given as a booster to adolescents.

    PubMed

    Theeten, H; Van Damme, P; Hoppenbrouwers, K; Vandermeulen, C; Leback, E; Sokal, E M; Wolter, J; Schuerman, L

    2005-02-10

    As aluminium in vaccines has been associated with the incidence of local side effects occurring after vaccination, this observer-blind randomised clinical trial was designed to evaluate the effect of lowering the aluminium content of a combined reduced-antigen-content dTpa vaccine on immunogenicity and safety when administered to healthy adolescents aged 10-18 years. A total of 647 subjects were enrolled, 224 (35%) received a dTpa formulation with 0.5 mg aluminium, 209 (32%) a formulation with 0.3 mg aluminium and 214 (33%) a formulation with 0.133 mg aluminium. One month after boostering, all subjects were seroprotected against diphtheria and tetanus toxoids. All subjects were seropositive for anti-FHA and anti-PRN but 4% of the initially seronegatives in both reduced aluminium groups did not seroconvert for anti-PT. Booster responses did not differ significantly between groups for any antibody, but post booster vaccination anti-PT GMC's differed significantly between groups and decreased when vaccine aluminium content decreased. No clear difference between study groups in local or general side effects was demonstrated. The most frequently reported symptoms after vaccination were injection site pain (89.5-90.7%), fatigue (42.1-47.4%) and headache (41.1-45.1%). This study showed that the aluminium content has a specific influence on the immunogenicity of this dTpa vaccine.

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

    PubMed

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

    2008-12-01

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

  6. Effects of molarity and solvents on the optical properties of the solutions of tris[4-(5-dicyanomethylidenemethyl-2-thienyl)phenyl]amine (TDCV-TPA) and structural properties of its film

    NASA Astrophysics Data System (ADS)

    Gündüz, Bayram

    2013-12-01

    The surface morphology of the tris[4-(5-dicyanomethylidenemethyl-2-thienyl)phenyl]amine (TDCV-TPA) film was investigated by high performance atomic force microscopy and the surface roughness parameters such as roughness average (sa), root mean square roughness (sq), surface skewness (ssk) and surface kurtosis (sku) of the TDCV-TPA film were obtained. The TDCV-TPA film indicated the low valleys with bumpy surface. The optical properties of the solutions of the small-molecule semiconducting dye TDCV-TPA for different high molarities, lower molarities and different solvents were investigated in detail. The molar extinction coefficient, optical band gap, angle values of refraction of the TDCV-TPA decreased with increasing molarity, while the absorbance at maximum absorption wavelengths, angle of incidence, electric susceptibility, real part values of the optical conductivity of the TDCV-TPA increased with increasing molarity. The maximum molar extinction coefficient (ɛmax) at λmax values (510 and 509 nm) of the solutions of the TDCV-TPA for 0.024 and 0.010 mM were found to be 1.175 × 105 and 1.931 × 105 L mol-1 cm-1, respectively. The maximum mass extinction coefficient (αmax) of the solutions of the TDCV-TPA for 0.024 and 0.010 mM were found to be 163.226 and 268.247 L g-1 cm-1, respectively. The optical band gap (Eg) values of the TDCV-TPA for 1, 2 and 3 mM were found to be 1.916, 1.898 and 1.892 eV, respectively. The absorption band edge for DCM varied from 1.882 to 1.997 eV, while the absorption band edge for chloroform varied from 1.923 to 2.027 eV. To obtain lower optical band gap of the TDCV-TPA can be prefered DCM solvent.

  7. 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.

  8. 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

  9. Displacement of submacular hemorrhage associated with age-related macular degeneration using vitrectomy and submacular tPA injection followed by intravitreal ranibizumab

    PubMed Central

    Sandhu, Sukhpal Singh; Manvikar, Sridhar; Steel, David Henry William

    2010-01-01

    Background/aims: To evaluate retrospectively the clinical outcomes of patients presenting with submacular hemorrhage (SMH) secondary to neovascular age-related macular degeneration (nAMD), treated by vitrectomy, submacular tissue plasminogen activator (tPA) injection and pneumatic displacement of SMH with air followed by postoperative intravitreal ranibizumab (RZB). Methods: Patients with SMH and nAMD had 25-guage vitrectomy and subretinal tPA (12.5 micrograms/0.1 mL) with fluid/air exchange. Intravitreal RZB was administered postoperatively to patients eligible for National Health Service (NHS) funded treatment. Results: Of the total of 16 patients, 11 (68.7%) had complete displacement of SMH. The remaining five had residual SMH, mainly subretinal pigment epithelium in location. Three of the four patients who previously had a failed expansile gas pneumatic displacement were successfully displaced with vitrectomy surgery. At presentation 5/16 (31.3%) patients were eligible for NHS funded intravitreal RZB. This increased to 12 patients after the vitrectomy procedure (75.0%). At 6 months postoperatively all improved by ≥1 line. Ten of the 16 patients (63%) improved by ≥2 lines, with 10 of the 12 patients (83%) treated with RZB improving by ≥2 lines. Conclusion: Vitrectomy/subretinal tPA/air to displace SMH followed by intravitreal RZB injection can stabilize/improve vision in patients with nAMD. This technique displaces hemorrhage not displaced by attempted expansile gas techniques. PMID:20668667

  10. 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

  11. 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.

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

    DOE PAGES

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

  13. Concordance of Hypermethylated DNA and the Tumor Markers CA 15-3, CEA, and TPA in Serum during Monitoring of Patients with Advanced Breast Cancer

    PubMed Central

    Kristiansen, Søren; Jørgensen, Lars Mønster; Hansen, Morten Høgh; Nielsen, Dorte; Sölétormos, György

    2015-01-01

    The serological protein tumor markers CA 15-3, CEA, and TPA are frequently used to monitor tumor burden among metastatic breast cancer patients. Breast cancer is associated with global DNA hypomethylation and hypermethylation of some promoter regions. No monitoring study has yet investigated the interrelationship between protein tumor markers, the global DNA hypomethylation, and hypermethylated genes in serum from patients with advanced disease. Twenty-nine patients with histologically proven advanced breast cancer received first-line chemotherapy with epirubicin. Samples were collected prior to each treatment and prospectively analyzed for CA 15-3, CEA, and TPA. The same samples were retrospectively analyzed for the concentration of hypermethylated RASSF1A and for global DNA hypomethylation using LINE-1. Among patients with elevated concentrations of the protein markers, concordance could be observed between serial changes of the hypermethylated RASSF1A gene and the protein markers. Among patients with lower concentrations, RASSF1A could only be detected periodically. There was discordance between changes of the hypomethylated LINE-1 as compared to the protein markers. Circulating hypermethylated RASSF1A and protein markers may have similar kinetics during monitoring of tumor burden. Further investigations are needed to determine whether any of the hypermethylated DNA genes may provide predictive information during monitoring. PMID:26339655

  14. The effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels are dependent on environmental context.

    PubMed

    Asselbergs, Folkert W; Williams, Scott M; Hebert, Patricia R; Coffey, Christopher S; Hillege, Hans L; Snieder, Harold; Navis, Gerjan; Vaughan, Douglas E; van Gilst, Wiek H; Moore, Jason H

    2007-11-01

    Thrombosis is a key factor in the pathophysiology of cardiovascular disease. Important biochemical constituents of the fibrinolytic system, affecting thrombosis, include tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Both t-PA and PAI-1 are determined by multiple genetic and environmental factors. We aimed to investigate whether the effects of polymorphism in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on t-PA or PAI-1 levels are dependent on environmental factors in a large population-based sample from the PREVEND study in Groningen, The Netherlands (n = 2,527). We found strong evidence (P t-PA in females and males and on PAI-1 in males. Only suggestive evidence (P t-PA and PAI-1 there was at least one BDKRB2-body size combination that exhibited suggestive (P t-PA and PAI-1 is dependent on the environmental context such as body size and alcohol use. The present study emphasizes the importance of including environmental factors in genetic analyses to fully comprehend the genetic architecture of a specific trait.

  15. 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

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

    PubMed

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

    2016-09-02

    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.

  17. Phorbol myristate acetate and dioctanoylglycerol inhibit transport in rabbit proximal convoluted tubule

    SciTech Connect

    Baum, M.; Hays, S.R. )

    1988-01-01

    The present in vitro microperfusion study examined the effect of protein kinase C activation on transport in the rabbit proximal convoluted tubule (PCT). PCT were perfused with an ultrafiltrate-like solution and were bathed in a serumlike albumin solution. Addition of phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibited volume absorption from 1.06 {plus minus} 0.10 to 0.77 {plus minus} 0.07 nl{center dot}mm{sup {minus}1}min{sup {minus}1}, and 0.76 {plus minus} 0.14 to 0.48 {plus minus} 0.08 nl{center dot}mm{sup {minus}1}{center dot}min{sup {minus}1}, respectively. Bath phorbol 12-myristate 13-acetate had no effect on volume absorption. In contrast, bath 4{alpha}-phorbol, an inactive phorbol that does not activate protein kinase C, had no effect on J{sub v}. Bath L-{alpha}-dioctanoylglycerol, another known activator of protein kinase C, inhibited volume absorption. A 10-fold lower concentration of L-{alpha}-dioctanoylglycerol had no effect on J{sub v}. Both 5 x 10{sup {minus}8} M phorbol 12-myristate 13-acetate and 10{sup {minus}4} M L-{alpha}-dioctanoylglycerol inhibited glucose, bicarbonate, and chloride transport in the PCT. These data are consistent with protein kinase C activation playing a role in the modulation of proximal tubular transport.

  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. 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

  2. Characterization of the human spr2 promoter: induction after UV irradiation or TPA treatment and regulation during differentiation of cultured primary keratinocytes.

    PubMed Central

    Gibbs, S; Lohman, F; Teubel, W; van de Putte, P; Backendorf, C

    1990-01-01

    We have isolated genomic clones from several members of the UV and TPA inducible human spr2 gene-family in order to analyse the regulation of these genes at a molecular level. From one of these members, the spr2-1 gene, we have identified and sequenced the regulatory region. By using CAT fusion plasmids and a liposome mediated transfection procedure we show that the isolated promoter region contains all the cis-elements necessary for induced expression after UV irradiation or phorbolester treatment of cultured human keratinocytes. Additionally the spr2-1 promoter is shown to be regulated aswell during the normal process of keratinocyte differentiation. This makes the spr2-1 promoter sequence an ideal tool to study the molecular mechanisms by which environmental agents such as UV radiation and chemical tumor promoters interfere with normal gene expression during cell proliferation and differentiation. Images PMID:2388825

  3. 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

  4. /sup 3/H-ouabain binding and sodium-pump activity measured in myocytes isolated from guinea-pig heart

    SciTech Connect

    Stemmer, P.

    1986-01-01

    Because of the toxicity of millimolar ouabain, non-specific /sup 3/H-ouabain binding was assessed by monitoring the dissociation of the bound drug. Analysis of specific /sup 3/H-ouabain binding to myocytes yielded non-linear Scatchard plots. Nonlinearity appears to result from reduced /sup 3/H-ouabain binding due to low intracellular Na/sup 2/ concentration. Addition of 2 ..mu..M monensin, A Na/sup +/ ionophore, significantly increased /sup 3/H-ouabain binding. Incubation in Ca/sup 2 +/-free solution (0.25 mM EGTA) stimulated /sup 3/H-ouabain binding to a greater degree than monensin and caused Scatchard plots to have two distinct linear components. Monensin had no significant effects when /sup 3/H-ouabain binding occurred in Ca/sup 2 +/-free solution. Effects of Ca/sup 2 +/-free incubation to increase /sup 3/H-ouabain binding suggest that Ca/sup 2 +/ has a direct effect on /sup 3/H-ouabain binding. Alternatively, Ca/sup 2 +/-free incubation may increase Na/sup +/ permeability of the sarcolemma. Isoproterenol, phenylephrine, TPA (phorbol 12-myristate 13-acetate), La/sup 3 +/, and the Ca/sup 2 +/-ionophore A23187 failed to cause significant changes in /sup 3/H-ouabain binding when myocytes were incubated in a solution containing 0.5 or 2.5 ..mu..M /sup 3/H-ouabain, 0.1 mM Ca/sup 2 +/ and 1 mM K/sup +/.

  5. Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea

    PubMed Central

    Uzlaner, Natalya; Priel, Zvi

    1999-01-01

    Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation.Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin.The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3′, 5′-cyclic monophosphothioate triethylamine (Rp-cAMPS).Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i.Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin.The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP. PMID:10066932

  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. Pathological VWF fibers resist tPA and ADAMTS13 while promoting the contact pathway and shear-induced platelet activation

    PubMed Central

    Herbig, Bradley A.

    2015-01-01

    Summary Background Under severe stenotic conditions, von Willebrand Factor (VWF) multimerizes into large insoluble fibers at pathological shear rates. Objective Evaluate the mechanics and biology of VWF fibers without the confounding effects of endothelium or collagen. Methods Within a micropost-impingement microfluidic device, >100 µm long VWF fibers multimerized on the post within 10 min using EDTA-treated PFP perfused at wall shear rates >5000 s−1. Results VWF fiber thickness increased to >10 µm by increasing shear rate to 10,000 s−1. In a stress-strain test, fibrous VWF had an elastic modulus of ~50 MPa. The insoluble VWF fibers were non-amyloid since they rapidly dissolved in trypsin, plasmin, or 2% SDS, but were resistant to 50 nM ADAMTS13 or 100 nM tPA in plasma. Following fiber formation, perfusion of low corn trypsin inhibitor (CTI)-treated (4 µg/ml), recalcified citrated plasma at 1500 s−1 caused fibrin formation on the VWF fibers, a result not observed with purified type 1 collagen or a naked micropost. During VWF fiber formation, contact pathway factors accumulated on VWF since the use of EDTA/PPACK/apixaban/high CTI-treated PFP during VWF fiber formation prevented subsequent fibrin production from low CTI, recalcified citrated PFP. VWF fibers displayed FXIIa-immunostaining. When PPACK-inhibited whole blood was perfused over VWF fibers, platelets rolled and arrested on the surface of VWF, but only displayed P-selectin if prevailing shear rates were pathological. Platelet arrest on VWF fibers was blocked with αIIbβ3 antagonist GR144053. Conclusions We report VWF fiber-contact pathway crosstalk and mechanisms of thrombolytic resistance in hemodynamic settings of myocardial infarction. PMID:26178390

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

    PubMed Central

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

    2016-01-01

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

  9. 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

  10. 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

  11. Differential expression of five protein kinase C isoenzymes in FAO and HepG2 hepatoma cell lines compared with normal rat hepatocytes.

    PubMed

    Ducher, L; Croquet, F; Gil, S; Davy, J; Féger, J; Bréhier, A

    1995-12-14

    We analyzed the expression of five protein kinase C (PKC) isoforms in cytosolic and membrane fractions from normal rat hepatocytes compared with those of two tumorigenic cell lines FAO and HepG2. Western blots with PKC-specific isoenzymes polyclonal antibodies provide evidences for the presence of the five isoforms alpha, beta II, delta, epsilon and zeta in normal rat hepatocytes. In hepatoma cells, we show differences in the level of expression, the molecular sizes and the responses to Phorbol 12-myristate 13-acetate (PMA).

  12. Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion.

    PubMed Central

    Esposito, F; Agosti, V; Morrone, G; Morra, F; Cuomo, C; Russo, T; Venuta, S; Cimino, F

    1994-01-01

    We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12-myristate 13-acetate-induced activation of the transcription factors AP-1 and Egr-1, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7519845

  13. Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain.

    PubMed

    Kumar, Yogesh; Vadivel, Kanagasabai; Schmidt, Amy E; Ogueli, Godwin I; Ponnuraj, Sathya M; Rannulu, Nalaka; Loo, Joseph A; Bajaj, Madhu S; Bajaj, S Paul

    2014-01-28

    Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2' residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin-KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin-KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes. PMID:24383758

  14. Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

    SciTech Connect

    Kato, Shigemi; Ben, T.L.; De Luca, L.M. )

    1988-11-01

    The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10{sup {minus}9} and 10{sup {minus}7} M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

  15. Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells.

    PubMed Central

    Anisowicz, A; Zajchowski, D; Stenman, G; Sager, R

    1988-01-01

    Previous studies of gro and related genes that are overexpressed in transformed fibroblasts suggest that gro may encode a specific growth regulator. However, DNA and protein sequence comparisons reveal relatedness to platelet factor 4 and other proteins involved in the inflammatory response. In this paper, both growth-related and cytokine-induced responses in gro gene expression are described. Human foreskin fibroblasts are shown to express approximately 10-fold elevated gro, myc, and fos mRNAs in response to serum and to phorbol 12-myristate 13-acetate stimulation, with early response kinetics indicative of growth regulation. In response to interleukin 1, however, in growing cells gro mRNA is elevated at least 100-fold but myc remains constant and fos is not expressed, suggesting a second regulatory pathway. In normal cultured mammary epithelial cells, gro is constitutively expressed, and elevated mRNA levels are induced by phorbol 12-myristate 13-acetate, but not by interleukin 1. However, most carcinoma cell lines examined do not express gro mRNA, suggesting a third function of gro as a negative growth regulator in epithelial cells. Images PMID:3264403

  16. p23/Tmp21 associates with protein kinase Cdelta (PKCdelta) and modulates its apoptotic function.

    PubMed

    Wang, HongBin; Xiao, Liqing; Kazanietz, Marcelo G

    2011-05-01

    There is emerging evidence that C1 domains, motifs originally identified in PKC isozymes and responsible for binding of phorbol esters and diacylglycerol, interact with the Golgi/endoplasmic reticulum protein p23 (Tmp21). In this study, we investigated whether PKCδ, a kinase widely implicated in apoptosis and inhibition of cell cycle progression, associates with p23 and determined the potential functional implications of this interaction. Using a yeast two-hybrid approach, we found that the PKCδ C1b domain associates with p23 and identified two key residues (Asp(245) and Met(266)) implicated in this interaction. Interestingly, silencing p23 from LNCaP prostate cancer cells using RNAi markedly enhanced PKCδ-dependent apoptosis and activation of PKCδ downstream effectors ROCK and JNK by phorbol 12-myristate 13-acetate. Moreover, translocation of PKCδ to the plasma membrane by phorbol 12-myristate 13-acetate was enhanced in p23-depleted LNCaP cells. Notably, a PKCδ mutant that failed to interact with p23 triggered a strong apoptotic response when expressed in LNCaP cells. In summary, our data compellingly support the concept that C1 domains have dual roles both in lipid and protein associations and provide strong evidence that p23 acts as an anchoring protein that retains PKCδ at the perinuclear region, thus limiting the availability of this kinase for activation in response to stimuli.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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...

  2. Biomimetic oxidation studies. 11: Alkane functionalization in aqueous solution utilizing in situ formed [Fe{sub 2}O({eta}{sup 1}-H{sub 2}O)({eta}{sup 1}-OAc)(TPA){sub 2}]{sup 3+}, as an MMO model precatalyst, embedded in surface-derivatized silica and contained in micelles

    SciTech Connect

    Neimann, K.; Neumann, R.; Rabion, A. |; Buchanan, R.M.; Fish, R.H.

    1999-07-26

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

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

    SciTech Connect

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

    1986-03-05

    In the present study, the effect of phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA), activation of protein kinase C on coronary vascular reactivity was studied in isolated dog coronary arteries. Addition of TPA (10-100 nM) produced a slow, time- and dose-dependent contraction reaching a maximum at approx 2-3 hrs and was essentially irreversible upon washing. Disruption of the endothelium(EC) greatly accelerated the development as well as increase the magnitude of TPA contraction (50-100%). Prior treatment of vessels with phentolamine (1..mu..M), cyproheptadine (1..mu..H) and ibuprofen (1..mu..g/ml) did not alter the TPA contraction. Furthermore, in contrast to previously reported calcium-dependence of TPA contraction in other vessels, complete removal of extracellular calcium (Ca/sub 0/) or addition of 1..mu..M nimodipine after TPA(30nM) resulted in only 32 +/- 4% and 25 +/- 3% reversal of TPA contraction, respectively. Addition of amiloride (10..mu..M to 1mM), however, resulted in a dose-dependent reversal of TPA contraction. The results of the present study indicate that a similar activation of protein kinase C by TPA leads to potent coronary vasoconstriction, which is not completely dependent on Ca/sub 0/. More importantly, these results further support their hypothesis that EC also functions as an inhibitory barrier to prevent circulating vasoconstrictors from exerting their deleterious constrictory effects.

  4. 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.

  5. 14-3-3 regulates the nuclear import of class IIa histone deacetylases

    SciTech Connect

    Nishino, Tomonori G.; Miyazaki, Masaya; Hoshino, Hideto; Miwa, Yoshihiro; Horinouchi, Sueharu; Yoshida, Minoru

    2008-12-19

    Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a manner dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.

  6. 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

  7. Clastogenic action of hydroperoxy-5,8,11,13-icosatetraenoic acids on the mouse embryo fibroblasts C3H/10T 1/2

    SciTech Connect

    Ochi, T.; Cerutti, P.A.

    1987-02-01

    Phorbol 12-myristate 13-acetate induces the release of a low molecular weight clastogenic factor from monocytes. Hydroperoxy-5,8,11,13-icosatetraenoic acids represent major components of clastogenic factor. The authors report that several isomeric hydroperoxy-5,8,11,13-icosatetraenoic acids efficiently induce DNA strand breakage and/or alkali-labile sites in the mouse embryo fibroblasts C3H/10T 1/2. Fe chelation by desferrioxamine suppresses breakage indicating the participation of Fe-catalyzed radical reactions. An additional 37% inhibition is observed upon addition of the Ca/sup 2 +/ chelators EGTA and quin-2. This result suggests that hydroxy-peroxy-5,8,11,13-icosatetraenoic acid may activate a Ca/sup 2 +/-dependent nuclease. The addition of the antioxidant enzymes CuZn-superoxide dismutase and catalase had no effect, while glutathione peroxidase suppressed strand breakage by 90%.

  8. 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.

  9. Involvement of PKC{alpha} in PMA-induced facilitation of exocytosis and vesicle fusion in PC12 cells

    SciTech Connect

    Xue Renhao; Zhao Yanying; Chen Peng

    2009-03-06

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

  10. Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

    SciTech Connect

    Davis, L.; Lipsky, P.E.

    1986-05-15

    The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4..beta..-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Results of cell cycle analysis support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen stimulated T cells through the first round of the cell cycle.

  11. Mitochondrial APE1/Ref-1 suppressed protein kinase C-induced mitochondrial dysfunction in mouse endothelial cells.

    PubMed

    Joo, Hee Kyoung; Lee, Yu Ran; Park, Myoung Soo; Choi, Sunga; Park, Kyoungsook; Lee, Sang Ki; Kim, Cuk-Seong; Park, Jin Bong; Jeon, Byeong Hwa

    2014-07-01

    Protein kinase C (PKC) induces mitochondrial dysfunction, which is an important pathological factor in cardiovascular diseases. The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) on PKC-induced mitochondrial dysfunction has not been variously investigated. In this study, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, induced mitochondrial hyperpolarization and reactive oxygen species generation and also increased mitochondrial translocation of APE1/Ref-1. APE1/Ref-1 overexpression suppressed PMA-induced mitochondrial dysfunction. In contrast, gene silencing of APE1/Ref-1 increased the sensitivity of mitochondrial dysfunction. Moreover, mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 more effectively suppressed PMA-induced mitochondrial dysfunctions. These results suggest that mitochondrial APE1/Ref-1 is contributed to the protective role to protein kinase C-induced mitochondrial dysfunction in endothelial cells.

  12. 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.

  13. 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.

  14. Inhibitory effects of [6]-gingerol on PMA-induced COX-2 expression and activation of NF-kappaB and p38 MAPK in mouse skin.

    PubMed

    Kim, Sue Ok; Chun, Kyung-Soo; Kundu, Joydeb Kumar; Surh, Young-Joon

    2004-01-01

    [6]-Gingerol, a major pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. Previous studies have demonstrated that [6]-gingerol inhibits mouse skin tumor promotion and anchorage-independent growth of cultured mouse epidermal cells stimulated with epidermal growth factor. Cyclooxygenase-2 (COX-2), a key enzyme in the prostaglandin biosynthesis, has been recognized as a molecular target for many anti-inflammatory as well as chemopreventive agents. Topical application of [6]-gingerol inhibited phorbol 12-myristate 13-acetate -induced COX-2 expression. One of the essential transcription factors responsible for COX-2 induction is NF-kappaB. [6]-Gingerol suppressed NF-kappaB DNA binding activity in mouse skin. In addition, [6]-gingerol inhibited the phoshorylation of p38 mitogen-activated protein kinase which may account for its inactivation of NF-kappaB and suppression of COX-2 expression. PMID:15630166

  15. [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

  16. Observation of phagocytosis of fullerene nanowhiskers by PMA-treated THP-1 cells

    NASA Astrophysics Data System (ADS)

    Nudejima, S.; Miyazawa, K.; Okuda-Shimazaki, J.; Taniguchi, A.

    2009-04-01

    Phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells (macrophage-like cells) were exposed to the C60 fullerene nanowhiskers (C60 NWs) with an average length of about 6.0 μm and an average diameter of about 660 run and observed with an inverted optical phase-contrast microscope for 48 h. The C60 NWs were well and stably dispersed onto the dishes of culture medium during the observation. The number of cells that internalised C60 NWs gradually increased after the exposure to C60 NWs. But no alteration of cellular morphology was observed compared to the control group without exposure to C60 NWs during this period in this pilot study.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA.

    PubMed Central

    Chanock, S J; Faust, L R; Barrett, D; Bizal, C; Maly, F E; Newburger, P E; Ruedi, J M; Smith, R M; Babior, B M

    1992-01-01

    The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every chronic granulomatous disease patient studied to date, an abnormality has been found in a gene encoding one of four components of the respiratory burst oxidase: the membrane protein p22phox or gp91phox, or the cytosolic protein p47phox or p67phox. We report here that O2- production was partly restored to phorbol 12-myristate 13-acetate-stimulated Epstein-Barr virus-transformed B lymphocytes from a patient with p47phox-deficient chronic granulomatous disease by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. No detectable O2- was produced by untransfected p47phox-deficient lymphocytes or by p47phox-deficient lymphocytes transfected with an antisense plasmid. The finding that O2- can be produced by p47phox-deficient B lymphocytes after the transfer of a p47phox cDNA into the deficient cells suggests that this system could be useful for studying the function of mutant p47phox proteins in whole cells. Images PMID:1332032

  2. Effects of an aqueous extract from leaves of Ligustrum vulgare on mediators of inflammation in a human neutrophils model.

    PubMed

    Czerwińska, Monika E; Granica, Sebastian; Kiss, Anna K

    2013-07-01

    Leaves of Ligustrum vulgare (common privet) have been used for treatment of oropharyngeal inflammations or as antirheumatic, diuretic, and hypotensive agents in folk medicine in southern Europe. Taking into account that neutrophils are involved in the inflammation, the aim of the study was to determine the effect of an aqueous extract prepared from leaves of Ligustrum vulgare on neutrophil functions. The extract was characterized by the HPLC-DAD-MSn method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase, metalloproteinase 9, and interleukin 8 production by neutrophils was measured by an enzyme-linked immunosorbent assay. Neutrophil elastase release was established spectrophotometrically. The expression of adhesion molecules on neutrophils was analyzed with flow cytometry. The main compounds detected were flavonoids, phenylpropanoids, hydroxycinnamates, and secoiridoids. The inhibition of oxidative burst by the extract was comparable in both stimuli models (formyl-met-leu-phenylalanine: IC50 = 18.2 ± 4.0 µg/mL; phorbol 12-myristate 13-acetate: IC50 = 19.8 ± 3.0 µg/mL). The extract in the concentration range of 5-50 µg/mL inhibited neutrophil elastase release by 23.9-34.1 % and myeloperoxidase release by 24.2-37.4 %. The inhibitory effect on metalloproteinase 9 and interleukin 8 production was around 20 %. The extract in the highest concentration modulated the expression of L-selectin and β2 integrin. Our results partly support the traditional use of common privet leaves as an anti-inflammatory agent.

  3. Activation of l-arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages

    PubMed Central

    Racké, Kurt; Hey, Claudia; Mössner, Jutta; Hammermann, Rainer; Stichnote, Christina; Wessler, Ignaz

    1998-01-01

    The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4β-Phorbol 12-myristate 13-acetate (PMA), but not 4α-phorbol 12-myristate 13-acetate (α-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This short-term effect was not affected by cycloheximide or actinomycin D. L-[3H]Arginine uptake and its stimulation by PMA was also observed in sodium-free medium. L-Leucine (0.1 mM) inhibited L-[3H]arginine uptake by 50 % in sodium-containing medium, but not in sodium-free medium. At 1 mM, L-leucine caused significant inhibition in sodium-free medium also. L-Leucine showed similar effects on PMA-treated cells. N-Ethylmaleimide (200 μm, 10 min) reduced L-[3H]arginine uptake by 70 % in control cells, but had no effect on PMA-treated (20 or 2 h) cells. In alveolar macrophages, multiple transport systems are involved in L-arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters. PMID:9714862

  4. Differential induction of apoptosis in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A

    SciTech Connect

    Kiguchi, Kaoru; Chubb, C.H.; Glesne, D.; Huberman, E. |; Fujiki, Hirota

    1994-09-01

    To investigate a possible relationship between apoptosis induction and protein phosphorylation in human breast carcinoma cells, the authors treated three such cell types, MB-231, MCF-7, and AU-565, wit okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, or phorbol 12 myristate 13-acetate, an activator of protein kinase C. They then examined these cells of the appearance of apoptosis markers. While OA caused multiplication arrest and cytotoxicity in all three cell lines, apoptosis was induced in MB-231 and MCF-7 cells but not in AU-565 cells. A similar cell-specific apoptosis induction was also observed after treatment with dinophysistoxin-1 (an active OA analogue) and with calyculin A (a structurally unrelated protein phosphatase inhibitor) but not with analogues that either ar inactive or penetrate epithelial cells poorly. Phorbol 12-myristate 13-acetate also inhibited cell multiplication but was without effect in inducing apoptosis in these cells. Levels of the apoptosis-inhibitory protein BCL2 were examined in these cells, but they did to correlate with this differential susceptibility. They additionally treated the three cell types with 1-{beta}-D-arabinofuranosylcytosine and genistein to determine whether the AU-565 cell line would also be resistant to apoptosis induction by other chemical stimuli. Both of these agents led to the induction of apoptosis in all three cell lines. These results indicate that the AU-565 cells are specifically resistant to apoptosis induction by inhibitors of protein phosphatases 1 and 2A. This cell-specific resistance may thus allow one to identify cellular mediators of apoptosis by comparing protein phosphorylation patterns in these cells before and after treatment with OA or related inhibitors.

  5. Effects of an aqueous extract from leaves of Ligustrum vulgare on mediators of inflammation in a human neutrophils model.

    PubMed

    Czerwińska, Monika E; Granica, Sebastian; Kiss, Anna K

    2013-07-01

    Leaves of Ligustrum vulgare (common privet) have been used for treatment of oropharyngeal inflammations or as antirheumatic, diuretic, and hypotensive agents in folk medicine in southern Europe. Taking into account that neutrophils are involved in the inflammation, the aim of the study was to determine the effect of an aqueous extract prepared from leaves of Ligustrum vulgare on neutrophil functions. The extract was characterized by the HPLC-DAD-MSn method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase, metalloproteinase 9, and interleukin 8 production by neutrophils was measured by an enzyme-linked immunosorbent assay. Neutrophil elastase release was established spectrophotometrically. The expression of adhesion molecules on neutrophils was analyzed with flow cytometry. The main compounds detected were flavonoids, phenylpropanoids, hydroxycinnamates, and secoiridoids. The inhibition of oxidative burst by the extract was comparable in both stimuli models (formyl-met-leu-phenylalanine: IC50 = 18.2 ± 4.0 µg/mL; phorbol 12-myristate 13-acetate: IC50 = 19.8 ± 3.0 µg/mL). The extract in the concentration range of 5-50 µg/mL inhibited neutrophil elastase release by 23.9-34.1 % and myeloperoxidase release by 24.2-37.4 %. The inhibitory effect on metalloproteinase 9 and interleukin 8 production was around 20 %. The extract in the highest concentration modulated the expression of L-selectin and β2 integrin. Our results partly support the traditional use of common privet leaves as an anti-inflammatory agent. PMID:23824550

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

    SciTech Connect

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

    1989-01-01

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

  7. Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

    PubMed Central

    Contor, L; Lamy, F; Lecocq, R; Roger, P P; Dumont, J E

    1988-01-01

    Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways. Images PMID:3261388

  8. Negative chronotropic actions of endothelin-1 on rabbit sinoatrial node pacemaker cells

    PubMed Central

    Tanaka, Hideo; Habuchi, Yoshizumi; Yamamoto, Taku; Nishio, Manabu; Morikawa, Junichiro; Yoshimura, Manabu

    1997-01-01

    AMP-resistant component (17.5±14.4%, n=20) was not affected by combined application of 8-Br cyclicAMP with 8-bromo cyclicGMP (500 μM), ryanodine (1 μM) or phorbol-12-myristate-13-acetate (TPA; 50 nM). In summary, ET-1 exerts negative chronotropic effects on the SA node via ETA-receptors. ET-1 inhibits both ICa(L) and IK, and increases background K+ current. The inhibition of ICa(L) by ET-1 is mainly due to reduction of the cyclicAMP levels via PTX-sensitive G protein, but some other mechanism(s) also seems to be operative. PMID:9313942

  9. Novel type of phorbol ester-dependent protein phosphorylation in the particulate fraction of mouse epidermis

    SciTech Connect

    Gschwendt, M.; Kittstein, W.; Marks, F.

    1986-06-13

    In a Triton X100-extract from the particulate fraction of mouse epidermis but also of other murine tissues, the phosphorylation of a protein with the relative molecular mass of 82,000 (p82) is found to be dependent on phosphatidyl serine and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Unlike protein kinase C-catalyzed phosphorylation, p82 phosphorylation is neither observed in the presence of high concentrations of Ca/sup 2 +/ and phosphatidyl serine alone nor after addition of exogenous protein kinase C. Dioctanoylglycerol and the incomplete promoter 12-O-retinoylphorbol-13-acetate are also capable of stimulating p82 phosphorylation, whereas the non-promoting phorbol ester 4-O-methyl-TPA is at least 100-fold less active in this respect.

  10. Cancer chemopreventive activity of carotenoids in the fruits of red paprika Capsicum annuum L.

    PubMed

    Maoka, T; Mochida, K; Kozuka, M; Ito, Y; Fujiwara, Y; Hashimoto, K; Enjo, F; Ogata, M; Nobukuni, Y; Tokuda, H; Nishino, H

    2001-10-30

    Capsanthin and related carotenoids isolated from the fruits of red paprika Capsicum annuum L. showed potent in vitro anti-tumor-promoting activity with inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Among them, capsanthin diester and capsorbin diester showed strong inhibitory effects. Furthermore, capsanthin , capsanthin 3'-ester and capsanthin 3,3'-diester , major carotenoids in paprika, exhibited potent anti-tumor-promoting activity in an in vivo mouse skin two-stage carcinogenesis assay using 7, 12-dimethylbenz[a]anthracene as an initiator and TPA as a promoter.

  11. 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.

  12. 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

  13. Ionic, electrical, and secretory effects of protein kinase C activation in mouse pancreatic B-cells: studies with a phorbol ester

    SciTech Connect

    Bozem, M.; Nenquin, M.; Henquin, J.C.

    1987-09-01

    The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to study the effects of protein kinase C activation on stimulus-secretion coupling in mouse pancreatic B-cells. At a nonstimulatory concentration of glucose (3 mM), 100 nM TPA, but not 10 nM TPA, slightly and slowly increased insulin release and /sup 45/Ca/sup 2 +/ efflux and decreased /sup 86/Rb/sup +/ efflux, but did not affect the membrane potential of B-cells. At a threshold concentration of glucose (7 mM), 100 nM TPA markedly increased insulin release without triggering electrical activity in B-cells. At a stimulatory concentration of glucose (10 mM), TPA caused a dose-dependent irreversible increase in insulin release, /sup 45/Ca/sup 2 +/ efflux, and /sup 86/Rb/sup +/ efflux and slightly augmented islet cAMP levels. Omission of extracellular Ca/sup 2 +/ abolished the effects of 10 nM TPA and partially inhibited those of 100 nM TPA on insulin release and /sup 45/Ca/sup 2 +/ efflux. In contrast, their effect on /sup 86/Rb/sup +/ efflux was paradoxically augmented. Glucose-induced electrical activity in B-cells was only marginally affected by TPA; the duration of the slow waves with spikes was not modified, but a small shortening of the polarized intervals raised their frequency and slightly increased the overall activity. This increase was significant only with 10 nM TPA, whereas only 100 nM TPA brought about a minute increase in /sup 45/Ca/sup 2 +/ influx. These results thus show that TPA induces insulin release or potentiates glucose-induced insulin release without mimicking or amplifying the initial ionic and electrical signals triggered by glucose. They suggest that protein kinase C activation affects stimulus-secretion coupling by modulating intracellular and/or nonelectrogenic membrane events.

  14. 12-tetradecanoyl-phorbol-13-acetate (PMA) produces injury to isolated rat lungs in the presence and absence of perfused neutrophils

    SciTech Connect

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

    1986-03-01

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

  15. Effect of phorbol esters on guniea pig skin in vivo.

    PubMed

    Bourin, M C; Delescluse, C; Fürstenberger, G; Marks, F; Schweizer, J; Klein-Szanto, A J; Prunieras, M

    1982-01-01

    When topically applied to guniea pig ear skin the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced inflammation and epidermal hyperproliferation which could be inhibited by indomethacin. This inhibition could be reversed both by prostaglandins E and F. Five minutes after TPA treatment an increase in the level of prostaglandin E but not of prostaglandin F was observed in the epidermis. The non-promoting phorbol ester 4-O-methyl-TPA also stimulated epidermal cell proliferation but this stimulation was not inhibited by indomethacin. The above results are in agreement with those already reported in the mouse system with these two compounds. Ornithine decarboxylase (ODC) activity has been evaluated in the epidermis of guniea pig ear after topical application of 20 nmol of TPA. No increase was noted. This is in contrast with the well documented activation of ODC in mouse skin treated with TPA. Since TPA acts as a promoter in the mouse whereas both croton oil and TPA have no promoting action in the guinea pig, the above result supports the view that ODC activationis related to promotion, and provides a possible explanation for the resistance of this animal species to promotion. This resistance is further documented by the fact that no "dark cells" were found in guinea pig ear skin.

  16. Protein modifications induced in mouse epidermis by potent and weak tumor-promoting hyperplasiogenic agents

    SciTech Connect

    Nelson, K.G.; Stephenson, K.B.; Slaga, T.J.

    1982-10-01

    Two-dimensional gel electrophoresis was used to compare the changes in mouse epidermal proteins induced by the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), by the moderate promoter mechanical abrasion, and by the weakly promoting hyperplasiogenic agents mezerein and ethylphenylpropiolate. Evidence is presented which indicates that TPA caused many changes in the epidermal protein profiles especially related to the keratins which are the major differentiation product of the epidermis. The criteria used for the identification of the keratins were extractability, isoelectric points, molecular weights, filament formation in vitro, immunological cross-reactivity, amino acid composition, and peptide mapping. Several other protein changes were evident in the more soluble epidermal proteins which were also prominent in the newborn epidermis. Mezerein and abrasion produced protein changes similar to those induced by TPA. Ethylphenylpropiolate-induced protein modifications not only occurred at later times compared with either mezerein or TPA but also were less in magnitude. However, although many of the protein modifications induced by TPA appear to be associated with the hyperplasiogenic properties of TPA, the major difference between a potent promoter like TPA and a weak promoter like ethylphenylpropiolate appeared to be related to the magnitude of the response and the time of appearance of the protein changes.

  17. Effect of phorbol esters on human erythrocyte morphological discocyte-echinocyte transitions

    SciTech Connect

    Jones, B.; Walker, T.F.; Chahwala, S.B.; Thompson, M.G.; Hickman, J.A.

    1987-02-01

    12-O-Tetradecanoylphorbol-13-acetate (TPA) (100 nM) when incubated with human erythrocytes under conditions of ATP depletion, delayed the onset of the morphological transition from discocytes to echinocytes so that at 2 h, when control incubations were estimated to contain 65% echinocytes, those treated with TPA contained 23% echinocytes. TPA did not alter the subsequent rate of the transition which was complete by 3 h in control cells and 5 h in TPA-treated cells. Addition of 100 nM TPA to ATP-depleted erythrocytes at 2.5 h for 0.5 h at 37/sup 0/C resulted in 17% reversal to a discocyte morphology, but as the time of incubation under conditions of ATP depletion was extended, the level of the reversal fell. TPA had no significant effect on the fall in ATP concentrations over the time course of the experiments (5 h). Preincubation of discocytes with TPA for 10 min also prevented, by approx. 50%, the echinocytosis induced by the calcium (0.2 mM) loading of discocytes using 5 ..mu..M A23187. Incubation of discocytes with the diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) (1-10 ..mu..M) had complex effects on morphology, and the ATP-induced morphological transition, ranging from stomatocyte formation to echinocyte formation, depending upon the concentration of the agent and the time of incubation.

  18. Tumor promoting phorbol diesters: substrates for diacylglycerol lipase

    SciTech Connect

    Cabot, M.C.

    1984-08-30

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

  19. Responding to edTPA: Transforming Practice or Applying Shortcuts?

    ERIC Educational Resources Information Center

    Denton, David W.

    2013-01-01

    Some states have used new teacher performance assessments in an attempt to improve teacher quality for more than two decades. New teacher performance assessments include performance expectations, scoring rubrics, and writing prompts, which are organized into subject-specific handbooks. Teacher candidates completing performance assessments assemble…

  20. 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 ...

  1. Carbachol regulates cholecystokinin receptor on pancreatic acinar cells

    SciTech Connect

    Honda, T.; Adachi, H.; Noguchi, M.; Sato, S.; Onishi, S.; Aoki, E.; Torizuka, K.

    1987-01-01

    The authors have examined the effect of carbamylcholine on the binding of cholecystokinin (CCK) to dispersed acini from rat pancreas. The CCK receptor on pancreatic acini possesses two classes of binding sites. Simultaneous addition of carbamylcholine inhibited binding of CCK binding sites. Atropine prevented the inhibitory effect of carbamylcholine, whereas calcium ionophore A23187 did not alter binding of CCK. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited binding of CCK in the same manner as carbamylcholine. Inhibition by carbamylcholine was reversible and the recovery was time dependent. By contrast, inhibition of binding of CCK by TPA did not reverse after a 60-min incubation without the agent. These findings, at least in part, account for the inhibition of the CCK-induced stimulation of amylase secretion by carbamylcholine. The action of TPA on binding of CCK suggests the possible involvement of the activation of protein kinase C in the inhibition of binding.

  2. 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.

  3. Curcumin suppresses activation of NF-kappaB and AP-1 induced by phorbol ester in cultured human promyelocytic leukemia cells.

    PubMed

    Han, Seong-Su; Keum, Young-Sam; Seo, Hyo-Joung; Surh, Young-Joon

    2002-05-31

    Many components that are derived from medicinal or dietary plants possess potential chemopreventive properties. Curcumin, a yellow coloring agent from turmeric (Curcuma longa Linn, Zingiberaceae), possesses strong antimutagenic and anticarcinogenic activities. In this study, we have found that curcumin inhibits the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced nuclear factor kB (NF-kappaB) activation by preventing the degradation of the inhibitory protein IkBalpa; and the subsequent translocation of the p65 subunit in cultured human promyelocytic leukemia (HL-60) cells. Alternatively, curcumin repressed the TPA-induced activation of NF-kappaB through direct interruption of the binding of NF-kappaB to its consensus DNA sequences. Likewise, the TPA-induced DNA binding of the activator protein-1 (AP-1) was inhibited by curcumin pretreatment. PMID:12297018

  4. Effects of 1-beta-D-arabinofuranosylcytosine and phorbol ester on differentiation of human K562 erythroleukemia cells.

    PubMed

    Watanabe, T; Mitchell, T; Sariban, E; Sabbath, K; Griffin, J; Kufe, D

    1985-06-01

    We have previously demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces hemoglobin synthesis in human K562 erythroleukemia cells. The present study extends these findings by demonstrating that ara-C treatment of K562 cells results in both increased heme synthesis and accumulation of alpha-, gamma-, epsilon-, and zeta-globin RNA. The results also demonstrate that ara-C enhances K562 cell surface expression of glycophorin. Furthermore, we demonstrate that phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) inhibits the effects of ara-C on heme production, accumulation of globin RNA, and glycophorin expression. The inhibitory effect occurs maximally when K562 cells are treated with TPA before undergoing ara-C-induced commitment to erythroid differentiation. These findings suggest that TPA inhibits an early step in the process required for ara-C to enhance expression of genes involved in the erythroid program.

  5. Inhibitory effect of topical application of a green tea polyphenol fraction on tumor initiation and promotion in mouse skin.

    PubMed

    Huang, M T; Ho, C T; Wang, Z Y; Ferraro, T; Finnegan-Olive, T; Lou, Y R; Mitchell, J M; Laskin, J D; Newmark, H; Yang, C S

    1992-06-01

    A green tea polyphenol fraction was evaluated for its ability to inhibit tumor initiation by polycyclic aromatic hydrocarbons and tumor promotion by a phorbol ester in the skin of CD-1 mice. Topical application of the green tea polyphenol fraction inhibited benzo[a]pyrene- and 7,12-dimethylbenz[a]-anthracene-induced tumor initiation as well as 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion. Topical application of the green tea polyphenol fraction also inhibited TPA-induced inflammation, ornithine decarboxylase activity, hyperplasia and hydrogen peroxide formation. Studies with individual polyphenolic compounds in green tea indicated that topical application of (-)-epigallocatechin gallate, (-)-epigallocatechin and (-)-epicatechin gallate inhibited TPA-induced inflammation in mouse epidermis.

  6. Resveratrol inhibits phorbol ester-induced expression of COX-2 and activation of NF-kappaB in mouse skin by blocking IkappaB kinase activity.

    PubMed

    Kundu, Joydeb Kumar; Shin, Young Kee; Kim, Sung Hoon; Surh, Young-Joon

    2006-07-01

    Aberrant expression of cyclooxygenase-2 (COX-2) has been implicated in tumor promotion. Resveratrol, a phytoalexin present in grapes, was reported to inhibit multistage mouse skin carcinogenesis. In the present study, we found that topically applied resveratrol significantly inhibited COX-2 expression induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Resveratrol-suppressed phosphorylation and subsequent degradation of IkappaBalpha, thereby inhibiting activation of nuclear factor-kappaB (NF-kappaB) in TPA-stimulated mouse skin. Pretreatment with resveratrol also suppressed TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Resveratrol blunted TPA-induced phosphorylation of p65 and its interaction with CBP/p300, rendering NF-kappaB transcriptionally inactive. To get further insights into the molecular basis of NF-kappaB inactivation by resveratrol, we examined the role of IkappaB kinase (IKK) in mediating TPA-induced activation of NF-kappaB and COX-2 expression. TPA treatment led to rapid induction of IKK activity in mouse skin, which was abolished either by resveratrol or an IKK inhibitor Bay 11-7082. Topical application of Bay 11-7082 also abrogated TPA-induced NF-kappaB activation and COX-2 expression, supporting the involvement of IKK in TPA-induced COX-2 expression. Taken together, the above findings suggest that resveratrol targets IKK in blocking TPA-induced NF-kappaB activation and COX-2 expression in mouse skin in vivo.

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

    SciTech Connect

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

    1984-11-01

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

  8. 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

  9. 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.

  10. 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.

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

    PubMed

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

    2014-12-01

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

  12. Expression and regulation of the lipoprotein lipase gene in human adrenal cortex.

    PubMed

    Staels, B; Martin, G; Martinez, M; Albert, C; Peinado-Onsurbe, J; Saladin, R; Hum, D W; Reina, M; Vilaro, S; Auwerx, J

    1996-07-19

    Lipoprotein lipase (LPL), an enzyme which hydrolyzes triglycerides and participates in the catabolism of remnant lipoproteins, plays a crucial role in energy and lipid metabolism. The goal of this study was to analyze the expression and regulation of the LPL gene in human adrenals. Reverse transcriptase-polymerase chain reaction amplification and sequence analysis demonstrated the presence of LPL mRNA in fetal and adult human adrenal cortex. Furthermore, the human adrenocortical carcinoma cell line, NCI-H295, expresses LPL mRNA and protein, which is localized to the outer cellular membrane as demonstrated by immunofluorescence confocal microscopy and can be released in the medium by heparin addition. To asses whether the LPL gene is regulated by agents regulating adrenal steroidogenesis, NCI-H295 cells were treated with activators of second messenger systems. Whereas the calcium-ionophore A23187 did not affect LPL gene expression, treatment with phorbol 12-myristate 13-acetate decreased LPL mRNA levels in a time- and dose-dependent manner. This decrease after phorbol 12-myristate 13-acetate was associated with diminished heparin-releasable LPL mass and activity in the culture medium. Addition of the cAMP analog 8-Br-cAMP to NCI-H295 cells resulted in a rapid, but transient dose-dependent induction of LPL mRNA. Treatment with the protein synthesis inhibitor cycloheximide gradually induced, whereas simultaneous addition of cAMP and cycloheximide superinduced LPL mRNA levels. Nuclear run-on analysis indicated that the effects of cAMP and cycloheximide occurred at the transcriptional and post-transcriptional level, respectively. Transient co-transfection assays demonstrated that the first 230 base pairs of the proximal LPL promoter contain a cAMP-responsive element activated by protein kinase A and transcription factors belonging to the CREB/CREM family. These data indicate that LPL is expressed in human adrenal cortex and regulated in NCI-H295 adrenocortical carcinoma

  13. The structural requirements for phorbol esters to enhance serotonin and acetylcholine release from rat brain cortex

    PubMed Central

    Iannazzo, L; Kotsonis, P; Majewski, H

    1999-01-01

    The effects of various phorbol-based protein kinase C (PKC) activators on the electrical stimulation-induced (S-I) release of serotonin and acetylcholine was studied in rat brain cortical slices pre-incubated with [3H]-serotonin or [3H]-choline to investigate possible structure-activity relationships. 4β-Phorbol 12,13-dibutyrate (4βPDB, 0.1–3.0 μM), enhanced S-I release of serotonin in a concentration-dependent manner whereas the structurally related inactive isomer 4α-phorbol 12, 13-dibutyrate (4αPDB) and phorbol 13-acetate (PA) were without effect. Another group of phorbol esters containing a common 13-ester substituent (phorbol 12,13-diacetate, PDA; phorbol 12-myristate 13-acetate, PMA; phorbol 12-methylaminobenzoate 13-acetate, PMBA) also enhanced S-I serotonin release with PMA being least potent. The deoxyphorbol monoesters, 12-deoxyphorbol 13-acetate (dPA), 12-deoxyphorbol 13-angelate (dPAng), 12-deoxyphorbol 13-phenylacetate (dPPhen) and 12-deoxyphorbol 13-isobutyrate (dPiB) enhanced S-I serotonin release but 12-deoxyphorbol 13-tetradecanoate (dPT) was without effect. The 20-acetate derivatives of dPPhen and dPAng were less effective in enhancing S-I serotonin release compared to the parent compounds. With acetylcholine release all phorbol esters tested had a far lesser effect when compared to their facilitatory action on serotonin release with only 4βPDB, PDA, dPA, dPAng and dPiB having significant effects. The effects of the phorbol esters on serotonin release were not correlated with their reported in vitro affinity and isozyme selectivity for PKC. A comparison across three transmitter systems (noradrenaline, dopamine, serotonin) suggests basic similarities in the structural requirements of phorbol esters to enhance transmitter release with short chain substituted mono- and diesters of phorbol being more potent facilitators of release than the long chain esters. Some compounds notably PDA, PMBA, dPPhen, dPPhenA had different potencies across

  14. 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.

  15. Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells.

    PubMed

    Chung, Myung-Hoon; Kim, Do-Hee; Na, Hye-Kyung; Kim, Jung-Hwan; Kim, Ha-Na; Haegeman, Guy; Surh, Young-Joon

    2014-10-01

    Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

  16. 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.

  17. Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9

    PubMed Central

    Hwang, Bo-Mi; Chae, Hee Suk; Jeong, Young-Ju; Lee, Young-Rae; Noh, Eun-Mi; Youn, Hyun Zo; Jung, Sung Hoo; Yu, Hong-Nu; Chung, Eun Yong; Kim, Jong-Suk

    2013-01-01

    The expression of matrix metalloproteinases (MMPs) produced by cancer cells has been associated with the high potential of metastasis in several human carcinomas, including breast cancer. Several pieces of evidence demonstrate that protein tyrosine phosphatases (PTP) have functions that promote cell migration and metastasis in breast cancer. We analyzed whether PTP inhibitor might control breast cancer invasion through MMP expression. Herein, we investigate the effect of 4-hydroxy-3,3-dimethyl-2H benzo[g]indole-2,5(3H)-dione (BVT948), a novel PTP inhibitor, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. The expression of MMP-9 and cell invasion increased after TPA treatment, whereas TPA-induced MMP-9 expression and cell invasion were decreased by BVT948 pretreatment. Also, BVT948 suppressed NF-κB activation in TPA-treated MCF-7 cells. However, BVT948 didn’t block TPA-induced AP-1 activation in MCF-7 cells. Our results suggest that the PTP inhibitor blocks breast cancer invasion via suppression of the expression of MMP-9. [BMB Reports 2013; 46(11): 533-538] PMID:24152909

  18. 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.

  19. 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.

  20. Inhibition of oncogene expression by green tea and (-)-epigallocatechin gallate in mice.

    PubMed

    Hu, G; Han, C; Chen, J

    1995-01-01

    The effects of tea drinking on the tobacco-specific nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung oncogene expression and the effect of topical application of the tea polyphenol component (-)-epigallocatechin-3-gallate (EGCG) on 12-O-tedradecanoylphorbol-13-acetate (TPA)-induced mouse skin oncogene expression were investigated. In the first experiment, mice were treated with NNK (1.3 mg/kg body wt ip) once a day for three days and were given 2% tea in drinking water during the whole experimental period. After four or eight weeks, the lung tissue of the mice treated with NNK displayed a significantly high level of expression in c-myc, c-raf, and c-H-ras oncogenes, and they were all inhibited by tea drinking with inhibitory rates of 50%, 20%, and 50%, respectively. In the second experiment, a single application of 10 nmol of TPA to mouse skin led to a marked increase in the transcripts' level of ornithine decarboxylase (ODC) gene, protein kinase C (PKC) gene, and c-myc oncogene at four hours after TPA administration. Topical application of EGCG (1 or 5 mumol) one hour before the application of TPA inhibited all TPA-induced gene expression in a dose-dependent fashion. These results confirm the anticarcinogenic effects of tea and suggest that a possible mechanism is the effect of tea on carcinogen-induced oncogene expression.

  1. Synthesis of novel polyphenols consisted of ferulic and gallic acids, and their inhibitory effects on phorbol ester-induced Epstein-Barr virus activation and superoxide generation.

    PubMed

    Nomura, Eisaku; Hosoda, Asao; Morishita, Hideko; Murakami, Akira; Koshimizu, Koichi; Ohigashi, Hajime; Taniguchi, Hisaji

    2002-04-01

    We prepared novel polyphenols which were esters composed of two naturally occurring products, ferulic and gallic acids, and investigated their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus (EBV) activation and superoxide (O2-) generation. Most of these compounds exhibited significant EBV activation suppression at a concentration of 20 microM and in particular, the ester 5f having 2-methyl-1-butyl group showed high activity. The suppressive effects on O2- generation were also observed in most of the esters.

  2. Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids.

    PubMed

    Belury, M A; Leyton, J; Patrick, K E; Cumberland, A G; Locniskar, M; Fischer, S M

    1991-09-01

    Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.

  3. 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

  4. Roles of protein kinase C on the mechanical activity of vascular smooth muscles.

    PubMed

    Itoh, T; Fujiwara, T; Kubota, Y; Nishiye, E; Kuriyama, H

    1990-08-01

    We investigated the role of protein kinase C in the mechanical responses evoked by high K or by acetylcholine (ACh) in intact vascular smooth muscle tissues, and by Ca in skinned vascular smooth muscle tissues. To activate protein kinase C, the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, or 1,2-diolein, plus phosphatidylserine (PS) was used. TPA enhanced or reduced the amplitude of the contraction evoked by increased concentrations of K below 39 mmol/L or over 90 mmol/L, respectively, but consistently enhanced the resting tension at any given concentration of high K. Similar effects of TPA were observed on the Ca-induced contraction in saponin skinned muscle tissues. The enhancing action of TPA on the K-induced contraction was not related to activation of either the voltage-dependent Ca channel or the sarcoplasmic reticulum, and did not occur in the case of Ca-independent contraction in skinned muscle tissues. During the enhancement of the contraction induced by TPA, the phosphorylation of myosin light chain and the shortening velocity of contraction as measured using the slack test, were enhanced with no remarkable change in the free Ca concentration in the cytosol. TPA consistently inhibited the ACH-induced contraction accompanied by a marked reduction in free Ca due to inhibition of the hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Under the assumption that TPA possesses the same action as DG, activation of protein kinase C increased the Ca sensitivity of contractile proteins in vascular smooth muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. 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.

  6. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  7. [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin.

    PubMed

    Kim, Sue Ok; Kundu, Joydeb Kumar; Shin, Young Kee; Park, Jin-Hong; Cho, Myung-Haing; Kim, Tae-Yoon; Surh, Young-Joon

    2005-04-01

    [6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. The present study was aimed at unraveling the molecular mechanisms underlying previously reported antitumor promoting effects of [6]-gingerol in mouse skin in vivo. One of the well-recognized molecular targets for chemoprevention is cyclooxygenase-2 (COX-2) that is abnormally upregulated in many premalignant and malignant tissues and cells. In our present study, topical application of [6]-gingerol inhibited COX-2 expression in mouse skin stimulated with a prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the transcription factor nuclear factor-kappaB (NF-kappaB) is known to regulate COX-2 induction, we attempted to determine the effect of [6]-gingerol on TPA-induced activation of NF-kappaB. Pretreatment with [6]-gingerol resulted in a decrease in both TPA-induced DNA binding and transcriptional activities of NF-kappaB through suppression of IkappaBalpha degradation and p65 nuclear translocation. Phosphorylation of both IkappaBalpha and p65 was substantially blocked by [6]-gingerol. In addition, [6]-gingerol inhibited TPA-stimulated interaction of phospho-p65-(Ser-536) with cAMP response element binding protein-binding protein, a transcriptional coactivator of NF-kappaB. Moreover, [6]-gingerol prevented TPA-induced phosphorylation and catalytic activity of p38 mitogen-activated protein (MAP) kinase that regulates COX-2 expression in mouse skin. The p38 MAP kinase inhibitor SB203580 attenuated NF-kappaB activation and subsequent COX-2 induction in TPA-treated mouse skin. Taken together, our data suggest that [6]-gingerol inhibits TPA-induced COX-2 expression in mouse skin in vivo by blocking the p38 MAP kinase-NF-kappaB signaling pathway. PMID:15735738

  8. PKCα activation down-regulates ATM and radio-sensitizes androgen-sensitive human prostate cancer cells in vitro and in vivo

    PubMed Central

    Truman, Jean-Philip; Rotenberg, Susan A.; Kang, Ji-Hye; Lerman, Gabriel; Fuks, Zvi; Kolesnick, Richard; Marquez, Victor E.; Haimovitz-Friedman, Adriana

    2009-01-01

    We previously demonstrated that treatment of human androgen-responsive prostate cancer cell lines LNCaP and CWR22-Rv1 with 12-O-tetradecanoylphorbol 13-acetate (TPA), a known protein kinase C (PKC) activator, decreases ATM protein levels, thus de-repressing the enzyme ceramide synthase (CS) and promoting apoptosis as well as radio-sensitizing these cells.1 Here we show that PKCα mediates the TPA effect on ATM expression, since ATM suppression and apoptosis induced by either TPA or diacylglycerol-lactone (DAG-lactone), both inducing PKCα activation,2 are abrogated in LNCaP cells following transfection of a kinase-dead PKCα mutant (KD-PKCα). Similarly, KD-PKCα blocks the apoptotic response elicited by combination of TPA and radiation, whereas expression of constitutively active PKCα is sufficient to sensitize cells to radiation alone, without a need to pre-treat the cells with TPA. These findings identify CS activation as a downstream event of PKCα activity in LNCaP cells. Similar results were obtained in CWR22-Rv1 cells with DAG-lactone treatment. Using the LNCaP orthotopic prostate model it is shown that treatment with TPA or DAG-lactone induces significant reduction in tumor ATM levels coupled with tumor growth delay. Furthermore, while fractionated radiation alone produces significant tumor growth delay, pretreatment with TPA or DAG-lactone significantly potentiates tumor cure. These findings support a model in which activation of PKCα downregulates ATM, thus relieving CS repression by ATM and enhancing apoptosis via ceramide generation. This model may provide a basis for the design of new therapies in prostate cancer. PMID:19029835

  9. 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.

  10. Varicella-zoster virus (VZV) mediates a delayed host shutoff independent of open reading frame (ORF) 17 expression.

    PubMed

    Waterboer, Tim; Rahaus, Markus; Wolff, Manfred H

    2002-01-01

    Varicella-zoster virus (VZV) open reading frame 17 (ORF 17) is the gene corresponding to Herpes simplex-virus (HSV) UL41. The UL41 gene encodes the virion host shutoff factor (vhs), a RNase that has been the object of detailed studies. In contrast to HSV, knowledge about VZV mediated shutoff effects and the role of ORF 17 is poor. We investigated the ORF 17 expression in infected cells and analyzed shutoff effects. ORF 17 expression could not be proven in infected human fibroblast cell lines and melanoma (MeWo) cells. Only after induction by Phorbol 12-myristate 13-acetate an ORF 17 expression became detectable in MeWo cells. Nevertheless, using stable expressed GAPDH mRNA as a marker for mRNA degradation, a VZV mediated shutoff, independent of ORF 17 expression, became measurable. Transfection experiments demonstrated that transient ORF 17 expression did not decrease the cellular GAPDH mRNA level. We examined whether the VZV shutoff factor is a tegument protein causing an early shutoff or whether it needs to be expressed (delayed shutoff). The GAPDH mRNA level in Actinomycin D pretreated and infected MeWo cells did not decrease even faster than the theoretical decay rate based on a half-life of 24 h. These findings lead to the conclusion that the VZV shutoff factor is not a mature protein localized in the virion and that VZV causes a delayed virion host shutoff effect.

  11. Block by gabapentin of the facilitation of glutamate release from rat trigeminal nucleus following activation of protein kinase C or adenylyl cyclase

    PubMed Central

    Maneuf, Yannick P; McKnight, Alexander T

    2001-01-01

    The effect of activation of protein kinase C (PKC) or adenylyl cyclase on release of glutamate has been investigated in a perfused slice preparation from the rat caudal trigeminal nucleus. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) produced a concentration-dependent increase in K+-evoked release of [2H]-glutamate (maximum increase 45%, EC50 11.8 nM), but in the presence of gabapentin (30 μM) the facilitation of release was blocked. The adenylyl cyclase activator forskolin (FSK) also induced a concentration-dependent increase in K+-evoked release of [3H]-glutamate (maximum increase 36%, EC50 2.4 μM), and again this facilitatory effect was blocked by gabapentin (30 μM). We suggest that these results may be of relevance to the antihyperalgesic properties of gabapentin, in conditions where concomitant release of substance P and CGRP produces activation of PKC and adenylyl cyclase respectively. PMID:11564640

  12. Clastogenic action of hydroperoxy-5,8,11,13-icosatetraenoic acids on the mouse embryo fibroblasts C3H/10T1/2.

    PubMed Central

    Ochi, T; Cerutti, P A

    1987-01-01

    Phorbol 12-myristate 13-acetate induces the release of a low molecular weight clastogenic factor from monocytes. Hydroperoxy-5,8,11,13-icosatetraenoic acids represent major components of clastogenic factor. We report that several isomeric hydroperoxy-5,8,11,13-icosatetraenoic acids efficiently induce DNA strand breakage and/or alkali-labile sites in the mouse embryo fibroblasts C3H/10T1/2. Fe chelation by desferrioxamine suppresses breakage by approximately equal to 42% indicating the participation of Fe-catalyzed radical reactions. An additional 37% inhibition is observed upon addition of the Ca2+ chelators EGTA and quin-2. This result suggests that hydroxyperoxy-5,8,11,13-icosatetraenoic acid may activate a Ca2+-dependent nuclease. The addition of the antioxidant enzymes CuZn-superoxide dismutase and catalase had no effect, while glutathione peroxidase suppressed strand breakage by 90%. To our knowledge, our results yield a first insight into the mechanism of action of monocyte clastogenic factor and the role of inflammation in tumor promotion. PMID:3469656

  13. Blockade of vascular angiogenesis by Aspergillus usamii var. shirousamii-transformed Angelicae Gigantis Radix and Zizyphus jujuba

    PubMed Central

    Kang, Sang-Wook; Choi, Jung-Suk; Bae, Ji-Young; Li, Jing; Kim, Dong Shoo; Kim, Jung-Lye; Shin, Seung-Yong; You, Hyun Ju; Park, Hyoung-Sook; Ji, Geun Eog

    2009-01-01

    The matrix metalloproteinases (MMP) play an important role in tumor invasion, angiogenesis and inflammatory tissue destruction. Increased expression of MMP was observed in benign tissue hyperplasia and in atherosclerotic lesions. Invasive cancer cells utilize MMP to degrade the extracellular matrix and vascular basement membrane during metastasis, where MMP-2 has been implicated in the development and dissemination of malignancies. The present study attempted to examine the antiangiogenic activity of the medicinal herbs of Aspergillus usamii var. shirousamii-transformed Angelicae Gigantis Radix and Zizyphus jujube (tAgR and tZj) with respect to MMP-2 production and endothelial motility in phorbol 12-myristate 13-acetate (PMA)- or VEGF-exposed human umbilical vein endothelial cells (HUVEC). Nontoxic tAgR and tZj substantially suppressed PMA-induced MMP-2 secretion. In addition, 25 µg/mL tAgR and tZj prevented vascular endothelial growth factor-stimulated endothelial cell transmigration and tube formation. The results reveal that tAgR and tZj dampened endothelial MMP-2 production leading to endothelial transmigration and tube formation. tAgR and tZj-mediated inhibition of endothelial MMP may boost a therapeutic efficacy during vascular angiogenesis. PMID:20016695

  14. Photon Counts Statistics in Leukocyte Cell Dynamics

    NASA Astrophysics Data System (ADS)

    van Wijk, Eduard; van der Greef, Jan; van Wijk, Roeland

    2011-12-01

    In the present experiment ultra-weak photon emission/ chemiluminescence from isolated neutrophils was recorded. It is associated with the production of reactive oxygen species (ROS) in the "respiratory burst" process which can be activated by PMA (Phorbol 12-Myristate 13-Acetate). Commonly, the reaction is demonstrated utilizing the enhancer luminol. However, with the use of highly sensitive photomultiplier equipment it is also recorded without enhancer. In that case, it can be hypothesized that photon count statistics may assist in understanding the underlying metabolic activity and cooperation of these cells. To study this hypothesis leukocytes were stimulated with PMA and increased photon signals were recorded in the quasi stable period utilizing Fano factor analysis at different window sizes. The Fano factor is defined by the variance over the mean of the number of photon within the observation time. The analysis demonstrated that the Fano factor of true signal and not of the surrogate signals obtained by random shuffling increases when the window size increased. It is concluded that photon count statistics, in particular Fano factor analysis, provides information regarding leukocyte interactions. It opens the perspective to utilize this analytical procedure in (in vivo) inflammation research. However, this needs further validation.

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

    PubMed

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

    2014-09-01

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

  16. Scavenger receptor of human monocytic leukemia cell line (THP-1) and murine macrophages for nonenzymatically glycosylated proteins.

    PubMed

    Takata, K; Horiuchi, S; Araki, N; Shiga, M; Saitoh, M; Morino, Y

    1989-11-17

    Long-term incubation of proteins with glucose undergo a series of nonenzymatic reactions to form advanced glycosylation end product (AGE) with fluorescence and brown color. The receptor for AGE-proteins was demonstrated in murine macrophages (Vlassara et al. (1985) Proc. Natl. Acad. Sci. USA 82. 5588). Our recent study with rat macrophages revealed that the receptor also recognized proteins modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, indicating its close identity to a scavenger receptor for aldehyde-modified proteins (Takata, K. et al. (1988) J. Biol. Chem. 263. 14819). This notion was tested in the present study with human monocytic leukemia cell line (THP-1 cells), human monocyte macrophages and murine peritoneal macrophages. Endocytic uptake of AGE-proteins and aldehyde-modified proteins was inhibited in a cross-competitive fashion. The receptor activities of THP-1 cells for AGE-albumin and aldehyde-modified proteins were induced synchronously by phorbol 12-myristate 13-acetate. Furthermore, upon reduction by NaBH4 of the Schiff base formed between proteins and glucose or aldehydes, no ligand activity was generated. However, once the ligand activity was generated, NaBH4 was no longer effective for the ligand activity. Thus, a structure in common between AGE-proteins and aldehyde-modified proteins may be crucial for recognition by the human macrophage receptor.

  17. The effect of electromagnetic field on reactive oxygen species production in human neutrophils in vitro.

    PubMed

    Poniedzialek, Barbara; Rzymski, Piotr; Nawrocka-Bogusz, Honorata; Jaroszyk, Feliks; Wiktorowicz, Krzysztof

    2013-09-01

    The present study was undertaken in order to determine the effect of low frequency electromagnetic field (EMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. We investigated how differently generated EMF and several levels of magnetic induction affect ROS production. To evaluate the level of ROS production, two fluorescent dyes were used: 2'7'-dichlorofluorscein-diacetate and dihydrorhodamine. Phorbol 12-myristate 13-acetate (PMA), known as strong stimulator of the respiratory burst, was also used. Alternating magnetic field was generated by means of Viofor JPS apparatus. Three different levels of magnetic induction have been analyzed (10, 40 and 60 μT). Fluorescence of dichlorofluorescein and 123 rhodamine was measured by flow cytometry. The experiments demonstrated that only EMF tuned to the calcium ion cyclotron resonance frequency was able to affect ROS production in neutrophils. Statistical analysis showed that this effect depended on magnetic induction value of applied EMF. Incubation in EMF inhibited cell activity slightly in unstimulated neutrophils, whereas the activity of PMA-stimulated neutrophils has increased after incubation in EMF.

  18. 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

  19. 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

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

    SciTech Connect

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

    1987-02-01

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

  1. Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones.

    PubMed

    Baker, Mark D

    2005-09-15

    The tetrodotoxin-resistant (TTX-r) persistent Na(+) current, attributed to Na(V)1.9, was recorded in small (< 25 mum apparent diameter) dorsal root ganglion (DRG) neurones cultured from P21 rats and from adult wild-type and Na(V)1.8 null mice. In conventional whole-cell recordings intracellular GTP-gamma-S caused current up-regulation, an effect inhibited by the PKC pseudosubstrate inhibitor, PKC19-36. The current amplitude was also up-regulated by 25 microM intracellular 1-oleoyl-2-acetyl-sn-glycerol (OAG) consistent with PKC involvement. In perforated-patch recordings, phorbol 12-myristate 13-acetate (PMA) up-regulated the current, whereas membrane-permeant activators of protein kinase A (PKA) were without effect. PGE(2) did not acutely up-regulate the current. Conversely, both PGE(2) and PKA activation up-regulated the major TTX-r Na(+) current, Na(V)1.8. Extracellular ATP up-regulated the persistent current with an average apparent K(d) near 13 microM, possibly consistent with P2Y receptor activation. Numerical simulation of the up-regulation qualitatively reproduced changes in sensory neurone firing properties. The activation of PKC appears to be a necessary step in the GTP-dependent up-regulation of persistent Na(+) current. PMID:16002450

  2. Cytokine expression in CD4(+) cells exposed to the monocyte locomotion inhibitory factor produced by Entamoeba histolytica.

    PubMed

    Rojas-Dotor, Sara; Rico, Guadalupe; Pérez, Julia; Velázquez, Juan; Silva, Raúl; Morales, Esther; Kretschmer, Roberto

    2006-04-01

    Entamoeba histolytica produces monocyte locomotion inhibitory factor (MLIF), a pentapeptide with in vitro and in vivo anti-inflammatory properties. MLIF may interfere with leukocyte migration, disturbing the balance of pro- and anti-inflammatory cytokines secreted by CD4(+) T lymphocytes. We evaluated the effect of MLIF on expression of pro- and anti-inflammatory cytokines in human CD4(+) T lymphocytes. Regulatory cytokines [interleukin-1 beta (IL-1beta), IL-2, interferon gamma (IFN-gamma), IL-5, IL-6, and IL-10] were studied by enzyme-linked immunosorbent assay method in CD4(+)-cell supernatant fluids. Proinflammatory cytokines were produced per se by MLIF (IL-1beta, IL-2, and IFN-gamma) and also anti-inflammatory cytokines (IL-5, IL-6, and IL-10) with 1-phorbol-12 myristate-13 acetate + MLIF; the IL-1beta, IFN-gamma, IL-5 and IL-6 production was inhibited but not that of IL-10 which disclosed increase in its expression. MLIF disturbs the pro- and anti-inflammatory balance, and it induces inhibition of IL-1beta (principal proinflammatory cytokine) and increases IL-10 (prototype of an anti-inflammatory cytokine).

  3. Cellular uptake and activity of heparin functionalised cerium oxide nanoparticles in monocytes.

    PubMed

    Ting, S R Simon; Whitelock, John M; Tomic, Romana; Gunawan, Cindy; Teoh, Wey Yang; Amal, Rose; Lord, Megan S

    2013-06-01

    Cerium oxide nanoparticles (nanoceria) are effective in scavenging intracellular reactive oxygen species (ROS). In this study nanoceria synthesized by flame spray pyrolysis (dXRD = 12 nm) were functionalised with heparin via an organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria were functionalised with approximately 130 heparin molecules per nanoparticle as determined by thermo gravimetric analysis. Heparin functionalised nanoceria were more effectively internalised by the human monocyte cell line, U937, and U937 cells that had been activated with phorbol 12 myristate 13-acetate (PMA) than bare nanoceria. The heparin functionalised nanoceria were also more effective in scavenging ROS than nanoceria in both activated and unactivated U937 cells. Heparin coupled nanoceria were found to be biologically active due to their ability to bind fibroblast growth factor 2 and signal through FGF receptor 1. Additionally, the heparin-coupled nanoceria, once internalised by the cells, were found to be degraded by 48 h. Together these data demonstrated that heparin enhanced the biological properties of nanoceria in terms of cellular uptake and ROS scavenging, while the nanoceria themselves were more effective at delivering heparin intracellularly than exposing cells to heparin in solution. PMID:23478040

  4. Cellular uptake and reactive oxygen species modulation of cerium oxide nanoparticles in human monocyte cell line U937.

    PubMed

    Lord, Megan S; Jung, MoonSun; Teoh, Wey Yang; Gunawan, Cindy; Vassie, James A; Amal, Rose; Whitelock, John M

    2012-11-01

    Cerium oxide nanoparticles (nanoceria) are promising materials for intracellular oxygen free radical scavenging providing a potential therapy for reactive oxygen species (ROS)-mediated inflammatory processes. In this study rhombohedral-shaped nanoceria were synthesized by flame spray pyrolysis with tuneable particle diameters between 3 and 94 nm by changing the liquid precursor flow rate. Monocytes and macrophages are major players in inflammatory processes as their production of ROS species has important downstream effects on cell signalling. Therefore, this study examined the ability of the nanoceria to be internalised by the human monocytic cell line, U937, and scavenge intracellular ROS. U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) were found to be more responsive to the nanoceria than U937 cells, which may not be surprising given the role of monocyte/macrophages in phagocytosing foreign material. The smaller particles were found to contain more crystal lattice defects with which to scavenge ROS, however a greater proportion of both the U937 and activated U937 cell populations responded to the larger particles. Hence all nanoceria particle sizes examined in this study were equally effective in scavenging intracellular ROS. PMID:22841920

  5. Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract.

    PubMed

    Roomi, M W; Ivanov, V; Kalinovsky, T; Niedzwiecki, A; Rath, M

    2006-01-01

    Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)-2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 microg/mL and MMP-9 at 500 microg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 microg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 microg/mL NM (P < 0.0001) and 1000 microg/mL NM (P < 0.0001), respectively. Our results suggest that the mixture of lysine, proline, arginine, ascorbic acid, and green tea extract has potential in the treatment of cervical cancer by inhibiting critical steps in cancer development and spread.

  6. In vitro effect of peas, Pisum pisum, and chickpeas, Cicer arietinum, on the immune system of gilthead seabream, Sparus aurata.

    PubMed

    Henry, M A; Nikolopoulou, D; Alexis, M N

    2012-08-01

    The future for a sustainable aquaculture relies on the formulation of feed including alternatives to fish meal and fish oil that do not impair fish growth and that improve fish health status. Grain legumes such as field peas and chickpeas offer good sources of proteins, carbohydrates, fibers, vitamins, and minerals. The effect of peas and chickpeas on the immune system of seabream was assessed in vitro in order to detect any potential immunosuppressing problem. Peas was determined to be a better fishmeal alternative than chickpeas as they induced higher respiratory burst measured by the nitro blue tetrazolium assay and primed the Phorbol 12-myristate 13-acetate (PMA)-stimulated intracellular respiratory burst whereas chickpeas neither directly stimulated respiratory burst nor primed it. However, when the intra- and extracellular respiratory burst activities were taken into account, high concentrations of peas inhibited the zymosan- and PMA-triggered chemiluminescence. This apparent reduction of the production of reactive oxygen species may reflect in fact the antioxidant activity of legumes. This, together with the absence of effect on the phagocytosis activity, suggested that peas are not immunosuppressing gilthead seabream. Further in vivo studies preferably comporting a bacterial challenge will have to ascertain the absence of immunosuppressing effect of these legumes.

  7. 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

  8. Variable DNA methylation changes during differentiation of human melanoma cells.

    PubMed

    Steigerwald, S D; Pfeifer, G P

    1988-09-01

    The DNA 5-methylcytosine content has been analyzed in the human melanoma cell line M21 at several time points after induction of differentiation by a variety of inducers. 5-Aza-2'-deoxycytidine reduces DNA methylation to about 50% of the control level and this demethylation occurs prior to the establishment of the differentiated phenotype. The DNA synthesis inhibitors cytosine arabinoside, aphidicolin, and hydroxyurea exert different effects on DNA methylation in these cells. Cytosine arabinoside induces an early DNA hypermethylation, which is however reversible and drops to the original level after 24 h. Hydroxyurea induces DNA hypermethylation after a lag period of more than 48 h and the DNA polymerase alpha inhibitor aphidicolin has no effect on the DNA methylation level. Treatment of cells with phorbol 12-myristate 13-acetate, another potent inducer of melanoma cell differentiation, does not result in a change of total DNA methylation over a period of 96 h. These results indicate that differentiation of human melanoma cells can be accompanied by variable changes of the DNA methylation pattern. These changes can be neither generally related to the differentiation process itself nor related to the effects of DNA synthesis inhibition on DNA methylation, but may more likely reflect a direct or indirect particular effect of the inducer on the DNA methylation process.

  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. Isolation of All CD44 Transcripts in Human Epidermis and Regulation of Their Expression by Various Agents.

    PubMed

    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 transcripts from normal human epidermis, and studied how their expressions are regulated. By RT-PCR, we found that a number of different CD44 transcripts were expressed in human epidermis, and we obtained all these transcripts from DNA bands in agarose and acrylamide gels by cloning. Detailed sequence analysis revealed 18 CD44 transcripts, 3 of which were novel. Next, we examined effects of 10 different agents on the expression of CD44 transcripts in cultured human keratinocytes, and found that several agents, particularly epidermal growth factor, hydrogen peroxide, phorbol 12-myristate 13-acetate, retinoic acid, calcium and fetal calf serum differently regulated their expressions in various patterns. Furthermore, normal and malignant keratinocytes were found to produce different CD44 transcripts upon serum stimulation and subsequent starvation, suggesting that specific CD44 isoforms are involved in tumorigenesis via different CD44-mediated biological pathways. PMID:27505250

  11. Withaferin A is an inhibitor of endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Ku, Sae-Kwang; Han, Min-Su; Bae, Jong-Sup

    2014-06-01

    Withaferin A (WFA), an active compound from Withania somnifera, has been widely researched for its anti-inflammatory and cardioactive properties and effects on the central nervous system. The endothelial cell protein C receptor (EPCR) plays important roles in blood coagulation and inflammation. EPCR activity is markedly changed by ectodomain cleavage and release as the soluble EPCR. EPCR is shed from the cell surface, mediated by tumor necrosis factor-α converting enzyme (TACE). In this study, we investigated the effects of WFA on the EPCR shedding in human umbilical vein endothelial cells (HUVECs) and in mice and the associated signaling pathways. WFA was found to induce inhibition of phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-induced EPCR shedding and WFA suppressed the expression and activity of TACE. In addition, treatment with WFA resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate a therapeutic potentiality of WFA as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

  12. Rosmarinic acid down-regulates endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Ku, Sae-Kwang; Yang, Eun-Ju; Song, Kyung-Sik; Bae, Jong-Sup

    2013-09-01

    The endothelial protein C receptor (EPCR) plays pivotal roles in coagulation and inflammation, however, its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). According to previous studies, there are approximately 100ng/ml sEPCR in human plasma and the levels increase in inflammatory diseases. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). We recently reported on the anti-inflammatory and barrier protective activities of rosmarinic acid (RA), an important component of the leaves of Perilla frutescens. However, little is known about the effects of RA on EPCR shedding. Here, we investigated this issue by monitoring the effects of RA on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Data showed that treatment with RA resulted in potent inhibition of PMA, TNF-α, IL-induced EPCR shedding by suppression of TACE expression. In addition, RA reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results suggest the potential for use of RA as an anti-sEPCR shedding reagent against PMA, TNF-α, IL-1β and CLP-mediated EPCR shedding.

  13. Inhibitory effects of oroxylin A on endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Ku, Sae-Kwang; Han, Min-Su; Lee, Min Young; Lee, You-Mie; Bae, Jong-Sup

    2014-06-01

    Endothelial cell protein C receptor (EPCR) plays important roles in blood coagulation and inflammation. EPCR activity is markedly changed by ectodomain cleavage and release as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). Oroxylin A (OroA), a major component of Scutellaria baicalensis Georgi, is known to exhibit anti-angiogenic, antiinflammation, and anti-invasive activities. However, little is known about the effects of OroA on EPCR shedding. Data showed that OroA induced potent inhibition of phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and on cecal ligation and puncture (CLP)-induced EPCR shedding through suppression of TACE expression and activity. In addition, treatment with OroA resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of OroA as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

  14. 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.

  15. Anti-inflammatory effects of hyperoside in human endothelial cells and in mice.

    PubMed

    Ku, Sae-Kwang; Zhou, Wei; Lee, Wonhwa; Han, Min-Su; Na, MinKyun; Bae, Jong-Sup

    2015-04-01

    High-mobility group box 1 (HMGB1) was recently shown to be an important extracellular mediator of systemic inflammation, and endothelial cell protein C receptor (EPCR) has been shown to be involved in vascular inflammation. Hyperoside is an active compound isolated from Rhododendron brachycarpum G. Don (Ericaceae) that was reported to have anti-oxidant, anti-hyperglycemic, anti-cancer, and anti-coagulant activities. Here, we show, for the first time, the anti-septic effects of hyperoside in HMGB1-mediated inflammatory responses and on the shedding of EPCR in vitro and in vivo. The data showed that hyperoside posttreatment suppressed lipopolysaccharide (LPS)-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangement. Hyperoside also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice and phorbol-12-myristate 13-acetate (PMA) of cecal ligation and puncture (CLP)-induced EPCR shedding. In addition, hyperoside inhibited the production of tumor necrosis factor-α (TNF-α) and the HMGB1-mediated activation of Akt, nuclear factor-κB (NF-κB), and extracellular regulated kinase (ERK) 1/2 in HUVECs. Hyperoside also reduced the CLP-induced release of HMGB1, the production of interleukin (IL)-1β, and septic mortality. Collectively, these results suggest hyperoside as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

  16. Anti-inflammatory effects of Baicalin, Baicalein, and Wogonin in vitro and in vivo.

    PubMed

    Lee, Wonhwa; Ku, Sae-Kwang; Bae, Jong-Sup

    2015-02-01

    Here, three structurally related polyphenols found in the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for its effects on inflammatory responses by monitoring the effects of baicalin, baicalein, and wogonin on lipopolysaccharide (LPS)-mediated vascular inflammatory responses. We found that each compound inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of monocytes to human endothelial cells. Each compound induced potent inhibition of phorbol-12-myristate 13-acetate and LPS-induced endothelial cell protein C receptor shedding. It also suppressed LPS-induced hyperpermeability and leukocytes migration in vivo. Furthermore, each compound suppressed the production of tumor necrosis factor-α or interleukin-6 and the activation of nuclear factor-κB or extracellular regulated kinases 1/2 by LPS. Moreover, treatment with each compound resulted in reduced LPS-induced lethal endotoxemia. These results suggest that baicalin, baicalein, and wogonin posses anti-inflammatory functions by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.

  17. 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

  18. 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

  19. cAMP post-transcriptionally diminishes the abundance of adrenodoxin reductase mRNA.

    PubMed Central

    Brentano, S T; Black, S M; Lin, D; Miller, W L

    1992-01-01

    Adrenodoxin reductase (AR; ferridoxin: NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein that mediates electron transport from NADPH to all known mitochondrial forms of cytochrome P450. AR mRNA was found in all human adult and fetal tissues examined; however, it was vastly more abundant in tissues that synthesize steroid hormones. The ratio of the 18- form of mRNA lacking 18 alternately spliced bases to the 18+ form was approximately 100:1 and remained constant irrespective of the tissue or hormonal manipulation, indicating that the alternate splicing is a passive nonregulated event. AR protein was unchanged by forskolin treatment of human JEG-3 cytotrophoblast cells for 24 h, but the mRNA diminished. Phorbol 12-myristate 13-acetate and cycloheximide had no effect, even though these agents had the expected effects on P450scc and adrenodoxin mRNAs. cAMP decreased the abundance of AR mRNA expressed from both transfected plasmids and the endogenous gene, indicating the effect was post-transcriptional. AR gene transcription in JEG-3 cells and promoter-chloramphenicol acetyltransferase constructs transfected into JEG-3 cells were unresponsive to forskolin. Powerful basal transcription elements were identified between -46 and -214 bases from the principal transcriptional initiation site, a region containing six elements closely resembling the binding site for transcription factor SP1. Images PMID:1315050

  20. 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

  1. 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.

  2. Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo.

    PubMed

    Kontos, C D; Wei, E P; Williams, J I; Kontos, H A; Povlishock, J T

    1992-10-01

    We used a cytochemical technique for the detection of superoxide in cerebral inflammation and ischemia-reperfusion in anesthetized cats. The technique is based on the oxidation of Mn2+ to Mn3+ by superoxide; Mn3+, in turn, oxidizes diaminobenzidine. The oxidized diaminobenzidine forms an osmiophilic electron-dense product that is detected by electron microscopy. The reagents, manganese chloride (2 mM) and diaminobenzidine (2 mg/ml), were placed topically on the brain surface of anesthetized cats equipped with cranial windows. Inflammation was induced by topical carrageenan with or without phorbol 12-myristate 13-acetate to activate leukocytes. In inflammation, superoxide was detected in the plasma membrane and in the phagocytic vacuoles of leukocytes. In ischemia-reperfusion, superoxide was identified in the meninges in association with blood vessels. It was located primarily in the extracellular space and occasionally in endothelial and vascular smooth muscle cells. In both inflammation and ischemia, the reaction product was eliminated by superoxide dismutase or by the omission of either manganese or diaminobenzidine. It was unaffected by sodium azide, which inhibits peroxidases. No superoxide was detected in the brain parenchyma. The findings confirm the generation of superoxide is cerebral ischemia-reperfusion and show that it is produced in cerebral vessels.

  3. Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells

    SciTech Connect

    Wilhelm, S.M.; Collier, I.E.; Kronberger, A.; Eisen, A.Z.; Marmer, B.L.; Grant, G.A.; Bauer, E.A.; Goldberg, G.I.

    1987-10-01

    The authors have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO/sub 4//PAGE of 60 and 57 kDa. Human stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively. Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. The data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.

  4. New lanostanes and naphthoquinones isolated from Antrodia salmonea and their antioxidative burst activity in human leukocytes.

    PubMed

    Shen, Chien-Chang; Shen, Yuh-Chiang; Wang, Yea-Hwey; Lin, Lie-Chwen; Don, Ming-Jaw; Liou, Kuo-Tong; Wang, Wen-Yen; Hou, Yu-Chang; Chang, Tun-Tschu

    2006-02-01

    Four new compounds were isolated from the basidiomata of the fungus Antrodia salmonea, a newly identified species of Antrodia (Aphyllophorales) in Taiwan. These new compounds are named as lanosta-8,24-diene-3beta,15alpha,21-triol (1), 24-methylenelanost-8-ene-3beta,15alpha,21-triol (2), 2,3-dimethoxy-5-(2',5'-dimethoxy-3',4'-methylenedioxyphenyl)-7-methyl-[1,4]-naphthoquinone (3), and 2,3-dimethoxy-6-(2',5'-dimethoxy-3',4'-methylenedioxyphenyl)-7-methyl-[1,4]-naphthoquinone (4), respectively. Their structures were elucidated by spectroscopic methods. An in vitro cellular functional assay was performed to evaluate their anti-oxidative burst activity in human leukocytes. They showed inhibitory effects against phorbol 12-myristate-13-acetate (PMA), a direct protein kinase C activator, induced oxidative burst in neutrophils (PMN) and mononuclear cells (MNC) with 50 % inhibitory concentration (IC(50)) ranging from 3.5 to 25.8 microM. The potency order of these compounds in PMA-activated leukocytes was as 1 > 3 > 4 > 2. They were relatively less effective in formyl-Met-Leu-Phe (fMLP), a G-protein coupled receptor agonist, induced oxidative burst, except for compounds 3 and 4 in fMLP-activated PMN. These results indicated that three (1, 3, and 4) of these four newly identified compounds displayed anti-oxidative effect in human leukocytes with different potency and might confer anti-inflammatory activity to these drugs.

  5. 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.

  6. Analysis of the mechanisms involved in the stimulation of neutrophil apoptosis by tumour necrosis factor-α

    PubMed Central

    Salamone, Gabriela; Trevani, Analía; Martínez, Diego; Vermeulen, Mónica; Gamberale, Romina; Fernández-Calotti, Paula; Raiden, Silvina; Giordano, Mirta; Geffner, Jorge

    2004-01-01

    We have previously reported that human neutrophils pretreated with tumour necrosis factor-α (TNF-α) and then exposed to a variety of agents such as immune complexes, zymosan, phorbol 12-myristate 13-acetate (PMA), C5a, fMLP, or granulocyte–macrophage colony-stimulating factor (GM-CSF), undergo a dramatic stimulation of apoptosis, suggesting that TNF-α is able to prime an apoptotic death programme which can be rapidly triggered by different stimuli. We report here that this response involves the participation of Mac-1 (CD11b/CD18), is dependent on caspases 3, 8 and 9, and is associated with both a loss of mitochondrial transmembrane potential and a down-regulation in expression of the anti-apoptotic protein, Mcl-1. Interestingly, we also found that the anti-apoptotic cytokine interleukin-1 (IL-1) improves the ability of TNF-α to promote apoptosis, supporting the notion than TNF-α, acting together with IL-1, may favour the depletion of neutrophils from the inflammatory areas during the course of acute inflammation. PMID:15500622

  7. 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

  8. 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.

  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. 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.

  11. 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.

  12. Characterization of antigen receptor response elements within the interleukin-2 enhancer.

    PubMed Central

    Durand, D B; Shaw, J P; Bush, M R; Replogle, R E; Belagaje, R; Crabtree, G R

    1988-01-01

    T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for IL-2 activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene. Images PMID:3260003

  13. 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.

  14. 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

  15. 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

  16. 7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils.

    PubMed

    Kabeya, Luciana M; Fuzissaki, Carolina N; Taleb-Contini, Silvia H; da C Ferreira, Ana Maria; Naal, Zeki; Santos, Everton O L; Figueiredo-Rinhel, Andréa S G; Azzolini, Ana Elisa C S; Vermelho, Roberta B; Malvezzi, Alberto; Amaral, Antonia T-do; Lopes, João Luis C; Lucisano-Valim, Yara Maria

    2013-10-25

    In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects. PMID:23994743

  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.

  18. 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.

  19. [Perioperative alterations in polymorphonuclear leukocyte function mediated by protein kinase C].

    PubMed

    Yokota, K; Nishihira, T; Shineha, R; Ueda, H; Mori, S

    1994-03-01

    To characterize the perioperative alterations in polymorphonuclear leukocytes (PMN) function mediated by protein kinase C, we studied twenty six patients undergoing gastrointestinal surgery. Seventeen patients with thoracic esophageal cancer were underwent total thoracic esophagectomy through a right thoracotomy (severe surgical stress group). Nine patients underwent cholecystectomy (slight surgical stress group). Measurement of O2- production capacity was used as a reflection of the activity of NADPH oxidase, and the activity of myeloperoxidase-H2O2-halide system was evaluated using luminol-dependent chemiluminescence. O2- production stimulated by phorbol 12-myristate 13-acetate (PMA) was suppressed, reaching a minimum on POD 3. On the other hand, luminol dependent chemiluminescence increased significantly after surgery, reached a maximum on POD 3. These alterations were more remarkable in the severely stressed patients. These results suggest that postoperative PMN signal transduction mechanisms, mediated by protein kinase C, may activate myeloperoxidase-H2-O2-halide system but suppress NADPH oxidase system dependently of the degree of surgical stress, revealing a differential effect of protein kinase C activation on PMN microbicidal activity.

  20. 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.

  1. 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.

  2. All-Trans Retinoic Acid Inhibits Human Colorectal Cancer Cells RKO Migration via Downregulating Myosin Light Chain Kinase Expression through MAPK Signaling Pathway.

    PubMed

    Zuo, Li; Yang, Xiaoping; Lu, Man; Hu, Ruolei; Zhu, Huaqing; Zhang, Sumei; Zhou, Qing; Chen, Feihu; Gui, Shuyu; Wang, Yuan

    2016-10-01

    All-trans-retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells. However, the underlying mechanism is unclear. Here, we demonstrate that ATRA inhibited colorectal cancer cells RKO (human colon adenocarcinoma cell) migration by downregulating cell movement and increasing cell adhesion. ATRA inhibited the expression and activation of myosin light chain kinase (MLCK) in RKO cells, while the expression level of MLC phosphatase (MLCP) had no change in RKO cells treated with or without ATRA. The expression and activity of MLC was also inhibited in RKO cells exposed to ATRA. Intriguingly, ATRA increased the expression of occludin messenger RNA (mRNA) and protein and its localization on cell membrane. However, ATRA did not change the expression of zonula occludens 1 (ZO-1), but increased the accumulation of ZO-1 on RKO cells membrane. ML-7, an inhibitor of MLCK, significantly inhibited RKO cell migration. Furthermore, knockdown of endogenous MLCK expression inhibited RKO migration. Mechanistically, we showed that MAPK-specific inhibitor PD98059 enhanced the inhibitory effect of ATRA on RKO migration. In contrast, phorbol 12-myristate 13-acetate (PMA) attenuated the effects of ATRA in RKO cells. Moreover, knocking down endogenous extracellular signal-regulated kinase (ERK) expression inhibited MLCK expression in the RKO cells. In conclusion, ATRA inhibits RKO migration by reducing MLCK expression via extracellular signal-regulated kinase 1/Mitogen-activated protein kinase (ERK1/MAPK) signaling pathway. PMID:27564600

  3. Stimulation of Ebola virus production from persistent infection through activation of the Ras/MAPK pathway.

    PubMed

    Strong, James E; Wong, Gary; Jones, Shane E; Grolla, Allen; Theriault, Steven; Kobinger, Gary P; Feldmann, Heinz

    2008-11-18

    Human infections with Ebola virus (EBOV) result in a deadly viral disease known as Ebola hemorrhagic fever. Up to 90% of infected patients die, and there is no available treatment or vaccine. The sporadic human outbreaks are believed to result when EBOV "jumps" from an infected animal to a person and is subsequently transmitted between persons by direct contact with infected blood or body fluids. This study was undertaken to investigate the mechanism by which EBOV can persistently infect and then escape from model cell and animal reservoir systems. We report a model system in which infection of mouse and bat cell lines with EBOV leads to persistence, which can be broken with low levels of lipopolysaccharide or phorbol-12-myristate-13-acetate (PMA). This reactivation depends on the Ras/MAPK pathway through inhibition of RNA-dependent protein kinase and eukaryotic initiation factor 2alpha phosphorylation and occurs at the level of protein synthesis. EBOV also can be evoked from mice 7 days after infection by PMA treatment, indicating that a similar mechanism occurs in vivo. Our findings suggest that EBOV may persist in nature through subclinical infection of a reservoir species, such as bats, and that appropriate physiological stimulation may result in increased replication and transmission to new hosts. Identification of a presumptive mechanism responsible for EBOV emergence from its reservoir underscores the "hit-and-run" nature of the initiation of human and/or nonhuman primate EBOV outbreaks and may provide insight into possible countermeasures to interfere with transmission. PMID:18981410

  4. Amphetamine and methamphetamine reduce striatal dopamine transporter function without concurrent dopamine transporter relocalization.

    PubMed

    German, Christopher L; Hanson, Glen R; Fleckenstein, Annette E

    2012-10-01

    Amphetamine (AMPH) and methamphetamine (METH) alter dopamine transporter (DAT) function. In vitro heterologous cell line and synaptosome studies demonstrate AMPH-induced DAT internalization, implicating relocalization in reduced DAT uptake following drug exposure. However, few studies have evaluated DAT localization following in vivo AMPH/METH administration. To determine DAT subcellular localization following drug administration, a centrifugation technique was developed to isolate striatal synaptosomal membrane and vesicle fractions. DAT was distributed between the synaptosomal membrane (60%) and endosomal vesicles (40%), and in vitro application of the protein kinase C activator phorbol 12-myristate 13-acetate to striatal synaptosomes caused DAT internalization into the vesicle fractions. In contrast, neither single nor repeated in vivo AMPH and/or METH administrations altered DAT localization 5, 15, 30, or 60 min post-treatment, despite reduced DAT uptake. Importantly, repeated METH injections uniformly decreased total DAT immunoreactivity within all fractions 7 days post-treatment. These findings suggest that factors other than internalization can contribute to the observed acute and persistent DAT dysfunction and dopaminergic deficits following in vivo AMPH or METH administration.

  5. 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.

  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.

  7. Suppressing effect of resveratrol on the migration and invasion of human metastatic lung and cervical cancer cells.

    PubMed

    Kim, Yoon Suk; Sull, Jae Woong; Sung, Ho Joong

    2012-09-01

    The antioxidant 3,4',5 tri-hydroxystilbene (resveratrol), a phytoalexin found in grapes, shows cancer preventive activities, including inhibition of migration and invasion of metastatic tumors. However, the molecular mechanism underlying the effect of resveratrol on tumor metastasis, especially in human metastatic lung and cervical cancers is not clear. A non-cytotoxic dosage of resveratrol causes a reduction in the generation of reactive oxygen species, and suppresses phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration in both A549 and HeLa cells. Resveratrol also decreases both the expression and the enzymatic activity of matrix metalloproteinase-9 (MMP-9), and the promoter activity of PMA-stimulated MMP-9 is also inhibited. However, resveratrol does not affect either the expression or the proteolytic activity of MMP-2. Our results also show that resveratrol suppresses the transcription of MMP-9 by the inhibition of both NF-κB and AP-1 transactivation. These results indicate that resveratrol inhibits both NF-κB and AP-1 mediated MMP-9 expression, leading to suppression of migration and invasion of human metastatic lung and cervical cancer cells. Resveratrol has potential for clinical use in preventing invasion by human metastatic lung and cervical cancers.

  8. Inhibitory effect of Trolox on the migration and invasion of human lung and cervical cancer cells.

    PubMed

    Sung, Ho Joong; Kim, Yoonseo; Kang, Hyereen; Sull, Jae Woong; Kim, Yoon Suk; Jang, Sung-Wuk; Ko, Jesang

    2012-02-01

    The antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) is implicated in migration and invasion of metastatic tumors. However, the molecular mechanism underlying the effect of Trolox on metastatic cancer cells is not known. We found that a non-cytotoxic dose of Trolox decreased phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration of both A549 and HeLa cancer cells. We also found that Trolox suppressed both the expression and the proteolytic activity of matrix metalloproteinase-9 (MMP-9), and that the promoter activity of PMA-induced MMP-9 was inhibited by Trolox. Our results show that Trolox inhibits the transcriptional activity of MMP-9 by suppression of NF-κB transactivation. These results indicate that Trolox inhibits NF-κB-mediated MMP-9 expression, leading to the suppression of migration and invasion in lung and cervical cancer cells. Trolox is a potential agent for clinical use in preventing the invasion and metastasis of human malignant lung and cervical cancers.

  9. 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

  10. Protein Kinase C Activity and Light Sensitivity of Single Amphibian Rods

    PubMed Central

    Xiong, W.-H.; Nakatani, K.; Ye, B.; Yau, K.-W.

    1997-01-01

    Biochemical experiments by others have indicated that protein kinase C activity is present in the rod outer segment, with potential or demonstrated targets including rhodopsin, transducin, cGMP-phosphodiesterase (PDE), guanylate cyclase, and arrestin, all of which are components of the phototransduction cascade. In particular, PKC phosphorylations of rhodopsin and the inhibitory subunit of PDE (PDE γ) have been studied in some detail, and suggested to have roles in downregulating the sensitivity of rod photoreceptors to light during illumination. We have examined this question under physiological conditions by recording from a single, dissociated salamander rod with a suction pipette while exposing its outer segment to the PKC activators phorbol-12-myristate,13-acetate (PMA) or phorbol-12,13-dibutyrate (PDBu), or to the PKC-inhibitor GF109203X. No significant effect of any of these agents on rod sensitivity was detected, whether in the absence or presence of a background light, or after a low bleach. These results suggest that PKC probably does not produce any acute downregulation of rod sensitivity as a mechanism of light adaptation, at least for isolated amphibian rods. PMID:9379174

  11. 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

  12. 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

  13. 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.

  14. Evidence for inhibition of nitric oxide and inducible nitric oxide synthase in Caco-2 and RAW 264.7 cells by a Maillard reaction product [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]-methanol.

    PubMed

    Chen, Xiu-Min; Kitts, David D

    2015-08-01

    We have recently isolated and characterized the chemical structure of a bioactive Maillard reaction product, [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]-methanol (F3-A), from an aqueous glucose (Glc) and lysine (Lys) Maillard reaction (MR) model system. Here, we investigate further the mechanisms for anti-inflammatory effects of this product in Caco-2 and RAW 264.7 cells. The anti-inflammatory capacity of F3-A recovered from Glc-Lys MR mixture and a synthesized product were compared with those of the specific inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), and the nuclear factor-kappa B (NF-κB) inhibitor, pyrrolidine dithiocarbamate (PDTC). F3-A produced a dose-dependent inhibition of extracellular nitric oxide (NO) production and iNOS translation in Caco-2 cells induced with interferon gamma (IFN-γ) and phorbol 12-myristate 13-acetate (PMA), and these effects were more potent than those obtained with AG. Moreover, a combination of F3-A and AG to attenuate intestinal inflammation was additive. However, F3-A inhibited only intracellular NO production in RAW 264.7 cells and did not inhibit COX-2 or NF-κB in either cell line. We conclude that the anti-inflammatory properties of F3-A are cell specific, working through different mechanism between macrophages and intestinal cells.

  15. Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

    PubMed

    Agoston, D V; Colburn, S; Krajniak, K G; Waschek, J A

    1992-05-25

    Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

  16. 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

  17. 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

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

    PubMed Central

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-01-01

    Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements. PMID:27322248

  19. Regulation of Cop9 signalosome activity by the EF-hand Ca2+-binding protein tescalcin

    PubMed Central

    Levay, Konstantin; Slepak, Vladlen Z.

    2014-01-01

    ABSTRACT The Ca2+-binding protein tescalcin is known to be involved in hematopoietic cell differentiation; however, this mechanism is poorly understood. Here, we identify CSN4 (subunit 4 of the COP9 signalosome) as a novel binding partner of tescalcin. The COP9 signalosome (CSN) is a multiprotein complex that is essential for development in all eukaryotes. This interaction is selective, Ca2+-dependent and involves the PCI domain of CSN4 subunit. We then investigated tescalcin and CSN activity in human erythroleukemia HEL and promyelocytic leukemia K562 cells and find that phorbol 12-myristate 13-acetate (PMA)-induced differentiation, resulting in the upregulation of tescalcin, coincides with reduced deneddylation of cullin-1 (Cul1) and stabilization of p27Kip1 – molecular events that are associated with CSN activity. The knockdown of tescalcin led to an increase in Cul1 deneddylation, expression of F-box protein Skp2 and the transcription factor c-Jun, whereas the levels of cell cycle regulators p27Kip1 and p53 decreased. These effects are consistent with the hypothesis that tescalcin might play a role as a negative regulator of CSN activity towards Cul1 in the process of induced cell differentiation. PMID:24659803

  20. Regulation of Cop9 signalosome activity by the EF-hand Ca2+-binding protein tescalcin.

    PubMed

    Levay, Konstantin; Slepak, Vladlen Z

    2014-06-01

    The Ca(2+)-binding protein tescalcin is known to be involved in hematopoietic cell differentiation; however, this mechanism is poorly understood. Here, we identify CSN4 (subunit 4 of the COP9 signalosome) as a novel binding partner of tescalcin. The COP9 signalosome (CSN) is a multiprotein complex that is essential for development in all eukaryotes. This interaction is selective, Ca(2+)-dependent and involves the PCI domain of CSN4 subunit. We then investigated tescalcin and CSN activity in human erythroleukemia HEL and promyelocytic leukemia K562 cells and find that phorbol 12-myristate 13-acetate (PMA)-induced differentiation, resulting in the upregulation of tescalcin, coincides with reduced deneddylation of cullin-1 (Cul1) and stabilization of p27(Kip1) - molecular events that are associated with CSN activity. The knockdown of tescalcin led to an increase in Cul1 deneddylation, expression of F-box protein Skp2 and the transcription factor c-Jun, whereas the levels of cell cycle regulators p27(Kip1) and p53 decreased. These effects are consistent with the hypothesis that tescalcin might play a role as a negative regulator of CSN activity towards Cul1 in the process of induced cell differentiation.

  1. Activation of protein kinase C in permeabilized human neuroblastoma SH-SY5Y cells.

    PubMed

    Larsson, C; Saermark, T; Mau, S; Simonsson, P

    1992-08-01

    The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.

  2. 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.

  3. 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

  4. A novel matrix metalloproteinase-2 inhibitor triazolylmethyl aziridine reduces melanoma cell invasion, angiogenesis and targets ERK1/2 phosphorylation.

    PubMed

    Romanchikova, Nadezhda; Trapencieris, Pēteris; Zemītis, Jānis; Turks, Māris

    2014-12-01

    A novel matrix metalloproteinase-2 (MMP-2) inhibitor JaZ-30, which belongs to the class of C(2)-monosubstituted aziridine - aryl-1,2,3-triazole conjugates, was developed. MTT and crystal violet assays were used to determine cytotoxicity- IC(50) values of compound JaZ-30 on melanoma cell line B16 4A5. Our study proves the anti-cancer properties of JaZ-30 with a wide spectrum of activities. JaZ-30 was revealed as selective inhibitor of matrix metalloproteinase-2. JaZ-30-mediated decrease of Vascular Endothelial Growth Factor (VEGF) secretion results in inhibition of angiogenesis, performed with the human umbilical vein endothelial cell line (HUVEC-2) on Matrigel. A novel inhibitor decreases invasive properties of melanoma cells measured in Matrigel chambers assay. JaZ-30 downregulates phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in melanoma cells stimulated by phorbol-12-myristate-13-acetate (PMA). Our findings propose a novel MMP-2 inhibitor JaZ-30 as an attractive potential agent for melanoma treatment.

  5. Carbamylcholine and phorbol esters desensitize muscarinic receptors by different mechanisms in rat pancreatic acini.

    PubMed

    Blanchard, L M; Paquette, B; Larose, L; Morisset, J

    1990-01-01

    Pretreatment of rat pancreatic acini with phorbol 12-myristate, 13-acetate (PMA), a protein kinase C (PK-C) activator, caused the desensitization of carbamylcholine (CBC)-induced amylase release in a concentration- and time-dependent fashion. The less potent phorbol-12, 13-dibutyrate (PDBu) also provoked a desensitization, but the inactive 4-alpha-phorbol-12,13-didecanoate had no effect. PMA or PDBu also significantly reduced subsequent amylase release induced by caerulein or secretin in contrast to CBC, which only reduced amylase release induced by CBC or secretin. Preincubation of acini with PMA did not lead to a decrease in PMA or A23187-stimulated amylase release. A 3 h resting period did not restore the desensitization induced by PMA or PDBu. Pretreatment with PMA did not cause changes in muscarinic receptor high- and low-affinity populations as observed with CBC pretreatment. The PK-C inhibitor H-7 completely prevented the desensitization induced by PDBu but not that induced by CBC. TMB-8, another PK-C inhibitor, also completely prevented the desensitization induced by PDBu but only partially that induced by CBC. These results suggest that phorbol esters can induce desensitization of muscarinic receptor-stimulated amylase release by a different mechanism than that involved in muscarinic agonist-induced desensitization.

  6. Inhibitory effects of Chikusetsusaponin IVa on lipopolysaccharide-induced pro-inflammatory responses in THP-1 cells.

    PubMed

    Wang, H; Qi, J; Li, L; Wu, T; Wang, Y; Wang, X; Ning, Q

    2015-09-01

    This study investigated anti-inflammatory effects and possible mechanisms of Chikusetsusaponin IVa (Chi IVa), one of the main bioactive components in saponins from Panacis japonica (SPJ), which is used in traditional Tujia and Hmong Chinese medicine. To this end, changes in the inflammatory profiles of lipopolysacchride (LPS)-stimulated phrobol 12-myristate 13-acetate(PMA)-differented THP-1 macrophages were evaluated following Chi IVa treatment. The results showed that Chi IVa markedly decreased the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) at both the mRNA and protein level, which proved to be dose-dependent. Further studies revealed that Chi IVa strongly suppressed NF-κB activation and downregulated the phosphorylation of ERK, p38, and JNK. Our present study demonstrates that Chi IVa suppresses the production of iNOS, COX-2, IL-1β, IL-6, and TNF-α in LPS-stimulated THP-1 cells likely by inhibiting NF-κB activation and ERK, JNK, and p38 signal pathway phosphorylation.

  7. Quantifying transient psychological stress using a novel technique: changes to PMA-induced leukocyte production of ROS in vitro.

    PubMed

    Shelton-Rayner, Graham K; Mian, Rubina; Chandler, Simon; Robertson, Duncan; Macdonald, David W

    2011-01-01

    Although much work has been conducted to quantify the long-term physiological effects of psychological stress, measures of short-term, low-level stress have been more elusive. This study assessed the effect of exposure of volunteers to a mild, brief, psychologically stressful event, on the functional ability of leukocytes in whole blood to respond to phorbol 12-myristate 13-acetate (PMA) in vitro. Volunteers operated a car electric window and adjusted it to 4 pre-determined positions. Between each operation the mechanism's polarity was covertly altered (group B) or remained unaltered (group A). For each treatment group 10 different subjects provided capillary blood samples pre- and post-stressor. Using a chemiluminescent technique termed leukocyte coping capacity, the ability of leukocytes to produce reactive oxygen species (ROS) in vitro was assessed. ROS release differed significantly at 10 min post-stressor between treatment groups, suggesting exposure to acute psychological stress leads to a reduced ability to respond to bacterial challenge.

  8. Thymus cell antigen 1 (Thy1, CD90) is expressed by lymphatic vessels and mediates cell adhesion to lymphatic endothelium.

    PubMed

    Jurisic, Giorgia; Iolyeva, Maria; Proulx, Steven T; Halin, Cornelia; Detmar, Michael

    2010-10-15

    The lymphatic vascular system plays an important role in inflammation and cancer progression, although the molecular mechanisms involved are poorly understood. As determined by comparative transcriptional profiling studies of ex vivo isolated mouse intestinal lymphatic endothelial cells versus blood vascular endothelial cells, thymus cell antigen 1 (Thy1, CD90) was expressed at much higher levels in lymphatic endothelial cells than in blood vascular endothelial cells. These findings were confirmed by quantitative PCR, and at the protein level by FACS and immunofluorescence analyses. Thy1 was also strongly expressed by tumor-associated lymphatic vessels, as evaluated in a B16 melanoma footpad model in mice. Blockade of Thy1 inhibited tumor cell adhesion to cultured mouse lymphatic endothelial cells. Importantly, treatment of human dermal microvascular endothelial cells with tumor necrosis factor or phorbol 12-myristate 13-acetate resulted in Thy1 upregulation in podoplanin-expressing lymphatic endothelial cells, but not in podoplanin-negative blood vascular endothelial cells. Moreover, adhesion of human polymorphonuclear and mononuclear leukocytes to human lymphatic endothelial cells was Thy1-dependent. Together, these results identify Thy1 as a novel lymphatic vessel expressed gene and suggest its potential role in the cell adhesion processes required for tumor progression and inflammation.

  9. Glycosaminoglycan sulfation in murine splenocytes

    SciTech Connect

    Rider, C.C.; Hart, G.W.

    1986-05-01

    The authors have studied the incorporation of /sup 35/sulfate into glycosaminoglycans (GAG) in splenocytes incubated in medium RPMI 1640 containing 3..mu..M sulfate. Addition of Concanavalin A (Con A) and phorbol 12-myristate 13-acetate (PMA) caused within 24 hr a 10- to 20-fold increase in incorporation into secreted GAG and a 2- to 4-fold increase in cell-retained GAG. PMA added alone caused only 2- to 4-fold increases in both fractions. Between 0 and 3 h however, PMA either alone or with Con A caused a substantial decrease in the incorporation of sulfate into the cellular GAG fraction, suggesting that an immediate effect of these agents is to cause the clearance of nascent GAG chains from the Golgi. The composition of newly sulfated lymphocyte GAG has been found to be approximately 75% chondroitin sulfate and 25% heparan sulfates in both secreted and non-secreted GAG irrespective of the presence of Con A and PMA. Amino column HPLC analysis of disaccharides released by chondroitinase ABC digestion indicates that both ..delta.. Di-4S and ..delta.. Di-6S are produced with the proportion of the latter increasing gradually from initially low levels such that at 24 h, equal proportions of the two are found. Possible mechanisms for this change in the position of sulfation will be discussed.

  10. Effect of phorbol and Bryostatin I on chondrogenic expression of chick limb bud, in vitro

    SciTech Connect

    Garrison, J.C.; Pettit, G.R.; Uyeki, E.M.

    1987-10-26

    The present paper describes the effects of PMA (phorbol 12-myristate 13 acetate) on in vitro chondrogenesis in non-passaged, embryonic limb bud cells, relative to the effects of Bryostatin I. This compound also activates C kinase and binds competitively to the phorbol ester receptor, yet does not affect cell differentiation. Levels of PMA as low as 10/sup -7/ M markedly reduced cartilage formation in 4-day cultures, as indicated by nodule count and Alcian blue staining for chondroitin sulfate. Coadministration of Bryostatin I at equimolar concentration prevented the PMA inhibitory effect on chondrocytic expression. This confirms other findings that phorbol activation of C kinase cannot exclusively account for the activity of phorbol on cell expression. Altering the time of PMA exposure demonstrated that PMA inhibited chondrocyte phenotypic expression, rather than cell commitment: early exposure to PMA had little inhibitor effect on the staining index, whereas, exposure from 49-96 h and 0-96 h had moderate and strong inhibitory effects, respectively, on cartilage synthesis. Further research on the phorbol/Bryostatin I interaction should add to their knowledge of the control processes involved in tumor promotion and cell differentiation. 21 references, 3 figures.

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

    SciTech Connect

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

    1989-09-05

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

  12. 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 transcripts from normal human epidermis, and studied how their expressions are regulated. By RT-PCR, we found that a number of different CD44 transcripts were expressed in human epidermis, and we obtained all these transcripts from DNA bands in agarose and acrylamide gels by cloning. Detailed sequence analysis revealed 18 CD44 transcripts, 3 of which were novel. Next, we examined effects of 10 different agents on the expression of CD44 transcripts in cultured human keratinocytes, and found that several agents, particularly epidermal growth factor, hydrogen peroxide, phorbol 12-myristate 13-acetate, retinoic acid, calcium and fetal calf serum differently regulated their expressions in various patterns. Furthermore, normal and malignant keratinocytes were found to produce different CD44 transcripts upon serum stimulation and subsequent starvation, suggesting that specific CD44 isoforms are involved in tumorigenesis via different CD44-mediated biological pathways. PMID:27505250

  13. 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.

  14. 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

  15. The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages.

    PubMed Central

    Hancock, J T; Jones, O T

    1987-01-01

    Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible. PMID:3036079

  16. 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

  17. Phosphorylation of farnesoid X receptor by protein kinase C promotes its transcriptional activity.

    PubMed

    Gineste, Romain; Sirvent, Audrey; Paumelle, Réjane; Helleboid, Stéphane; Aquilina, Alexis; Darteil, Raphaël; Hum, Dean W; Fruchart, Jean-Charles; Staels, Bart

    2008-11-01

    The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKCalpha phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKCalpha-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor gamma coactivator 1alpha. In conclusion, these findings show that the phosphorylation of FXR induced by PKCalpha directly modulates the ability of agonists to activate FXR.

  18. 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

  19. 7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils.

    PubMed

    Kabeya, Luciana M; Fuzissaki, Carolina N; Taleb-Contini, Silvia H; da C Ferreira, Ana Maria; Naal, Zeki; Santos, Everton O L; Figueiredo-Rinhel, Andréa S G; Azzolini, Ana Elisa C S; Vermelho, Roberta B; Malvezzi, Alberto; Amaral, Antonia T-do; Lopes, João Luis C; Lucisano-Valim, Yara Maria

    2013-10-25

    In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.

  20. Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel.

    PubMed

    Kinsella, J L; Grant, D S; Weeks, B S; Kleinman, H K

    1992-03-01

    Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.

  1. 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.

  2. Functional Differences of Very-Low-Density Lipoprotein Receptor Splice Variants in Regulating Wnt Signaling

    PubMed Central

    Chen, Qian; Takahashi, Yusuke; Oka, Kazuhiro

    2016-01-01

    The very-low-density lipoprotein receptor (VLDLR) negatively regulates Wnt signaling. VLDLR has two major alternative splice variants, VLDLRI and VLDLRII, but their biological significance and distinction are unknown. Here we found that most tissues expressed both VLDLRI and VLDLRII, while the retina expressed only VLDLRII. The shed soluble VLDLR extracellular domain (sVLDLR-N) was detected in the conditioned medium of retinal pigment epithelial cells, interphotoreceptor matrix, and mouse plasma, indicating that ectodomain shedding of VLDLR occurs endogenously. VLDLRII displayed a higher ectodomain shedding rate and a more potent inhibitory effect on Wnt signaling than VLDLRI in vitro and in vivo. O-glycosylation, which is present in VLDLRI but not VLDLRII, determined the differential ectodomain shedding rates. Moreover, the release of sVLDLR-N was inhibited by a metalloproteinase inhibitor, TAPI-1, while it was promoted by phorbol 12-myristate 13-acetate (PMA). In addition, sVLDLR-N shedding was suppressed under hypoxia. Further, plasma levels of sVLDLR-N were reduced in both type 1 and type 2 diabetic mouse models. We concluded that VLDLRI and VLDLRII had differential roles in regulating Wnt signaling and that decreased plasma levels of sVLDLR-N may contribute to Wnt signaling activation in diabetic complications. Our study reveals a novel mechanism for intercellular regulation of Wnt signaling through VLDLR ectodomain shedding. PMID:27528615

  3. Chemopreventive properties of phytosterols and maslinic acid extracted from Coleus tuberosus in inhibiting the expression of EBV early-antigen in Raji cells.

    PubMed

    Mooi, Lim Yang; Wahab, Norhanom Abdul; Lajis, Nordin Haji; Ali, Abdul Manaf

    2010-05-01

    Bioassay-guided fractionation of a MeOH extract of tubers of Coleus tuberosus afforded the active anti-tumor-promoting compounds identified as the triterpenoid 2alpha,3beta-dihydroxyolean-12-en-28-oic acid (maslinic acid; CT2) and a phytosterol mixture (CT1). CT1 consists of stigmasterol (32%), beta-sitosterol (40.3%), and campesterol (27.7%) as determined by capillary gas chromatography. CT1 and CT2 showed very strong anti-tumor-promoting activities at IC(50) 0.7 microg/ml and 0.1 microg/ml, respectively, in a convenient, short-term in vitro assay, i.e., the inhibition of Epstein-Barr virus (EBV) activation induced by phorbol 12-myristate 13-acetate (PMA) and sodium butyrate. We report for the first time the anti-tumor-promoting activity of 2alpha,3beta-dihydroxyolean-12-en-28-oic acid and show that a mixture of stigmasterol, beta-sitosterol, and campesterol is more potent than the individual components in inhibiting tumor-promoting activity.

  4. Identification of 1,8-cineole, borneol, camphor, and thujone as anti-inflammatory compounds in a Salvia officinalis L. infusion using human gingival fibroblasts.

    PubMed

    Ehrnhöfer-Ressler, Miriam M; Fricke, Kristina; Pignitter, Marc; Walker, Joel M; Walker, Jessica; Rychlik, Michael; Somoza, Veronika

    2013-04-10

    Drinking or gargling Salvia officinalis L. infusion (sage infusion) is thought to soothe a sore throat, tonsillitis, and inflamed, red gums, although structure-based scientific evidence for the key anti-inflammatory compounds in sage infusion is scarce. Human gingival fibroblasts (HGF-1) were treated with sage infusion (SI) or SI fractions containing either its volatile components and water (aqueous distillate, AD) or its dry matter (DM) for six hours. SI, AD, and DM reduced a mean phorbol-12-myristate-13-acetate/ionomycin (PMA/I)-stimulated release of the pro-inflammatory interleukins IL-6 and IL-8 by more than 50% (p < 0.05). Cellular uptake experiments and subsequent GC-MS analysis using stable-isotope-labeled internal standards revealed the presence of 1,8-cineole, borneol, camphor, and α-/β-thujone in SI-treated cells; LC-MS analysis demonstrated the presence of rosmarinic acid. A significant, more than 50% mean inhibition of PMA/I-induced IL-6 and IL-8 release was demonstrated for the volatile compounds 1,8-cineole, borneol, camphor, and thujone, but not for the nonvolatile rosmarinic acid when applied in concentrations representative of sage infusion. Therefore, the volatile compounds were found to be more effective than rosmarinic acid. 1,8-Cineole, borneol, camphor, and α-/β-thujone chiefly contribute to the anti-inflammatory activity of sage infusion in human gingival fibroblasts.

  5. 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

  6. Characterization of phorbol ester-stimulated serine phosphorylation of the human insulin receptor.

    PubMed Central

    Feener, E P; Shiba, T; Hu, K Q; Wilden, P A; White, M F; King, G L

    1994-01-01

    Phorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of the human insulin receptor (IR) was characterized and compared in two cell types of different lineage: normal rat kidney epithelial (NRK) cells and Chinese hamster ovary (CHO) fibroblasts. PMA stimulation increased IR beta-subunit phosphorylation to 252 +/- 43 and 25- +/- 47% (+/- S.D.) of the unstimulated control in NRK and CHO cells respectively. Tryptic phosphopeptide analysis by Tricine/SDS/PAGE revealed significant differences in the PMA-stimulated phosphorylation of the IR in these two cell types. This phosphorylation of the IR was predominantly located in two tryptic phosphopeptides, and these phosphopeptides were absent in an IR mutant truncated by 43 C-terminal amino acids. The major PMA-stimulated tryptic phosphopeptide from in vivo-labelled CHO/IR was immunoprecipitated with an antibody against residues Ser1315 to Lys1329, and this precipitation was blocked with excess unlabelled peptide containing this sequence. Radiosequencing by manual Edman degradation revealed that this tryptic phosphopeptide was phosphorylated at Ser1315. This PMA-stimulated phosphorylation did not inhibit autophosphorylation of the IR in vivo. These results demonstrate that PMA-stimulated phosphorylation of the IR can exhibit significant differences when expressed in different cell types, and that Ser1315 is a major PMA-stimulated phosphorylation site on the human IR. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7945263

  7. Induction of T cell adhesion to extracellular matrix or endothelial cell ligands by soluble or matrix-bound interleukin-7.

    PubMed

    Ariel, A; Hershkoviz, R; Cahalon, L; Williams, D E; Akiyama, S K; Yamada, K M; Chen, C; Alon, R; Lapidot, T; Lider, O

    1997-10-01

    The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human, IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state. PMID:9368611

  8. The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

    PubMed

    Frankel, G; Lider, O; Hershkoviz, R; Mould, A P; Kachalsky, S G; Candy, D C; Cahalon, L; Humphries, M J; Dougan, G

    1996-08-23

    Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin. PMID:8702771

  9. Inhibition of arachidonate release from rat peritoneal macrophage by biflavonoids.

    PubMed

    Lee, S J; Son, K H; Chang, H W; Kang, S S; Kim, H P

    1997-12-01

    Biflavonoid is one of unique classes of naturally-occurring bioflavonoid. Previously, certain biflavonoids were found to possess the inhibitory effects on phospholipase A(2) activity and lymphocytes proliferation(1) suggesting their anti-inflammatory/immunoregulatory potential. In this study, effects of several biflavonoids on arachidonic acid release from rat peritoneal macrophages were investigated, because arachidonic acid released from the activated macrophages is one of the indices of inflammatory conditions. When resident peritoneal macrophages labeled with [(3)H]arachidonic acid were activated by phorbol 12-myristate 13-acetate (PMA) or calcium ionophore, A23187, radioactivity released in the medium was increased approximately 4.1 approximately 7.3 fold after 120 min incubation compared to the spontaneous release in the control incubation. In this condition, biflavonoids (10 uM) such as ochnaflavone, ginkgetin and isoginkgetin, showed inhibition of arachidonate release from macrophages activated by PMA (32.5 approximately 40.0% inhibition) or A23187 (21.7 approximately 41.7% inhibition). Amentoflavone showed protection only against PMA-induced arachidonate release, while apigenin, a monomer of these biflavonoids, did not show the significant inhibition up to 10 uM. Staurosporin (1 uM), a protein kinase C inhibitor, showed an inhibitory effect only against PMA-induced arachidonate release (96.8% inhibition). Inhibition of arachidonate release from the activated macrophages may contribute to an anti-inflammatory potential of biflavonoidsin vivo.

  10. 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

  11. 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.

  12. Nonrandom duplication of the chromosome bearing a mutated Ha-ras-1 allele in mouse skin tumors

    SciTech Connect

    Bianchi, A.B.; Aldaz, C.M.; Conti, C.J. )

    1990-09-01

    The authors analyzed the normal/mutated allelic ratio of the Ha-ras-1 gene in mouse skin squamous cell carcinomas induced by initation with dimethylbenz(a)anthracene and promotion with phorbol 12-myristate 13-acetate. DNA for these studies was obtained from short-term tumor cultures (24-72 hr) to eliminate the contribution of stromal and inflammatory cells to the sample. The alelotypic analysis was performed in 25 squamous cell carcinomas by quantitative radio-analysis of the Xba I restriction fragment length polymorphism as detected by BS9, a v-Ha-ras probe, and rehybridization of the Southern blots with probes for chromosomes 7 and 9. Approximately 85% of the tumors presented overrepresentation of the mutated allele in the form of 1 normal/2 mutated (12 tumors), 0 normal/3 mutated (4 tumors), 0 normal/2 mutated (3 tumors), and gene amplification (3 tumors). No tumor was found with a 2 normal/1 mutated allelic ratio. These results support their previous cytogenetic studies, indicating that trisomy of chromosome 7 is present in themajority of these tumors show that nonrandom duplication of the chromosome carrying the mutated Ha-ras-1 allel appears to be a major mechanism by which the mutated gene is overrepresented.

  13. 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

  14. The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

    PubMed Central

    1985-01-01

    Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl- phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C. PMID:2859292

  15. 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.

  16. Posttranslational regulation of neurofibromin content in melanocytes of neurofibromatosis type 1 patients.

    PubMed

    Kaufmann, D; Bartelt, B; Hoffmeyer, S; Müller, R

    1999-06-01

    Neurofibromatosis type 1 (NF1) is a common autosomal dominantly inherited disorder characterized by neurofibromas and café-au-lait macules. Most of the NF1 gene germline mutations result in a reduction in the level of neurofibromin. As shown recently, the neurofibromin level can be regulated posttranslationally through alteration of the protein half-life. This raises the question as to whether this type of regulation is also operating in cultured melanocytes of NF1 patients especially in melanocytes derived from café-au-lait macules. In melanocytes cultured without phorbol 12-myristate 13-acetate (PMA) the neurofibromin half-lives were 24 h (healthy controls, MC), 26 h (apparently healthy skin of NF1 patients, MNFS) and 25 h (café-au-lait macules of NF1 patients, MNFC). In PMA-stimulated cells the neurofibromin half-lives were 68 h (MC) and 73 h (MNFS) whereas it was 45 h in melanocytes derived from NF1 café-au-lait macules. The amount of NF1 mRNA was not altered under these culture conditions as shown by competitive RT-PCR. We speculate that this regulation is involved in the formation of some NF1 symptoms, for instance in the formation of café-au-lait macules.

  17. Increased melanogenesis in cultured epidermal melanocytes from patients with neurofibromatosis 1 (NF 1).

    PubMed

    Kaufmann, D; Wiandt, S; Veser, J; Krone, W

    1991-06-01

    Melanocyte cultures from the normally pigmented skin of patients with neurofibromatosis 1 (NF 1) have a higher melanin content than those from the skin of healthy donors. An additional increase in the amount of melanin per cell was found in 5 out of 6 lines of melanocytes derived from café au lait macules of NF 1 patients. Omission of the tumor promoter phorbol-12-myristate-13-acetate from the culture medium brings about a comparable increase in the melanin content in all three kinds of melanocyte cultures. Cultures of NF 1 melanocytes show a higher tyrosine hydroxylase activity than those of control melanocytes, and incorporate larger amounts of dihydroxyphenylalanine than the latter. We conclude that melanogenesis in epidermis melanocytes is affected by defective alleles of the NF 1 gene. Our findings do not contradict the hypothesis that the defect underlying NF 1 impairs the inhibition of a wild-type RAS oncogene by interfering with the GTPase-activating function of the NF 1 gene product.

  18. 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

  19. 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

  20. 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.

  1. Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

    PubMed Central

    Reiser, H; Freeman, G J; Razi-Wolf, Z; Gimmi, C D; Benacerraf, B; Nadler, L M

    1992-01-01

    We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex. Images PMID:1370349

  2. Fc gamma receptor type III (CD16) is included in the zeta NK receptor complex expressed by human natural killer cells.

    PubMed Central

    Anderson, P; Caligiuri, M; O'Brien, C; Manley, T; Ritz, J; Schlossman, S F

    1990-01-01

    We recently reported that CD3- natural killer (NK) cells express the zeta chain of the T-cell receptor complex (zeta NK) in association with higher molecular weight structures whose expression differs between individual NK cell clones. Because NK cell cytolytic activity is known to be triggered by perturbation of the type III Fc gamma receptor (CD16), we sought to determine whether this activating molecule is included in the zeta NK molecular complex. Biochemical evidence for a physical association between CD16 and zeta NK was obtained by comparing immunoprecipitates formed using monoclonal antibodies reactive with each of these molecules by SDS/polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping. In both clonal and polyclonal populations of CD3- NK cells, CD16 and zeta NK specifically associated with one another. Functional evidence for a specific association between CD16 and zeta NK in intact cells was obtained by demonstrating a coordinate down-modulation of both of these molecules induced by either phorbol 12-myristate 13-acetate or monoclonal antibodies reactive with CD16. Our results suggest that Fc gamma receptor type III (CD16) is included in the zeta NK complex and that this complex is likely to play an important role in NK cell activation. Images PMID:2138330

  3. Antinociceptive activity of CP-101,606, an NMDA receptor NR2B subunit antagonist

    PubMed Central

    Taniguchi, Kana; Shinjo, Katsuhiro; Mizutani, Mayumi; Shimada, Kaoru; Ishikawa, Toshihisa; Menniti, Frank S; Nagahisa, Atsushi

    1997-01-01

    The analgesic activity of CP-101,606, an NR2B subunit-selective N-methyl-D-aspartate (NMDA) receptor antagonist, was examined in carrageenan-induced hyperalgesia, capsaicin- and 4β-phorbol-12-myristate-13-acetate (PMA)-induced nociceptive tests in the rat. CP-101,606 30 mg kg−1, s.c., at 0.5 and 2.5 h after carrageenan challenge suppressed mechanical hyperalgesia without any apparant alternations in motor coordination or behaviour in the rat. CP-101,606 also inhibited capsaicin- and PMA-induced nociceptive responses (licking behaviour) with ED50 values of 7.5 and 5.7 mg kg−1, s.c., respectively. These results suggest that inhibition of the NR2B subunit of the NMDA receptor is effective in vivo at modulating nociception and hyperalgesia responses without causing the behavioural side effects often observed with currently available NMDA receptor antagonists. PMID:9384494

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

    PubMed

    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.

  5. Tetrahydrophthalazine derivative "sodium nucleinate" exert its anti-inflammatory effects through inhibition of oxidative burst in human monocytes.

    PubMed

    Jukić, Tomislav; Ihan, Alojz; Jukić, Dubravko

    2012-06-01

    We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit, is 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the oxidative burst responses of whole blood human monocytes and polimorphonuclear cells (PMC) stimulated with phorbol 12-myristate 13-acetate (PMA) or E. coli suspension in vitro. SN did not inhibit the proportion of neutrophils and monocytes phagocytosing E. coli. Oxidative burst responses of monocytes stimulated with PMA were strongly inhibited at SN concentration ranging from 10-500 mg/ml, less efficient inhibitor was SN in E. coli stimulated monocytes (inhibition range was from 50-500 mg/ml SN). SN inhibited PMC oxidative burst only in range 100-500 mg/ml SN. In conclusion, we found SN as an efficient inhibitor of oxidative burst in monocytes. Since ROS generation in monocytes/macrophages has been found to be important for LPS-driven production of several proinflammatory cytokines, SN may exsert its antiinflammatory effects through monocyte/macrophage oxidative burst inhibition.

  6. Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes.

    PubMed

    Kalgraff, Cathrine A K; Wergeland, Heidrun I; Pettersen, Eirin Fausa

    2011-09-01

    The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.

  7. 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

  8. 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

  9. Neutrophil function and apoptosis in patients with chronic hepatitis C treated with pegylated interferon α and ribavirin.

    PubMed

    Jabłonowska, Elżbieta; Wójcik, Kamila; Kur, Barbara; Lewkowicz, Przemysław; Nocuń, Marek

    2012-02-01

    The role of neutrophils in the pathogenesis of chronic hepatitis C as well as the effect of pegylated interferon α (PEG-IFN-α) and ribavirin treatment on neutrophil function is not precisely known. The study included 32 patients with CCH aged between 19 and 58 years (mean 33.5 years). Before and after 12 weeks of treatment with Peg-IFN-α and ribavirin, intracellular reactive oxygen species (ROS) level, expression of adhesion molecules CD11b/MAC-1, CD16, CD18 and CD62L on neutrophils, as well as apoptosis and necrosis of these cells were analyzed with the use of flow cytometry. During antiviral therapy, a statistically significant decrease of mean fluorescence intensity for CD16 high and CD62 and increase for CD11b/MAC-1 along with the increased apoptosis and decreased necrosis of neutrophils were observed. After 12 weeks of treatment, intracellular ROS production by unstimulated neutrophils did not change, but after stimulation with phorbol 12-myristate 13-acetate, statistically significant increase of ROS level was observed. During PEG-IFN-α and ribavirin treatment, activation of neutrophil function and increased ROS production were reported, which possibly resulted in accelerated apoptosis of these cells.

  10. 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.

  11. Pro-oxidative interactions of cobalt with human neutrophils.

    PubMed

    Ramafi, Grace J; Theron, Annette J; Anderson, Ronald

    2004-08-01

    The primary objectives of this study were to investigate the effects of cobalt(II) chloride (Co, 1.5-25 microM) on the reactivity of hydrogen peroxide (H2O2, 100 microM) or oxidants generated by activated human neutrophils. The prooxidative interactions of Co with H2O2 or cells were measured by luminol-enhanced chemiluminescence (LECL), and according to the extent of oxidative inactivation of added alpha-1-proteinase inhibitor (API). Cobalt dramatically potentiated the oxidation of luminol and API by both H2O2 and neutrophils activated with phorbol 12-myristate 13-acetate (5 ng/ml), without affecting the assembly of NADPH oxidase or the magnitude of oxygen consumption by the cells. Using 5,5-dimethyl-pyrolline 1-oxide-based electron spin resonance spectroscopy we were unable to detect hydroxyl radical formation by Co in the presence of either H2O2 or activated neutrophils, while the corresponding LECL responses were unaffected by the hydroxyl radical scavengers benzoate and mannitol (50 mM). These observations indicate that Co potentiates the reactivity of neutrophil-derived oxidants, primarily H2O2, which if operative in vivo during exposure to the heavy metal may pose the risk of oxidant- and protease-mediated tissue injury.

  12. Limited effect of selected organic pollutants on cytokine production by peripheral blood leukocytes.

    PubMed

    Devos, Sabrina; Van Den Heuvel, Rosette; Hooghe, Robert; Hooghe-Peters, Elisabeth L

    2004-01-01

    To test the hypothesis that some persistent organic pollutants contribute to the increased prevalence of allergic disease, the effects of selected compounds on cytokine production by PBMC from control and allergic donors were evaluated. Cells were cultured for six days in the presence of a xenobiotic (PCB 153, hexachlorobenzene, pentachlorobenzene, pentachlorophenol, lindane, atrazine or DMSO vehicle) with phytohemagglutinin (PHA) or Dermatophagoides pteronyssinus extract, then for one day in the presence of PHA + phorbol 12-myristate 13-acetate. PCB 153 reduced the levels of IL-10, IFN-gamma and TNF-alpha. Hexachlorobenzene reduced the levels of IL-5, IL-10 and IFN-gamma. Pentachlorobenzene reduced IL-6 levels. Pentachlorophenol reduced IL-5 levels. Lindane and atrazine reduced both IL-5 and IFN-gamma. In addition, lindane reduced TNF-alpha levels. As these compounds had similar effects on cells from allergic and non-allergic donors, and as these effects were, in all cases, very limited indeed, the potential deleterious impact of the xenobiotics tested on the allergic response seems unlikely.

  13. 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.

  14. Characterization of noradrenaline-stimulated cyclic GMP formation in brain astrocytes in culture.

    PubMed Central

    Agulló, L; García, A

    1992-01-01

    Cyclic GMP accumulation induced by noradrenaline in astrocyte-enriched primary cultures from rat cerebrum involves synthesis of NO, as evidenced by the competitive inhibition exerted by the NO synthase inhibitor NG-monomethyl-L-arginine (IC50 = 3 microM). Furthermore, the noradrenaline effect was potently inhibited by haemoglobin (IC50 = 25 nM) and potentiated by superoxide dismutase, indicating that NO synthesis and cyclic GMP formation may occur in different subsets of astrocytes. Investigation of the receptors implicated by using selective adrenoceptor agonists and antagonists indicates that about 75% of the NO-dependent noradrenaline response is mediated by alpha 1-adrenoceptors and the rest by beta-adrenoceptors, with no evidence for potentiating effects between the two receptor types. This noradrenaline effect appears to require Ca2+ entry, since it is strongly dependent on extracellular Ca2+ but is not affected by conditions that will abolish intracellular Ca2+ mobilization (incubation with neomycin or pretreatment with carbachol). Inhibition by pretreatment with pertussis toxin is in agreement with involvement of the alpha 1A-adrenoceptor subtype in this Ca(2+)-dependent effect. However, implication of an unknown alpha 1-adrenoceptor subtype cannot be disregarded, because a similar inhibition is exerted by the presumably selective alpha 1B- and alpha 1C-adrenoceptor blocking agent chloroethylclonidine. Treatment of the cultures with the protein kinase C activator phorbol 12-myristate 13-acetate inhibits to a great extent the noradrenaline-induced cyclic GMP formation. PMID:1334410

  15. Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons.

    PubMed

    Hourez, Raphaël; Azdad, Karima; Vanwalleghem, Gilles; Roussel, Céline; Gall, David; Schiffmann, Serge N

    2005-10-19

    Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP(3)) receptors on voltage-dependent sodium current. Cellular dialysis with phorbol 12-myristate 13-acetate, an activator of PKC, significantly reduced peak sodium current amplitude, while adenophostin A, an activator of IP(3) receptors, significantly increased peak sodium current amplitude. This effect of adenophostin was abolished by calcium chelation or by FK506, an inhibitor of calcineurin. These results suggest an antagonistic role of PKC and IP(3) in the modulation of striatal voltage-gated sodium channels, peak current amplitude being decreased through phosphorylation by PKC and increased through dephosphorylation by calcineurin.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. A new human breast cancer cell line, KPL-3C, secretes parathyroid hormone-related protein and produces tumours associated with microcalcifications in nude mice.

    PubMed Central

    Kurebayashi, J.; Kurosumi, M.; Sonoo, H.

    1996-01-01

    Parathyroid hormone-related protein (PTHrP) is the main cause of humoral hypercalcaemia of malignancy (HHM). We recently established a new human breast cancer cell line, designated KPL-3C, from the malignant effusion of a breast cancer patient with HHM. Morphological, cytogenetic and immunohistochemical analyses indicated that the cell line is derived from human breast cancer. The KPL-3C cells stably secrete immunoreactive PTHrP measured by a two-site immunoradiometric assay, possess both oestrogen and progesterone receptors and are tumorigenic in female nude mice. The addition of phorbol-12-myristate-13-acetate to the medium significantly increased PTHrP secretion from the cells. In contrast, hydrocortisone, medroxyprogesterone acetate and 22-oxacalcitriol decreased PTHrP secretion in a dose-dependent manner. Unexpectedly, a number of microcalcifications were observed in the transplanted tumours. Radiographical examination indicated that the microcalcifications in the tumours are very similar to those commonly observed in human breast cancer. These findings suggest that this KPL-3C cell line may be useful for studying the regulatory mechanisms of PTHrP secretion and the mechanisms that lead to the deposition of microcalcifications in breast cancer. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 PMID:8688322

  2. 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

  3. Silver nanoparticles impede phorbol myristate acetate-induced monocyte-macrophage differentiation and autophagy.

    PubMed

    Xu, Yingying; Wang, Liming; Bai, Ru; Zhang, Tianlu; Chen, Chunying

    2015-10-14

    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. PMID:26372376

  4. 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

  5. S-Allylcysteine, a garlic compound, increases ABCA1 expression in human THP-1 macrophages.

    PubMed

    Malekpour-Dehkordi, Zahra; Javadi, Ebrahim; Doosti, Mahmood; Paknejad, Maliheh; Nourbakhsh, Mitra; Yassa, Narguess; Gerayesh-Nejad, Siavash; Heshmat, Ramin

    2013-03-01

    ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cholesterol efflux to apoA-I in lipid-loaded macrophages, which is the first step of reverse cholesterol transport in vivo and a critical step in preventing atherosclerosis. Enhanced ABCA1 expression may inhibit foam cell formation and consequently reduce atherogenic risk. The purpose of this study was to investigate the effect of S-allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, on the expression of ATP-binding cassette transporter A1 in human THP-1 macrophages. The human monocyte THP-1 cells were differentiated to macrophage cells in the presence of phorbol 12-myristate13-acetate (PMA). Macrophage cells were then treated with different concentrations (10, 20 and 40 mM) of SAC for 24 h. Total RNA of treated macrophages was extracted and analyzed with real-time RT-PCR. ABCA1 protein expression was also analyzed with western blotting. Results showed that SAC increased the ABCA1 mRNA (1.82-, 2.07- and 2.23-fold) and protein (1.37-, 1.55- and 2.08-fold) expression in macrophage THP-1 cells compared with control (untreated cells). Results suggested that SAC can increase ABCA1 expression in macrophages and may be beneficial in promoting reverse cholesterol efflux. PMID:22610793

  6. 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.

  7. 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

  8. 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

  9. 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

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

    PubMed

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

    2013-08-20

    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

  12. 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.

  13. Identification of a 57-kilodalton selenoprotein in human thyrocytes as thioredoxin reductase and evidence that its expression is regulated through the calcium-phosphoinositol signaling pathway.

    PubMed

    Howie, A F; Arthur, J R; Nicol, F; Walker, S W; Beech, S G; Beckett, G J

    1998-06-01

    Human thyrocytes incubated with the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-5)-10(-8) mol/L) and the calcium ionophore A23187 (10(-5)-10(-8) mol/L) showed a marked increase in the expression of a 57-kDa selenoprotein identified as thioredoxin reductase (TR). After the addition of A23187 with PMA, a significant induction in TR expression was observed after 6 h, with maximal induction occurring by 24 h. The addition of 8-bromo-cAMP (10(-4) mol/L) or TSH (10 U/L) alone had no effect on TR expression, nor did these agents influence the induction of TR brought about by the addition of A23187 and PMA. These data show that the calcium-phosphoinositol second messenger cascade that controls hydrogen peroxide generation in the human thyrocyte is also an important stimulator of TR expression. The role of TR in the thyrocyte is unclear, but the selenoenzyme has a high capacity to detoxify compounds, such as hydrogen peroxide and lipid hydroperoxides, that are produced in high concentration during thyroid hormone synthesis.

  14. 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.

  15. 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.

  16. 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

  17. High-throughput Protease Activity Cytometry Reveals Dose-dependent Heterogeneity in PMA-mediated ADAM17 Activation†

    PubMed Central

    Wu, Lidan; Claas, Allison M.; Sarkar, Aniruddh; Lauffenburger, Douglas A.; Han, Jongyoon

    2015-01-01

    As key components of autocrine signaling, pericellular proteases, A Disintegrin and Metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases.1-3 There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases. PMID:25832727

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

    SciTech Connect

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

    1988-07-01

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

  19. Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling

    SciTech Connect

    Kilfeather, S.A.; Stein, M.; O'Malley, K. )

    1991-01-01

    Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate for 10 minutes enhanced cyclic AMP accumulation in human neutrophils under basal conditions and in response to the beta-adrenergic receptor agonist isoproterenol (ISO, 1{mu}M) and the adenylate cyclase activator forskolin (FSK, 10mM). Potentiation of responses to ISO by PMA was dose-dependent between 0.1 and 100nM PMA. The diacylglycerol analogue, 1-oleoyl-2-actylgylcerol (OAG) (50 {mu}M) also elevated beta-receptor responses, but 4beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 {mu}M) also elevated neutrophil cyclic AMP accumulation. All potentiating effects of PMA on cyclic AMP production were inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H{sub 7}). PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalized compartments, or the capacity of ISO to induce beta-receptor internalization. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation int he presence of PMA were not elevated by PMA.

  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. 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. 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.

  3. Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells

    SciTech Connect

    Sesko, A.; Mossman, B. ); Cabot, M. )

    1990-10-01

    Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with ({sup 3}H)diacylglycerol. In contrast, crocidolite-exposed cells labeled with ({sup 3}H)myristic acid or ({sup 3}H)choline did not display elevated production of ({sup 3}H)diacylglycerol or release of ({sup 3}H)choline metabolites (i.e., evidence of phosphatidylcholine hydrolysis). The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-({sup 3}H)Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in ({sup 3}H)inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.

  4. Epidermal growth factor (EGF)-receptor is phosphorylated at threonine-654 in A431 cells following EGF addition

    SciTech Connect

    Whiteley, B.; Glaser, L.

    1986-05-01

    It has been shown that activation of protein kinase C by tumor-promoting phorbol diesters causes phorphorylation of the EGF-receptor at threonine-654 and is believed to thereby regulate the EGF receptor tyrosine kinase and EGF binding activity. In their present studies, /sup 32/P-labeled A431 cells were treated with and without 10 nM phorbol 12-myristate 13-acetate (PMA), or with 200 ng/ml EGF. Analysis of /sup 32/P-labeled EGF receptor tryptic phosphopeptides by reverse-phase HPLC confirmed the known effects of PMA and revealed that EGF caused phosphorylation at threonine-654 as well as various tyrosine residues. This effect occurred as early as 1 minute after EGF addition and was maximal after 5 minutes. The magnitude of the response appears to be 50% of a 15 minute treatment with 10 nM PMA. Direct measurement of diacylglycerol using an E. coli diacylglycerol kinase confirmed that EGF-stimulated phosphoinositide turnover could cause very rapid activation of protein kinase C. These results imply that protein kinase C is playing a role in negative modulation of EGF-receptor activity following EGF addition to A431 cells.

  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. 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

  7. 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.

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

    PubMed

    Tahanian, Elizabeth; Sanchez, Luis Arguello; Shiao, Tze Chieh; Roy, René; Annabi, Borhane

    2011-01-01

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

  9. Recombinant human interferon-gamma reconstitutes defective phagocyte function in patients with chronic granulomatous disease of childhood.

    PubMed Central

    Sechler, J M; Malech, H L; White, C J; Gallin, J I

    1988-01-01

    Monocytes from 19 of 30 patients with the classic phenotype of chronic granulomatous disease of childhood (CGD) responded to 3 days of treatment in culture with recombinant human interferon-gamma (rHuIFN-gamma) at 100 units/ml by producing superoxide after stimulation with phorbol 12-myristate 13-acetate. Cells from 15 of 16 patients with cytochrome b-positive CGD (15 with autosomal and 1 with X chromosome-linked inheritance) and cells from 4 of 14 patients with cytochrome b-negative CGD (13 with X chromosome-linked and 1 with autosomal recessive inheritance) responded. Subcutaneous rHuIFN-gamma (0.01-0.05 mg/m2) administered as a single dose, daily or every other day, for five or six doses to 3 patients whose phagocytes responded to rHuIFN-gamma in vitro resulted in significant improvement in phagocyte bactericidal activity against Staphylococcus aureus and increases in superoxide production. Studies on 1 patient's cells indicated the increases in superoxide production correlated with increased membrane cytochrome b. The effects of rHuIFN-gamma persisted for more than a week following cessation of therapy. Thus, we have demonstrated a partial correction in vivo of these CGD patients' phagocyte defect with rHuIFN-gamma. Moreover, the data suggest that a significant proportion of patients with CGD will respond to rHuIFN-gamma with augmentation of phagocyte microbicidal function. Images PMID:2838849

  10. 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.

  11. Myristic Acid, A Side Chain of Phorbol Myristate Acetate (PMA), Can Activate Human Polymorphonuclear Leukocytes to Produce Oxygen Radicals More Potently than PMA

    PubMed Central

    Tada, Mika; Ichiishi, Eiichiro; Saito, Rumiko; Emoto, Natsumi; Niwano, Yoshimi; Kohno, Masahiro

    2009-01-01

    Myristic acid (MyA), which is a saturated fatty acid (C14:0) and a side chain of phorbol 12-myristate 13-acetate (PMA), was examined if MyA stimulates human polymorphonuclear leukocytes (PMNs) to release oxygen radicals comparable to PMA by applying electron paramagnetic resonance (EPR)-spin-trapping method. When MyA was added to isolated human PMNs, spin adducts of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH and DMPO-OOH were time-dependently observed. The amounts of these spin adducts were larger than those of PMNs stimulated by PMA. These results clearly show that MyA is more potent agent to prime human PMNs than PMA, in a point of view of not only O2·− but also ·OH production. This fact calls attention that too much intake of MyA that is known to be contained vegetable oils can lead to crippling effect through uncontrolled production of reactive oxygen species. PMID:19902021

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

    PubMed

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-06-16

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

  13. 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.

  14. OSBP-related protein 3 (ORP3) coupling with VAMP-associated protein A regulates R-Ras activity.

    PubMed

    Weber-Boyvat, Marion; Kentala, Henriikka; Lilja, Johanna; Vihervaara, Terhi; Hanninen, Raisa; Zhou, You; Peränen, Johan; Nyman, Tuula A; Ivaska, Johanna; Olkkonen, Vesa M

    2015-02-15

    ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3-VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and β1-integrin activity were enhanced by ORP3-VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3-VAPA complexes stimulate R-Ras signaling.

  15. Tumor necrosis factor alpha induces proteins that bind specifically to kappa B-like enhancer elements and regulate interleukin 2 receptor alpha-chain gene expression in primary human T lymphocytes.

    PubMed Central

    Lowenthal, J W; Ballard, D W; Böhnlein, E; Greene, W C

    1989-01-01

    We have investigated the biochemical basis for the activation of interleukin 2 receptor alpha-subunit (IL-2R alpha) gene expression in primary human T lymphocytes by a cytokine (tumor necrosis factor alpha), a T-cell mitogen (phorbol 12-myristate 13-acetate), and the transactivator protein (Tax) from the type I human T-cell leukemia virus. Using in vivo transfection techniques specificially designed for these primary T cells in conjunction with in vitro gel retardation and DNA footprinting assays, we found that activation of the IL-2R alpha promoter by each of these agents involves the induction of nuclear proteins that specifically interact with a kappa B-like enhancer element (i.e., an element resembling the immunoglobulin kappa-chain enhancer sequence recognized by transcription factor NF-kappa B). DNA-protein crosslinking studies revealed that primary T cells express at least three different inducible DNA-binding proteins (50-55, 70-75, and 80-90 kDa) that specifically interact with this IL-2R alpha kappa B element. Images PMID:2494663

  16. Activation of the fructose 1,6-bisphosphatase gene by 1,25-dihydroxyvitamin D3 during monocytic differentiation.

    PubMed Central

    Solomon, D H; Raynal, M C; Tejwani, G A; Cayre, Y E

    1988-01-01

    Cells from the human leukemia cell line HL-60 undergo terminal monocyte-like differentiation after exposure to either the active circulating form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or phorbol 12-myristate 13-acetate. Little is known about the genes that regulate monocytic differentiation. Using clonal variant cells of HL-60 origin, we constructed a cDNA library enriched for genes that are induced by 1,25-(OH)2D3. We now report that in HL-60, the fructose 1,6-bisphosphatase (FBPase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) gene is activated during 1,25-(OH)2D3-induced monocytic differentiation. This gene encodes two closely related mRNAs; one, activated by 1,25-(OH)2D3 at an early stage of HL-60 differentiation, encodes a protein that has homology to mammalian FBPase, a key enzyme in gluconeogenesis, although it does not exhibit its classical enzymatic activity. A second mRNA is activated by 1,25-(OH)2D3 mainly in peripheral blood monocytes. This mRNA is present in kidney as a unique transcript and encodes a protein with FBPase activity. Our data also show that this FBPase-encoding mRNA can be activated during monocytic maturation since it was detected in human alveolar macrophages. Images PMID:2842796

  17. Effects of an inhaled corticosteroid, budesonide, on alveolar macrophage function in smokers.

    PubMed Central

    Bergstrand, H; Björnson, A; Blaschke, E; Brattsand, R; Eklund, A; Larsson, K; Linden, M

    1990-01-01

    Selected functions of alveolar macrophages obtained by bronchoalveolar lavage of 12 healthy smokers were examined before and after eight weeks' treatment with an inhaled glucocorticosteroid, budesonide (400 micrograms twice daily). After budesonide treatment spontaneous as well as opsonised zymosan triggered prostaglandin E2 (PGE2) secretion from harvested cells was reduced; no such reduction in opsonised zymosan triggered leukotriene B4 (LTB4) production was observed. Neither the capacity to phagocytose opsonised yeast particles nor the superoxide radical generation triggered by the calcium ionophore A23187, 4 beta-phorbol 12-myristate 13-acetate (PMA), or opsonised zymosan ex vivo were more than marginally affected by the glucocorticosteroid treatment in vivo. Lavage fluid concentrations of angiotensin converting enzyme (ACE), however, after treatment were twice those before treatment and concentrations of fibronectin were reduced to half. Albumin concentrations in lavage fluid were not affected by the glucocorticosteroid treatment. In separate experiments treatment of alveolar macrophages with 10(-7) or 10(-6) M budesonide overnight in vitro did not affect their superoxide radical or PGE2 generation but significantly blocked LTB4 release. These data indicate that inhaled gluco-corticosteroid treatment may affect synthesis or release (or both) of ACE and fibronectin by alveolar macrophages from healthy smokers whereas other functions of these cells, such as the generation of reactive oxygen derived products ex vivo, are only marginally affected. PMID:2166359

  18. The beetroot component betanin modulates ROS production, DNA damage and apoptosis in human polymorphonuclear neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Kostrzewa, Artur; Łuczak, Michał; Jagodziński, Paweł P; Baer-Dubowska, Wanda

    2012-06-01

    The aim of this study was to evaluate the effect of betanin, one of the beetroot major components, on ROS production, DNA damage and apoptosis in human resting and stimulated with phorbol 12-myristate13-acetate polymorphonuclear neutrophils, one of the key elements of the inflammatory response. Incubation of neutrophils with betanin in the concentration range 2-500 µM resulted in significant inhibition of ROS production (by 15-46%, depending on the ROS detection assay). The antioxidant capacity of betanin was most prominently expressed in the chemiluminescence measurements. This compound decreased also the percentage of DNA in comet tails in stimulated neutrophils, but only at the 24 h time point. In resting neutrophils an increased level of DNA in comet tails was observed. Betanin did not affect the activity of caspase-3, in resting neutrophils, but significantly enhanced the enzyme activity in stimulated neutrophils. The western blot analysis showed, however, an increased level of caspase-3 cleavage products as a result of betanin treatment both in resting and stimulated neutrophils. The results indicate that betanin may be responsible for the effect of beetroot products on neutrophil oxidative metabolism and its consequences, DNA damage and apoptosis. The dose and time dependent effects on these processes require further studies.

  19. CD107a as a marker of activation in chicken cytotoxic T cells.

    PubMed

    Wattrang, Eva; Dalgaard, Tina S; Norup, Liselotte R; Kjærup, Rikke B; Lundén, Anna; Juul-Madsen, Helle R

    2015-04-01

    The study aimed to evaluate cell surface mobilisation of CD107a as a general activation marker on chicken cytotoxic T cells (CTL). Experiments comprised establishment of an in vitro model for activation-induced CD107a mobilisation and design of a marker panel for the detection of CD107a mobilisation on chicken CTL isolated from different tissues. Moreover, CD107a mobilisation was analysed on CTL isolated from airways of infectious bronchitis virus (IBV)-infected birds direct ex vivo and upon in vitro stimulation. Results showed that phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin was a consistent inducer of CD107a cell surface mobilisation on chicken CTL in a 4h cell culture model. In chickens experimentally infected with IBV, higher frequencies of CTL isolated from respiratory tissues were positive for CD107a on the cell surface compared to those from uninfected control chickens indicating in vivo activation. Moreover, upon in vitro PMA+ ionomycin stimulation, higher proportions of CTL isolated from the airways of IBV-infected chickens showed CD107a mobilisation compared to those from uninfected control chickens. Monitoring of CD107a cell surface mobilisation may thus be a useful tool for studies of chicken CTL cytolytic potential both in vivo and in vitro.

  20. 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 ar