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1
Hamdaoui, O., M. Ouchefoun, and M. Zerdaoui. "Inhibition de la corrosion d'un acier au carbone par le Kemazur 1620." Revue des sciences de l'eau 13, no. 1 (April 12, 2005): 47–54. http://dx.doi.org/10.7202/705380ar.
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Dans cette étude, des mesures électrochimiques ont été utilisées pour caractériser l'efficacité inhibitrice du produit commercial Kemazur 1620, employé pour le traitement des eaux des circuits de refroidissement. L'influence de la concentration de ce composé ainsi que l'effet de la température du milieu ont été étudiés.Les courbes de polarisation ont été obtenues à l'aide d'un montage à trois électrodes. La méthode électrochimique a permis de déterminer la vitesse de corrosion en l'absence et en présence d'inhibiteur et par conséquent, le taux de protection. Ainsi, le Kemazur 1620 présente une très bonne efficacité pour une concentration de 2000 ppm. Entre 1000 et 2000 ppm, l'efficacité inhibitrice augmente de 56 à 91 %. En outre, l'efficacité inhibitrice du composé a été comparée à celle du nitrite de sodium.
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Peings, Vanessa, Albéric Andrin, Mickael Le Bechec, Sylvie Lacombe, Jérôme Frayret, and Thierry Pigot. "Couplage photocatalyse-oxydation par le ferrate (VI) pour le traitement du colorant rhodamine 6G." Revue des sciences de l’eau 30, no. 1 (June 8, 2017): 35–39. http://dx.doi.org/10.7202/1040061ar.
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Un facteur limitant de l’oxydation photocatalytique du dioxyde de titane (TiO2) sous irradiation UV est la recombinaison des électrons de la bande de conduction (e-cb) avec les trous d’électron (h+vb) à la surface de TiO2. Le couplage du ferrate(VI), Fe(VI), connu comme un oxydant respectueux de l’environnement, avec la photocatalyse UV/TiO2 pourrait conduire à une synergie oxydative de par le piégeage de e-cb par Fe(VI) et la consécutive formation de l’espèce hautement réactive Fe(V). Cette étude décrit les résultats du couplage TiO2 commercial P25 avec Fe(VI) sous forme pure ou sous forme d’une matière synthétisée dans notre laboratoire (produit solide nommé Fe(VI) matter) pour l’abattement du colorant rhodamine 6G (R6G). Les cinétiques de transformation de R6G ([R6G]0 = 10‑5 M; pH = 8,00 ± 0,05), en présence de TiO2 ([P25] = 0,1 g∙L‑1) illuminé sous UV et/ou de Fe(VI) ([Fe(VI)]0 = 10‑4 M) sont suivies par spectrophotométrie. Une synergie est mise en évidence lors du traitement de R6G par UV/TiO2/Fe(VI) pur, conduisant à une accélération de la transformation de R6G et à une minéralisation plus importante. Cependant, cet abattement n’est pas atteint lors du couplage UV/TiO2/Fe(VI) matter. Une étude de l’impact de sels inorganiques présents dans Fe(VI) matter sur l’activité photocatalytique est présentée. Le sulfate, SO42‑, et le Fe(OH)3 en particulier mènent à une forte inhibition de l’activité de TiO2. Le suivi de la production des radicaux hydroxyles (OH•) montre une inhibition physique de leur production due à la formation d’une couche de sels inorganiques à la surface de TiO2 et au piégeage de radicaux OH• dans la solution.
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Blache, Denis, Laurence Gesquière, Nadine Loreau, and Phillipe Durand. "Oxidant stress: the role of nutrients in cell-lipoprotein interactions." Proceedings of the Nutrition Society 58, no. 3 (August 1999): 559–63. http://dx.doi.org/10.1017/s0029665199000737.
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Oxidant stress is increasingly becoming an important hypothesis to explain the genesis of several pathologies, including cancer, atherosclerosis and also ageing. Beside a few rare genetic defects, dietary factors are thought to play a key role in the regulation of the production of reactive oxygenated species. An imbalance between nutrients, and in particular those involved in antioxidant status, could explain the onset of an enhanced production of free radicals. We will briefly review information concerning oxidation of lipids and lipoproteins which lead to atherothrombosis. We also present new findings supporting a role for blood platelets in generating oxidant species. New data are also described concerning the role of oxygenated derivatives of cholesterol, oxysterols, in cellular cholesterol efflux and NO production. Also, new developments relating to the influence of direct effects of free radicals on cellular cholesterol homeostasis are presented. Finally, the in vitro effects of butyrate, a natural short-chain fatty acid produced by bacterial fermentation, in the protection against free radical-mediated cytotoxicity are discussed. These data provide information on the mechanisms of dietary antioxidants in preventing oxidant stress.Résumé Au côté des rares cas d’origine génétique, les facteurs nutritionnels (déséquilibres alimentaires, déficience en nutriments antioxydants) jouent des rôles cruciaux dans la modulation de la production d’espèces actives de l’oxygène, conduisant à l’établissement d’un stress oxydant, situation métabolique de plus en plus reconnue comme susceptible d’être à l’origine de nombreuses pathologies comme les cancers, l’athérosclérose et également le vieillissement. Après avoir brièvement rappelé les données concernant l’oxydation des lipides et des lipoprotéines susceptibles de conduire au développement de l’athéro-thrombogenèse, nous présentons des données récentes et originales indiquant que les plaquettes sont en fait capables à l’instar d’autres cellules, de produire des formes actives de l’oxygène susceptibles de modifier les LDL. Des résultats originaux sont également exposés concernant l’effets des oxystérols, produits d’oxydation du cholestérol générés au cours de l’oxydation des LDL ou présents dans l’alimentation, sur deux paramètres importants comme l’efflux du cholestérol cellulaire et la production de monoxyde d’azote. De plus, des données nouvelles relatives à l’effets du stress oxydant et son inhibition par des antioxydants d’origine nutritionnelle sont exposées sur l’homéostasie du cholestérol cellulaire. Enfin, dans ce contexte, les effets potentiellement antiathérogènes d’un acide gras à courte chaîne produit par la fermentation bactérienne, le butyrate, sont décrits sur la protection de cellules en culture vis-à-vis d’un stress oxydant in vitro. Ces éléments contribuent à apporter de nouvelles informations renforçant la notion de fonctionnalité des nutriments dans la protection du stress oxydant en relation avec la pathogenèse.
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Baila, Stefano, Christian Furlan Freguia, Nicholas Iacobelli, Danielle Dunn, Joerg Schuettrumpf, Federico Mingozzi, Patricia Andrade-Gordon, and Valder R. Arruda. "Protease−Activated Receptor 2 (PAR−2) as a Novel Target To Prevent Inhibitor Formation to FIX." Blood 108, no. 11 (November 16, 2006): 763. http://dx.doi.org/10.1182/blood.v108.11.763.763.
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Abstract Direct intramuscular injection (IM) of adeno−associated viral (AAV) serotype 2 in humans with hemophilia B (HB) is a promising therapeutic strategy since muscle biopsies obtained >3 years after vector injection demonstrated stable local gene expression. However to achieve therapeutic FIX levels using AAV−2 would required hundreds IM injections. The use of alternate AAV serotypes is an attractive strategy since AAV−1 or AAV−6, resulted in a >10−fold increase in transgene levels compared to AAV−2 in large animals, but the immune response to the transgene product has been consistently demonstrated as a major limitation of this strategy. There is growing evidence that blood proteases play an important role in modulating inflammatory and immune responses through activation of PARs. Mice lacking PAR−1(−/−) or PAR−2(−/−) alleles presented amelioration of immune− or infection−mediated diseases. Here we sought to determine whether inhibition of PARs could be used as a strategy to prevent immune responses to the FIX following AAV−mediated gene transfer to skeletal muscle. We used PAR−1 and PAR−2 knockout mice on C57Bl/6 background and littermate mice received IM injection of AAV1−CMV−hFIX. At dose 5x1011vg/kg, PAR−2 (−/−) mice (n=5) exhibited circulating FIX levels of 500± 99ng/ml (8–10%) which remained stable for the duration of the experiment (10 weeks), and no antibodies for FIX were detected (n=5). In contrast, all PAR−2 (+/+) mice (n=4) developed antibodies to FIX which inhibits FIX clotting activity, as determined by Bethesda assay (2.1± 0.6 BU). However, when similar vector doses were delivered to PAR−1(−/−) or PAR−1(+/+) (n=4/genotype) mice, antibodies to FIX developed in all animals. We next tested a higher vector dose in PAR−2 models. At dose 1x1012vg/kg, PAR−2(−/−) mice(n=7) resulted in FIX levels of 1,500±353ng/ml, and again no antibodies for FIX were detected. At the same dose, 6 out of 10 mice of PAR−2 (+/+)/(+/−) developed inhibitory antibodies (1.8± 0.7 BU). Further increase in the vector dose to 5 x 1012 vg/kg resulted in the development of inhibitor to FIX in both PAR−2 (−/−) (4/11 mice, 36%) and PAR−2(+/+)/(+/−) (10/17 mice, 60%). This suggests a threshold value in the protective effect in the PAR−2 (−/−) model. We sought to assay for FIX−specific T−cell by ELISPOT assay to quantify IFN−γ secretion from splenocytes of PAR−2 (−/−) and PAR−2 (+/+) mice injected at 5x1011 or 1x1012vg/kg. No difference in IFN−γ secretion was observed between PAR−2 (−/−) and their controls. Moreover, upon repeated challenges with FIX protein following vector injection antibody to FIX was detected in only 1/4 PAR−2 (+/+) mouse and none of 5 PAR−2 (−/−). Thus, PAR−2 inhibition does not compromise the tolerance to FIX. In a different model, intravenous injection of FIX protein into normal mice upon simultaneous activation of PAR−2 by using specific agonist peptide the rates of FIX antibody formation were comparable with those of a control peptide group. Thus, PAR−2−mediating antibody formation to FIX may differ among distinct immunologic challenges. Together, these data suggest that PARs play a role in the immune response to FIX and that inhibition of PAR−2 (but not PAR−1) could be a novel target in preventing inhibitor formation in hemophilia gene therapy and potentially for protein−based therapy.
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Deans, N. L., R. D. Allison, and D. L. Purich. "Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase." Biochemical Journal 286, no. 1 (August 15, 1992): 243–51. http://dx.doi.org/10.1042/bj2860243.
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The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.
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Ohtsuki, M., and J. Massagué. "Evidence for the involvement of protein kinase activity in transforming growth factor-beta signal transduction." Molecular and Cellular Biology 12, no. 1 (January 1992): 261–65. http://dx.doi.org/10.1128/mcb.12.1.261.
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Transforming growth factor-beta 1 (TGF-beta 1) rapidly increases the expression of junB transcription factor and plasminogen activator inhibitor-1 (PAI-1) and prevents the cell cycle-dependent phosphorylation of the RB retinoblastoma susceptibility gene product during late G1 phase in Mv1Lu lung epithelial cells. These responses are shown in this report to be blocked by the potent serine/threonine protein kinase inhibitor, H7, added with TGF-beta 1. Added alone, H7 does not alter the basal junB or PAI-1 mRNA levels, the deposition of PAI-1 into the extracellular matrix, or the phosphorylation of RB in late G1 phase, suggesting that this inhibitor does not have a general nonspecific effect on the cell. The analogs H8 and H9, which are preferential inhibitors of cyclic nucleotide-dependent protein kinases, are fivefold less potent than H7 as inhibitors of the TGF-beta response. The PAI-1 response to TGF-beta 1 is not affected by the simultaneous addition of staurosporine, which is a protein kinase C inhibitor, or by the prolonged preincubation of cells with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C. The results suggest the possibility that H7 and its analogs block various early TGF-beta responses by inhibiting a protein serine/threonine kinase(s).
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Ohtsuki, M., and J. Massagué. "Evidence for the involvement of protein kinase activity in transforming growth factor-beta signal transduction." Molecular and Cellular Biology 12, no. 1 (January 1992): 261–65. http://dx.doi.org/10.1128/mcb.12.1.261-265.1992.
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Transforming growth factor-beta 1 (TGF-beta 1) rapidly increases the expression of junB transcription factor and plasminogen activator inhibitor-1 (PAI-1) and prevents the cell cycle-dependent phosphorylation of the RB retinoblastoma susceptibility gene product during late G1 phase in Mv1Lu lung epithelial cells. These responses are shown in this report to be blocked by the potent serine/threonine protein kinase inhibitor, H7, added with TGF-beta 1. Added alone, H7 does not alter the basal junB or PAI-1 mRNA levels, the deposition of PAI-1 into the extracellular matrix, or the phosphorylation of RB in late G1 phase, suggesting that this inhibitor does not have a general nonspecific effect on the cell. The analogs H8 and H9, which are preferential inhibitors of cyclic nucleotide-dependent protein kinases, are fivefold less potent than H7 as inhibitors of the TGF-beta response. The PAI-1 response to TGF-beta 1 is not affected by the simultaneous addition of staurosporine, which is a protein kinase C inhibitor, or by the prolonged preincubation of cells with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C. The results suggest the possibility that H7 and its analogs block various early TGF-beta responses by inhibiting a protein serine/threonine kinase(s).
8
Zhang, Wenlin, Gongwei Li, Fei Jin, Yu Huo, Tengfei Sun, and Chunli Li. "Synthesis and characterization of an ionic liquid–carboxylic acid copolymer scale inhibitor and its scale inhibition performance." Water Supply 19, no. 5 (January 22, 2019): 1463–72. http://dx.doi.org/10.2166/ws.2019.011.
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Abstract A phosphorus-free scale inhibitor (ionic liquid–carboxylic acid copolymer) was successfully synthesized by the reaction of 1-sulfobutyl-3-vinylimidazolium hydrogen sulfate (SVIS) and acrylic acid (AA). The structure of the product was characterized by Fourier transform infrared spectroscopy (FTIR), hydrogen nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR). Then the scale inhibition efficiency of 1-sulfobutyl-3-vinylimidazolium hydrogen sulfate-acrylic acid (SVIS-AA) copolymer against CaCO3 and CaSO4 was determined. The results indicated that SVIS-AA copolymer showed better scale inhibition efficiency than poly (acrylic acid) (PAA). After that, the effects of temperature and Ca2+ concentration on the scale inhibition efficiency against CaCO3 were studied. Results showed that when the temperature reached 90 °C, the scale inhibition efficiency could still remain 91% at a concentration of 18 mg L−1. When the concentration of Ca2+ reached 1,200 mg L−1, the scale inhibition efficiency could remain 70% at a concentration of 20 mg L−1. At last, the effect of SVIS-AA copolymer on the morphologies of CaCO3 and CaSO4 scale was studied by scanning electron microscopy (SEM) and X-ray diffraction (XRD).
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Xiao, Guang-Hui, Ryan Gallagher, Justin Shetler, Kristine Skele, Deborah A. Altomare, Richard G. Pestell, Suresh Jhanwar, and Joseph R. Testa. "The NF2 Tumor Suppressor Gene Product, Merlin, Inhibits Cell Proliferation and Cell Cycle Progression by Repressing Cyclin D1 Expression." Molecular and Cellular Biology 25, no. 6 (March 15, 2005): 2384–94. http://dx.doi.org/10.1128/mcb.25.6.2384-2394.2005.
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ABSTRACT Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.
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Benallaoua, S., P. Nguyen Van, M. P. De Meo, J. Coulon, G. Dumenil, and R. Bonaly. "Recherches sur le mode d'action d'un antifongique non polyénique (désertomycine) produit par une souche de Streptomyces spectabilis." Canadian Journal of Microbiology 36, no. 9 (September 1, 1990): 609–16. http://dx.doi.org/10.1139/m90-106.
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A metabolite with antifungal activity, of non polyenic macrolide structure, was extracted and purified from the culture supernatant of a soil-isolated Streptomyces spectabilis strain, BT 352. This product was found to be related to (or being) desertomycin. Six yeast and five filamentous fungus strains were used to determine minimum concentration of the metabolite that inhibits growth by 80% (IMC); it was established at 50 μg/mL for the fungi and at 100 μg/mL or more for the yeasts tested. Short-term genotoxicity tests showed no antifungal effect on the bacterial genome, and desertomycin at concentration levels of 100 μg/mL or more affected protein synthesis. The antifungal metabolite had no immediate inhibiting effect upon yeast respiration, even at high concentrations; however, the respiration activity of cells grown in the presence of subinhibiting doses and collected during their growth phase was reduced by as much as 40%. Saccharomyces uvarum spheroplast regeneration in a liquid medium containing desertomycin was inhibited at doses fivefold weaker than the IMC determined with intact cells. Contrary to amphotericin B, desertomycin subinhibiting doses do not modify, and if so lightly, the yeast latent phase or the spheroplast wall regeneration phase, thus indicating a fungicidal action. Moreover, following a 30-min contact with desertomycin subinhibiting and inhibiting doses, yeasts liberated potassium in large amounts, indicating that plasma membranes were affected. Key words: Streptomyces spectabilis, desertomycin, antifungal activity, genotoxicity, yeast. [Translated by the journal]
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Dalaklioglu, Selvinaz, Pinar Sahin, Ece Gungor Ordueri, Ciler Celik-Ozenci, and Arda Tasatargil. "Potential Role of Poly(ADP-Ribose) Polymerase (PARP) Activation in Methotrexate-Induced Nephrotoxicity and Tubular Apoptosis." International Journal of Toxicology 31, no. 5 (August 22, 2012): 430–40. http://dx.doi.org/10.1177/1091581812457430.
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Nephrotoxicity is one of the serious dose-limiting complications of methotrexate (MTX) when used in the treatment of various malignancies and nononcological diseases. The aim of this study was to investigate the role of poly(adenosine diphosphate ribose) polymerase (PARP) activity in MTX-induced nephrotoxicity. Rats were divided into 4 groups as control, MTX treated (MTX, 7 mg/kg per d, intraperitoneally [ip], once daily for 3 consecutive days), MTX plus 1,5-isoquinelinediol (ISO, a PARP inhibitor, 3 mg/kg per d, i.p.) treated, or ISO treated. Histopathology of kidneys was evaluated by light microscopy. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay was used to analyze apoptosis in kidney sections. Blood urea nitrogen (BUN), serum creatinine, and urinary N-acetyl-β-d-glucosaminidase (NAG) were used as biochemical markers of MTX-induced renal injury. Our results showed that MTX administration significantly increased BUN, serum creatinine, and urinary NAG levels. The PARP-1 and PAR (a product of PARP activity) expression and apoptotic cell death were also markedly increased in renal tubules after MTX administration. The ISO treatment attenuated MTX-induced renal injury, as indicated by BUN and serum creatinine levels, urinary NAG excretion, and renal histology. The PARP inhibitor treatment reduced PARP-1 and PAR expression to levels similar to that of controls. These results revealed that ISO may have a protective effect against the nephrotoxic effects of MTX by inhibiting PARP activation. This is the first study that demonstrates the role of PARP activation in MTX-induced nephrotoxicity and tubular apoptosis.
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Strehaiano, Pierre, M. Mota, and Gérard Goma. "Inhibitions non conventionnelles des croisssances levuriennes : effets interspécifiques et essais de levée d'inhibition." OENO One 19, no. 2 (June 30, 1985): 97. http://dx.doi.org/10.20870/oeno-one.1985.19.2.1322.
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<p style="text-align: justify;">Les phénomènes d'inhibition observés en fermentation alcoolique levurienne sont imputables à l'alcool mais aussi à des co-métabolites produits par la levure.</p><p style="text-align: justify;">Les aptitudes de quatre souches à inhiber leur propre développement ainsi que celui des autres souches sont discutées.</p><p style="text-align: justify;">Nous montrons qu'il est également possible d'éliminer ces inhibiteurs par adsorption sur divers adjuvants, sans toutefois parvenir à une réactivation complète.</p><p style="text-align: justify;">+++</p><p style="text-align: justify;">Along with ethanol, secondary products of yeast metabolism inhibit the alcoholic fermentation.</p><p style="text-align: justify;">The abilities of four strains to inhibit their own development likewise that of others strains are discussed.</p><p style="text-align: justify;">We demonstrate the possibility to eliminate these inhibitors by adsorption on different materials, howewer whithout succeeding in an entire reactivation.</p>
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Ménard, Luc, Michel Laviolette, and Pierre Borgeat. "Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol-2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes." Canadian Journal of Physiology and Pharmacology 70, no. 6 (June 1, 1992): 808–13. http://dx.doi.org/10.1139/y92-108.
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We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol-2-yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or forrnyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its Ω-oxidation products, and 6-trans-isomers with IC50 values of 2.8–4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 μM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its Ω-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5–11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3–0.9, 3.7–5.3, and 8.5–17.3 nM, respectively. In neutrophils primed with granulocyte – macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7–8.7 nM. At the concentration of 1 μM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.Key words: 5-lipoxygenase inhibition, colony-stimulating factor, leukotriene, neutrophil, eosinophil, monocyte, macrophage, platelet-activating factor.
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Goolyam Basavaraj, Manjunath, and Sriram Krishnaswamy. "The Affinity of Factor X for the Intrinsic Xase Is Determined By Exosite Interactions Between the Substrate and the Enzyme Complex." Blood 126, no. 23 (December 3, 2015): 1065. http://dx.doi.org/10.1182/blood.v126.23.1065.1065.
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Abstract Distinctive protein substrate specificities are achieved by the membrane-bound enzyme complexes of coagulation, despite the comparable active site geometries of the structurally homologous, trypsin-like coagulation proteinases. In contrast to long-standing ideas in the field, there is increasing evidence for the prevalence of a binding strategy in which binding specificity for the protein substrate is achieved by exosite interactions between sites on the substrate removed from the cleavage site and sites on the enzyme complex distant from the active site of the proteinase. This is evident as a marked disconnect between the kinetic signatures of reversible inhibition of proteinase active site function versus protein substrate cleavage. Despite the widespread clinical interest in factor X (FX) activation by intrinsic Xase composed of the activated antihemophilic factors VIIIa and IXa (FIXa) assembled on membranes, the molecular basis for substrate specificity of the enzyme complex remains poorly understood. This partly reflects the difficulties in assessing active site function of FIXa within intrinsic Xase in a kinetically informative way and in the absence of added alcohols. Here, we exploit the observation that the fluorogenic peptidyl substrate H-D-Leu-Phenylglycine-Arg-aminomethylcoumarin (PF-3688) exhibits an experimentally accessible Km for FIXa or intrinsic Xase (220 µM and 560 µM, respectively) in the absence of alcohols. We have probed the contribution of active site interactions to intrinsic Xase function using the reversible inhibitor 4-aminobenzamidine (PAB) along with recombinant variants of FX. As expected but never previously shown, PAB acted as a classical competitive inhibitor (Ki ~ 100 µM) of PF-3688 cleavage by intrinsic Xase. The signature features of classical competitive inhibition are that increasing concentrations of inhibitor systematically increase Km without affecting Vmax and that inhibition can be completely overcome at saturating substrate concentrations. This implies that PAB and PF-3688 bind in a mutually exclusive way to the active site of FIXa within intrinsic Xase and is fully expected given the established ability of PAB to reversibly occlude the primary specificity pocket of trypsin-like serine proteinases and the limited way that PF3688 is expected to engage the active site of FIXa. On the other hand, PAB was found to act as a noncompetitive inhibitor for FX activation with increasing concentrations of inhibitor decreasing the Vmax for FXa formation and minimally affecting the Km for FX. In contrast to the peptidyl substrate, this indicates that binding interactions between FX and the active site of FIXa within intrinsic Xase contributes in a minor way to protein substrate affinity. Because FX must dock at the active site for it to be cleaved, the data suggest a multistep binding pathway in which the initial interaction between FX and intrinsic Xase occurs at exosites distant from the active site followed by active site docking and bond cleavage. The initial binding step dominates substrate affinity and the second docking step contributes to the Vmax. These ideas were tested using FXS195A as an alternate protein substrate in which the catalytic triad Serine is replaced with Alanine to yield a catalytically inactive product, and using FXR15Q in which the P1 Arginine is replaced with Glutamine to yield an uncleavable substrate that cannot engage the active site. Both variants were found to act as classical competitive inhibitors of FX activation with Ki ~ Km. Thus, competitive inhibition of FX activation requires the occlusion of exosite binding by FX and can be accomplished even by a FX variant that cannot engage the active site of FIXa within intrinsic Xase. These findings establish exosite binding as a primary determinant of the affinity and binding specificity of FX for intrinsic Xase. Whether this is entirely accomplished by protein-protein contacts between the substrate and the enzyme complex or also involves contributions from FX binding to membranes remains to be established. Our findings provide new mechanistic insights into intrinsic Xase function that might be exploited to modulate FXa formation through the intrinsic pathway of coagulation in human disease. Disclosures No relevant conflicts of interest to declare.
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Doudican, Nicole A., Shih Ya Wen, Amitabha Mazumder, and Seth J. Orlow. "Identification of the Black Tea Polyphenol Theaflavin-3, 3'-Digallate From Screen of Natural Product Inducers of Endoplasmic Reticulum Stress." Blood 120, no. 21 (November 16, 2012): 5021. http://dx.doi.org/10.1182/blood.v120.21.5021.5021.
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Abstract Abstract 5021 Background: A distinguishing characteristic of myeloma plasma cells is the large quantity of paraprotein that they synthesize and secrete, rendering them especially sensitive to the effects of endoplasmic reticulum (ER) stress. Consistent with this notion, the proteasome inhibitor bortezomib disrupts protein equilibrium in the ER by preventing misfolded proteins from being properly degraded. Given the clinically validated importance of targeting ER stress mediated pathways in the treatment of multiple myeloma (MM), we sought to identify natural products that modulate pathways known to be an effective therapeutic target for MM for potential use to inhibit progression of asymptomatic MM to symptomatic MM without the limiting side effects of current targeted therapies. Methods: Using decreased protein processing in the secretory pathway as a measurable hallmark of ER stress, our screen employed the naturally secreted Gaussia luciferase (Gluc) as a reporter that can be easily monitored through extracellular release of luciferase activity in real time. KMS11 and ARP-1 MM cells expressing Gluc were exposed to compounds in our natural products library in order to identify those which potentially induce ER stress as measured by inhibition of Gluc secretion. The growth inhibitory activity of theaflavin-3, 3'–digallate (TF3) was further characterized by MTS assay. Mechanistic studies of ER stress related pathways including the unfolded protein response (UPR) and apoptotic cascades were analyzed by standard Western blotting techniques. Results: Our screen identified the black tea polyphenol TF3 as a significant inhibitor of GLUC secretion in ARP-1 and KMS-11 cells. TF3 at 0. 5 μM inhibits GLUC secretion by 73 and 68% in ARP-1 and KMS-11 cells, respectively. This inhibition observed is on par with that observed for bortezomib and tunacamycin (a well known inducer of ER stress). TF-3 effectively inhibits cellular proliferation and induces apoptosis in a panel of MM cell lines at physiologically achievable concentrations. Apoptotic induction is at least partially mediated by ER stress mediated pathways as upregulation of the protein chaperone HSP90 and phosphorylation of eIF2-a, a key mediator of the UPR pathway, occurs prior to caspase and PARP cleavage. Conclusions: Our results suggest that TF-3 inhibits protein secretion and MM cell growth through promotion of ER stress generating mechanisms. Based upon these promising results, further mechanistic evaluation and characterization of this safe, natural product as a prophylactic agent in the treatment of asymptomatic conditions like monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) is warranted. Disclosures: No relevant conflicts of interest to declare.
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Colucci, Mario, Loreto Gesualdo, Pasqualina Montemurro, Luca G. Cavallo, Massimo Conese, Elisa Mascolo, Elena Ranieri, Salvatore Di Paolo, Francesco P. Schena, and Nicola Semeraro. "Cultured Human Mesangial Cells Produce both Type 1 and Type 2 Plasminogen Activator Inhibitors." Thrombosis and Haemostasis 74, no. 06 (1995): 1516–20. http://dx.doi.org/10.1055/s-0038-1649975.
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SummaryCultured human mesangial cells (HMC) derived from normal kidneys have been shown to synthesize tissue-type plasminogen activator (t-PA) and excess amounts of PA inhibitor type 1 (PAI-1). Conflicting results have been obtained concerning the production of urokinase-type PA (u-PA) and efforts to show PA inhibitor 2 (PAI-2) met with failure. We evaluated the fibrinolytic profile of cultured HMC lines obtained from 12 patients with renal carcinoma and one cadaveric kidney donor. Subconfluent cells (third passage) were incubated overnight in serum-free medium. t-PA, u-PA, PAI-1 and PAI-2 antigens were assayed by ELISA methods and PA and PAI activities by amidolytic methods both in conditioned medium (CM) and cell extracts (CE). Besides PAI-1, PAI-2 antigen was detected in all but one HMC lines. At variance with the former, which was largely released in the culture medium, PAI-2 was mainly cell-associated. t-PA antigen was found in all but two cell lines while u-PA antigen was detected in relatively high concentrations in 8 cell lines. PA activity, identified as u-PA by functional and immunological criteria, was measured in CM of six of the eight u-PA producing cell lines, whereas PAI activity was undetectable or very low in CM of all cell lines, suggesting that PAI-1 was largely inactive. Functional assays of cell extracts demonstrated the presence of PA activity, again identified as u-PA, only in samples (five lines) containing u-PA antigen in excess over PAI-2. PAI activity was found instead in the extracts in which the inhibitor was higher than the activator (six lines) and was identified as PAI-2, as it inhibited u-PA but not single-chain t-PA and was neutralized by a polyclonal anti-PAI-2 antibody. The heterogeneous fibrinolytic pattern of HMC lines was confirmed by mRNA analysis of three representative lines. Results were similar when HMC lines at passage five were used, except that the u-PA content was significantly reduced both in CM and CE. These findings indicate that the fibrinolytic profile of cultured HMC is more complex than previously reported. The production of large amounts of PAI-2 may represent an additional control mechanism of proteinase activity.
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Robbie, Linda, Seonag Kinghorn, Rachel Exley, Nuala Booth, and Helen Ritchie. "Monocyte Plasminogen Activator Inhibitor 2 (PAI-2) Inhibits u-PA-mediated Fibrin Clot Lysis and Is Cross-linked to Fibrin." Thrombosis and Haemostasis 81, no. 01 (1999): 96–103. http://dx.doi.org/10.1055/s-0037-1614425.
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SummaryPlasminogen activator inhibitor 2 (PAI-2) is a major product of activated human monocytes. Here we show that monocytes inhibited u-PA- but not t-PA-mediated fibrinolysis, by secreting PAI-2 into an overlying fibrin clot. Extracts of arterial and venous human thrombi were found to contain active PAI-2. PAI-2 was cross-linked to fibrin in a reaction catalyzed by two major transglutaminases (TG), tissue TG and factor XIII. The activity of PAI-2 was not affected by such cross-linking. Cross-linking of PAI-2 to fibrin was inhibited by Tridegin, a specific inhibitor of TG, and also by EDTA and iodoacetamide. The use of competitive peptides mimicking the loop between helices C and D of PAI-2 identified Gln 83 and 86 as residues important in cross-linking. This study defines a mechanism by which PAI-2 is localized to fibrin, where it acts as an effective inhibitor of u-PA-mediated fibrinolysis.
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Hazra, Sugata, Yagna P. R. Jarajapu, Li Liu, Sergio Caballero, Valerie Stepps, Ashay Bhatwadekar, Michael E. Boulton, Paul J. Higgins, Stephen H. Bartelmez, and Maria B. Grant. "Inhibition of Plasminogen Activator Inhibitor (PAI)-1 Corrects Diabetic CD34+ Dysfunction." Blood 116, no. 21 (November 19, 2010): 1601. http://dx.doi.org/10.1182/blood.v116.21.1601.1601.
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Abstract Abstract 1601 Objective: The dysfunction of human diabetic CD34+ endothelial progenitor cells limits their utility in autologous cell therapy for vascular complications. Previously, we showed that transient inhibition of transforming growth factor-beta 1 (TGF-β1) enhances vascular reparative function of human CD34+ cells isolated from diabetics (Bhatwadekar et al, 2010). Expression of PAI-1, the major gene product of TGF-β1 activation, is increased by high glucose and insulin exposure in endothelial cells and PAI-1 has been shown to be increased in the serum of diabetics. We asked whether the beneficial effects of TGF-β1 blockade on CD34+ cells function were mediated by inhibition of PAI-1 and whether blocking of PAI-1 could correct diabetes associated dysfunction of these cells. Research Design and Methods: Plasma determinations of PAI-1 and TGF-β1 (both measured by ELISA) were compared in type 2 (n=17) and type 1 (n=7) diabetic patients. CD34+ cells from these individuals were isolated and analyzed for cell survival (in the presence and absence of growth factors), cell proliferation, cell cycle analysis and migration. The effect of TGF-β1 phosphorodiamidate morpholino oligomers (PMO) treatment on PAI-1 level was determined in CD34+ cells. In CD34+ cells, PAI-1 was blocked using either lentivirus expressing PAI-1 shRNA or PAI-1 siRNA. In vivo homing ability of PAI-1 inhibited CD34+ cells was assessed using an ocular model of ischemia/reperfusion (I/R) Injury. Results: Plasma PAI-1 level was increased in type 2 diabetic patients compared to type 1 (p<0.05) and directly correlated with TGF-β1 plasma levels (r= 0.44). TGF-β1 PMO treatment resulted in a reduction of PAI-1 mRNA expression (p=0.0018 in diabetic, p=0.05 in non-diabetic). PAI-1 blockade promoted EPC proliferation in vitro and bypassed the inhibitory effect of TGF-β1 on cell survival (p<0.001) even in the absence of growth factors. PAI-1 blockade enhanced the migration of these cells in response to SDF-1α in (p<0.01) compared to cells treated with scrambled siRNA and improved the in vivo re-endothelialization by CD34+ cells in the I/R model. Conclusions: Our results suggest that the cytostatic activity of TGF-β1 in CD34+ cells is mediated largely through PAI-1. Blocking PAI-1 corrects multiple defects in CD34+ cells from type 2 diabetic patients. This approach may offer a promising therapeutic strategy for restoring vascular reparative function in diabetic cells and facilitate their use in autologous cell therapy. Disclosures: No relevant conflicts of interest to declare.
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Molle, Virginie, Robert C. Reynolds, Luke J. Alderwick, Gurdyal S. Besra, Alain J. Cozzone, Klaus Fütterer, and Laurent Kremer. "EmbR2, a structural homologue of EmbR, inhibits the Mycobacterium tuberculosis kinase/substrate pair PknH/EmbR." Biochemical Journal 410, no. 2 (February 12, 2008): 309–17. http://dx.doi.org/10.1042/bj20071384.
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EmbR is a transcriptional regulator that is phosphorylated by the cognate mycobacterial STPK (serine/threonine protein kinase) PknH. Recent studies demonstrated that PknH-dependent phosphorylation of EmbR enhances its DNA-binding activity and activates the transcription of the embCAB genes encoding arabinosyltransferases, which participate in arabinan biosynthesis. In the present study, we identified a genomic region of 4425 bp, which is present in Mycobacterium tuberculosis CDC1551, but absent from M. tuberculosis H37Rv, comprising the MT3428 gene, which is homologous with embR. Homology modelling of the MT3428 gene product illustrated its close relationship (56% identity) to EmbR, and it was hence termed EmbR2. In marked contrast with EmbR, EmbR2 was not phosphorylated by PknH, although it is a substrate of other M. tuberculosis kinases, including PknE and PknF. Tryptophan fluorescence emission of EmbR2 was monitored in the presence of three different PknH-derived phosphopeptides and demonstrated that EmbR2 binds to at least two of the threonine sites known to undergo autophosphorylation in PknH. We observed that the capacity of EmbR2 to interact physically with PknH without being phosphorylated was a result of EmbR2-mediated inhibition of kinase activity: incubation of PknH with increasing concentrations of EmbR2 led to a dose–response inhibition of the autokinase activity, similarly to O6-cyclohexylmethylguanine, a known inhibitor of eukaryotic cyclin-dependent kinases. Moreover, EmbR2 inhibited PknH-dependent phosphorylation of EmbR in a dose-dependent manner. Together, these results suggest that EmbR2 is a regulator of PknH activation, thus directly participating in the control of the PknH/EmbR pair and potentially in mycobacterial physiology/virulence of M. tuberculosis CDC1551.
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Cohen, David, Patrick J. Brennwald, Enrique Rodriguez-Boulan, and Anne Müsch. "Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton." Journal of Cell Biology 164, no. 5 (February 23, 2004): 717–27. http://dx.doi.org/10.1083/jcb.200308104.
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Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.
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Lafont, M. C., N. Pebere, F. Moran, and P. Bleriot. "Inhibition de la corrosion d'un acier au carbone par des produits dérivés de phosphonates en association avec des sels de zinc." Revue des sciences de l'eau 6, no. 1 (April 12, 2005): 97–112. http://dx.doi.org/10.7202/705168ar.
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Dans cette étude, des mesures électrochimiques ont été utilisées pour caractériser l'efficacité inhibitrice de produits dérivés de phosphonates associés à des sels de zinc, employés pour le traitement des eaux des circuits de refroidissement. L'influence de la concentration de cette formulation ainsi que l'effet du pH du milieu ont été étudiés. Les courbes courant-tension stationnaires et les diagrammes d'impédance électrochimique ont été obtenus avec des électrodes tournantes en acier au carbone dans une solution de chlorure de sodium à 200 mg l-1. Ce milieu a été choisi car sa faible conductivité électrique est proche de celle rencontrée dans les eaux naturelles. La méthode stationnaire (relevé des courbes courant-tension) a permis de déterminer la vitesse de corrosion en l'absence et en présence de l'inhibiteur et par conséquent, le taux de protection. Ainsi, le composé présente une très bonne efficacité dès les faibles concentrations (50 mg · l-1). Entre 50 et 200 mg · l-1, l'efficacité inhibitrice augmente de 95 à 98 %. Pour la concentration de 100 mg · l-1 il est efficace dans un large domaine de pH (de 5,5 à 9). Cette efficacité apparaît légèrement supérieure à pH = 7 et à pH = 8. Les valeurs des résistances de polarisation mesurées à partir des diagrammes d'impédance confirment les résultats obtenus à partir des courbes stationnaires. En outre, l'efficacité inhibitrice du composé a été comparée à celle de produits déjà testés au Laboratoire pour des utilisations identiques. Le chlorure de zinc, le monofluorophosphate de zinc et l'association d'une amine grasse et d'acide phosphonique présentent des efficacités moindres que le mélange testé ici à base de produits dérivés de phosphonates associés aux sels de zinc.
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Ismail, Abdul R., Noorul M. N. A. Mohd, Nurul F. Basir, Jeffrey O. Oseh, Issham Ismail, and Shafeeg O. Blkoor. "Improvement of rheological and filtration characteristics of water-based drilling fluids using naturally derived henna leaf and hibiscus leaf extracts." Journal of Petroleum Exploration and Production Technology 10, no. 8 (September 29, 2020): 3541–56. http://dx.doi.org/10.1007/s13202-020-01007-y.
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Abstract Biodegradable additives are required to minimize the environmental hazards from drilling fluid wastes. This study explores the feasibility of the applications of henna leaf extracts (HLE) and hibiscus leaf extracts (HBLE) as ecological benign products in water-based drilling fluids (WBDFs). Rheological and filtration characterizations were carried out on the WBDFs to detect the effects of different concentrations (1, 2, 10, 20, 30, and 40 g) of these plant extracts at 78 and 300 °F. The results of 1 and 2 g of the plant extracts were compared with those of low-viscosity polyanionic cellulose (PAC LV). Compatibility test was carried out using 25 g/L of the green additives on base fluid (A-0), and the swelling rate of sodium bentonite in distilled water was also considered using 1, 10, and 20 g of the green additives. The findings showed that HLE and HBLE significantly reduced the filtrate loss between 62% and 67% and between 64% and 76%, respectively, and improved the rheological characteristics of the WBDF system between 10 and 40 g. PAC LV showed a greater effect on the rheological properties than the green additives in equal amounts (1 and 2 g), but it exhibited flat high and progressive gels which can lead to mechanical pipe sticking. The test data also showed that the inclusion of HLE and HBLE in the WBDF demonstrated larger impact on the mud cake than PAC LV. The cake thickness of the WBDF was reduced in the following order: 30–32% (by HLE), 32–33% (by HBLE), and 24–27% (by PAC LV). This interprets the outstanding filtration characteristics of green additives. Further, compatibility test data confirmed that the green additives are compatible with the other base fluid additives and the swelling behavior of sodium bentonite verified that the green plants are effective in inhibiting bentonite swelling. Here, the Henna extracts displayed higher inhibition property than the Hibiscus product. Notwithstanding, both products showed excellent inhibition property and a strong viscosity enhancing effect on the WBDF system.
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Berger, Patrick, J. Manuel Tunon-De-Lara, Jean-Pierre Savineau, and Roger Marthan. "Selected Contribution: Tryptase-induced PAR-2-mediated Ca2+signaling in human airway smooth muscle cells." Journal of Applied Physiology 91, no. 2 (August 1, 2001): 995–1003. http://dx.doi.org/10.1152/jappl.2001.91.2.995.
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Tryptase, the major mast cell product, is considered to play an important role in airway inflammation and hyperresponsiveness. Tryptase produces different, sometimes opposite, effects on airway responsiveness (bronchoprotection and/or airway contraction). This study was designed to examine the effect of human lung tryptase and activation of protease-activated receptor (PAR)-2 by synthetic activated peptide (AP) SLIGKV-NH2 on Ca2+ signaling in human airway smooth muscle (HASM) cells. Immunocytochemistry revealed that PAR-2 was expressed by HASM cells. Tryptase (7.5–30 mU/ml) induced a concentration-dependent transient relative rise in cytoplasmic Ca2+ concentration ([Ca2+]i) that reached 207 ± 32 nM ( n = 10) measured by indo 1 spectrofluorometry. The protease inhibitors leupeptin or benzamidine (100 μM) abolished tryptase-induced [Ca2+]iincrease. Activation of PAR-2 by AP (1–100 μM) also induced a concentration-dependent transient rise in [Ca2+]i, whereas the reverse peptide produced no effect. There was a homologous desensitization of the [Ca2+]i response on repeated stimulation with tryptase or AP. U-73122, a specific phospholipase C (PLC) antagonist, xestospongin, an inositol trisphosphate (IP3)-receptor antagonist, or thapsigargin, a sarcoplamic Ca2+-ATPase inhibitor, abolished tryptase-induced [Ca2+]iresponse, whereas Ca2+ removal, in the additional presence of EGTA, had no effect. Calphostin C, a protein kinase C inhibitor, increased PAR-2 [Ca2+]i response. Our results indicate that tryptase activates a [Ca2+]iresponse, which appears as PAR-2 mediated in HASM cells. Signal transduction implicates the intracellular Ca2+ store via PLC activation and thus via the IP3 pathway. This study provides evidence that tryptase, which is increasingly recognized as an important mediator in airway inflammation and hyperresponsiveness, is also a potent direct agonist at the site of airway smooth muscle.
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Kasari, Villu, Kristi Kurg, Tõnu Margus, Tanel Tenson, and Niilo Kaldalu. "The Escherichia coli mqsR and ygiT Genes Encode a New Toxin-Antitoxin Pair." Journal of Bacteriology 192, no. 11 (March 16, 2010): 2908–19. http://dx.doi.org/10.1128/jb.01266-09.
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ABSTRACT Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells.
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Loh, Yen Siew, George Li, Kei Fan, Iyad Ahmed, Basil Roufogalis, and Daniel Sze. "Kaempferide Targets Side Population, the Putative Cancer Stem Cell, In Myeloma and Induced Apoptosis In Dose-Dependant Manner." Blood 116, no. 21 (November 19, 2010): 5029. http://dx.doi.org/10.1182/blood.v116.21.5029.5029.
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Abstract Abstract 5029 Multiple myeloma (MM), cancer of the plasma cells, remains incurable despite advancement in therapeutic regiments. Studies showed that the ‘side population (SP)’, a subpopulation enriched with CSC in various cancers, has higher drug transporter activity than the bulk tumor; suggesting the reason why these cells survived despite post-chemotherapy. The increasing evidence of cancer stem cells (CSC) suggests that anti-cancer drugs targeting this subpopulation may lead to eradication of the root of cancer recurrence. In this study, we explored the capability of kaempferide (KFD), a flavanoid in propolis (bee glue) that can reverse drug transporter activity, to combat SP cells in myeloma. KFD was one of the compounds in Brazilian propolis that reduced SP percentage in myeloma cell lines. We report for the first time that KFD is able to induce apoptosis in the SP cells in myeloma. The ability of KFD to inhibit growth of unfractionated KMS-11 cells was first investigated. Parthenolide (PAR), a natural product shown to inhibit growth of putative CSC in acute and chronic myelogenous leukemia was included as control. Unfractionated cells were seeded at 20000 cells per well in a 96 well plate. After 24h of incubation with various concentration of KFD, PAR, and DMSO (vehicle control), MTS solution was added and absorbance at 490 nm was determined. The IC50 of KFD and PAR in KMS-11 cells was 26 μM and 5 μM with 95% confidence intervals between 17.7 to 33.3 μM and 4.0 to 5.8 μM respectively. This is shown in the dose-response curve in figure 1A. No significant growth inhibition was observed in DMSO (0.1% to 0.5 % v/v) treated cells. We then examined if the KFD causes apoptosis in the sorted-SP cells. Sorted-SP cells were treated with 26 μM KFD, 5 μM PAR, or 0.2% v/v DMSO as mentioned above. After 1, 3, and 6 hour of incubation, cells were harvested and stained with Annexin V and propidium iodide and then analyzed using FACS Calibur. Result showed that percentage of Annexin V+ apoptotic cells in KFD-treated cells increased in a time series manner (1, 3, 6 h) (figure 1B). Because KFD was reported to be capable to reverse the activity of drug efflux transporter, further studies to investigate the synergistic effect of KFD with conventional drugs to treat myeloma will be carried out. Alternative medicines employing natural products have become increasingly sought after when conventional drugs cause immense side effects. Component in propolis that was reported to have anti-cancer properties, has low toxicity at high concentration, and spare normal cells, appeals to be a potential compound to combat myeloma. Exploitation of KFD that has the dual effect to induce apoptosis in putative CSC in myeloma and reverse drug transporter activity offers great opportunity in cancer drug development and future clinical trials in patients with myeloma. A B Annexin V-FITC (FL-1) Propidium iodied (FL-3) Figure 1. (A) Growth inhibition by KFD and PAR after 24h of incubation. Experiment was repeated at least 3 times. (B) KFD induced apoptosis in KMS-11 in a time-series manner. Cells were treated with 26 μM KFD or 5 μM PAR for 1, 3, and 6 hour and was stained with Annexin V and propidium iodide. KFD PAR Figure 1. (A) Growth inhibition by KFD and PAR after 24h of incubation. Experiment was repeated at least 3 times. (B) KFD induced apoptosis in KMS-11 in a time-series manner. Cells were treated with 26 μM KFD or 5 μM PAR for 1, 3, and 6 hour and was stained with Annexin V and propidium iodide. . / KFD . / PAR Disclosures: No relevant conflicts of interest to declare.
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Sodeoka, Mikiko, Kosuke Dodo, Yuou Teng, Katsuya Iuchi, Yoshitaka Hamashima, Eriko Iwasa, and Shinya Fujishiro. "Synthesis and biological activities of chaetocin and its derivatives." Pure and Applied Chemistry 84, no. 6 (April 2, 2012): 1369–78. http://dx.doi.org/10.1351/pac-con-11-10-31.
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Chaetocin, a natural product isolated from fungi of Chaetomium species, is a member of the epipolythiodiketopiperazines (ETPs), which have various biological activities, including cytostatic and anticancer activities. Recently, the inhibitory activity toward histone methyltransferases (HMTs) was discovered for chaetocin. We previously reported the first total synthesis of chaetocin and various derivatives. During studies on the structure–activity relationship for HMT inhibition, we found that the enantiomer of chaetocin (ent-chaetocin) is a more potent apoptosis inducer than natural chaetocin in human leukemia HL-60 cells. Mechanistic studies showed that ent-chaetocin induces apoptosis through the caspase-8/caspase-3 pathway.
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LEGGATE, JOHANNA, and BURTON W. BLAIS. "An Internal Amplification Control System Based on Primer-Dimer Formation for PCR Product Detection by DNA Hybridization." Journal of Food Protection 69, no. 9 (September 1, 2006): 2280–84. http://dx.doi.org/10.4315/0362-028x-69.9.2280.
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The detection of PCR products by DNA hybridization techniques can suffer from inhibition of the amplification process by sample matrix components. We have designed a simple internal control system for PCR based on the incorporation of a primer pair with complementary 3′ ends, resulting in the generation of a unique “primer-dimer” detectable by hybridization with a specific capture probe immobilized on polyester cloth as part of an array of amplicon-specific probes. The inclusion of this primer pair did not adversely affect the amplification and subsequent detection of target gene sequences by hybridization with immobilized probes in either single gene amplification or multiplex PCR systems. The failure to amplify target gene sequences because of the presence of inhibitors was mirrored by a failure to amplify the internal control primer-dimer, demonstrating the efficacy of this system in identifying the presence of DNA amplification inhibitors.
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Xiong, Sang, Jianlin Sun, Yang Xu, and Xundong Yan. "Adsorption behavior of tautomeric forms of 2-aminino-5-mercato-1,3,4 – thiadizole as corrosion inhibitor on copper surface." Anti-Corrosion Methods and Materials 63, no. 6 (November 7, 2016): 452–60. http://dx.doi.org/10.1108/acmm-01-2015-1483.
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Purpose The purpose of this study is to evaluate the effect of the four tautomeric forms of 2-amino-5-mercatpo-1,3,4-thiadizole (AMT) absorbed on copper surface by the polar or non-polar groups. Polar group of AMT is mostly electronegative with larger N and S atoms as central atoms. 5-amino-1,3,4-thiadiazole-2(3H)-thion (AMT-c) has the highest adsorption energy and is easy to react with copper. The interaction between AMT-c and copper conforms to chemisorption, which is to be further verified by the experiment on the weight loss measurement. Design/methodology/approach Adsorption behavior of AMT as corrosion inhibitor on copper surface in oil field was studied by weight loss measurement, and the corrosion inhibition mechanism was analyzed. Reactive sites and distributions of tautomeric forms of AMT as inhibitor on Cu(100) crystal plane were calculated by density functional theory. Findings All atoms of AMT are in the same plane, and AMT is an aromatic ring structure by large p-chain adsorbed on the metal surface by a plane configuration. AMT-c has the highest adsorption energy and also the most stable isomerized product. The determinate locations of AMT on the Cu(100) surface are the bridge and the hollow sites using molecular dynamics. Corrosion of copper can be effectively inhibited by AMT, which is a kind of excellent corrosion inhibitor, and this property is attributed to the polar groups and non-polar groups of AMT that play a role as absorption and shielding on copper surface, respectively. Inhibition efficiency is increased with the increase in the concentration of the inhibitor. The maximum efficiency of 92 per cent is obtained for 50 ppm AMT concentration at 373 K, which is attributed to the presence of extensively delocalized electrons of the phenyl rings, planarity and the presence of lone pair of electrons on N and S atoms, which favored a greater adsorption of inhibitors on copper surface. Originality/value Corrosion of copper can be effectively inhibited by AMT, which is a kind of excellent corrosion inhibitor, and this property is attributed to the polar groups and non-polar groups of AMT that play a role as absorption and shielding on copper surface, respectively. Adsorption of AMT as corrosion inhibitor on copper surface obeys Langmuir isotherm. The interaction between AMT and copper conforms to chemisorption, which is to be further verified by the experiment on the weight loss measurement.
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Lucas-Osma, Ana M., Yaqing Li, Shihao Lin, Sophie Black, Rahul Singla, Karim Fouad, Keith K. Fenrich, and David J. Bennett. "Extrasynaptic α5GABAA receptors on proprioceptive afferents produce a tonic depolarization that modulates sodium channel function in the rat spinal cord." Journal of Neurophysiology 120, no. 6 (December 1, 2018): 2953–74. http://dx.doi.org/10.1152/jn.00499.2018.
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Activation of GABAA receptors on sensory axons produces a primary afferent depolarization (PAD) that modulates sensory transmission in the spinal cord. While axoaxonic synaptic contacts of GABAergic interneurons onto afferent terminals have been extensively studied, less is known about the function of extrasynaptic GABA receptors on afferents. Thus, we examined extrasynaptic α5GABAA receptors on low-threshold proprioceptive (group Ia) and cutaneous afferents. Afferents were impaled with intracellular electrodes and filled with neurobiotin in the sacrocaudal spinal cord of rats. Confocal microscopy was used to reconstruct the afferents and locate immunolabelled α5GABAA receptors. In all afferents α5GABAA receptors were found throughout the extensive central axon arbors. They were most densely located at branch points near sodium channel nodes, including in the dorsal horn. Unexpectedly, proprioceptive afferent terminals on motoneurons had a relative lack of α5GABAA receptors. When recording intracellularly from these afferents, blocking α5GABAA receptors (with L655708, gabazine, or bicuculline) hyperpolarized the afferents, as did blocking neuronal activity with tetrodotoxin, indicating a tonic GABA tone and tonic PAD. This tonic PAD was increased by repeatedly stimulating the dorsal root at low rates and remained elevated for many seconds after the stimulation. It is puzzling that tonic PAD arises from α5GABAA receptors located far from the afferent terminal where they can have relatively little effect on terminal presynaptic inhibition. However, consistent with the nodal location of α5GABAA receptors, we find tonic PAD helps produce sodium spikes that propagate antidromically out the dorsal roots, and we suggest that it may well be involved in assisting spike transmission in general. NEW & NOTEWORTHY GABAergic neurons are well known to form synaptic contacts on proprioceptive afferent terminals innervating motoneurons and to cause presynaptic inhibition. However, the particular GABA receptors involved are unknown. Here, we examined the distribution of extrasynaptic α5GABAA receptors on proprioceptive Ia afferents. Unexpectedly, these receptors were found preferentially near nodal sodium channels throughout the afferent and were largely absent from afferent terminals. These receptors produced a tonic afferent depolarization that modulated sodium spikes, consistent with their location.
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Cruz-Izquierdo, Álvaro, Lambertus A. M. van den Broek, Juan L. Serra, María J. Llama, and Carmen G. Boeriu. "Lipase-catalyzed synthesis of oligoesters of 2,5-furandicarboxylic acid with aliphatic diols." Pure and Applied Chemistry 87, no. 1 (January 1, 2015): 59–69. http://dx.doi.org/10.1515/pac-2014-1003.
Full textAbstract:
Abstract2,5-Furandicarboxylic acid is a platform chemical for the production of biobased polymers and materials. This study reports the synthesis of furan oligoesters via polytransesterification of dimethyl furan-2,5-dicarboxylate and linear α, ω-aliphatic diols with chain length ranging from C2 to C12, using immobilized lipase B from Candida antarctica (Novozym 435) in dry organic solvents. Dimethyl furan-2,5-dicarboxylic acid (A) and 1,4-butanediol (B) were used as model substrates under different conditions producing a mixture of cyclic (CEOs) and linear (LEOs) ester oligomers up to decamers and dodecamers, respectively, with high yield. The size of the oligomers and distribution of the products is controlled by the initial concentration of substrates and temperature. While the shortest CEOs are the main cyclic compounds at 20 mM, the longest CEOs are formed at 175 mM. The chain length of the aliphatic diol co-monomers strongly influences the yield and the type of oligoesters formed. High substrate conversion of 90–95 % was obtained for C4–C12 diols, while in the case of ethylene glycol and 1,3-propanediol the conversion was moderate (i.e., 75 %). The product of the reaction between dimethyl furan-2,5-dicarboxylate and ethylene glycol (C2) and 1,3-propanediol (C3), respectively, consisted only of linear oligoesters. Longer oligoesters were obtained for alkyl chains higher than C4. The chain length and the abundance of oligoesters increases in the order: C2<C12<C10<C3<C8<C4 <C6. No substrate or product inhibition was observed in the production of furan-based oligoesters. The present biobased oligoesters are obtained via a green process and have potential application as macromonomers.
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Touqui, L., M. Hatmi, and B. B. Vargaftig. "Human platelets stimulated by thrombin produce platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when the degrading enzyme acetyl hydrolase is blocked." Biochemical Journal 229, no. 3 (August 1, 1985): 811–16. http://dx.doi.org/10.1042/bj2290811.
Full textAbstract:
It has been shown [Touqui, Jacquemin & Vargaftig (1983) Thromb. Haemostasis 50, 163; Touqui, Jacquemin & Vargaftig (1983) Biochem. Biophys. Res. Commun. 110, 890-893; Alam, Smith & Melvin (1983) Lipids 18, 534-538; Pieroni & Hanahan (1983) Arch. Biochem. Biophys. 224, 485-493] that rabbit platelets inactivate exogenous PAF (platelet-activating factor, PAF-acether) by a deacetylation-reacylation mechanism. The deacetylation step is catalysed by an acetyl hydrolase sensitive to the serine-hydrolase inhibitor PMSF (phenylmethanesulphonyl fluoride) [Touqui, Jacquemin, Dumarey & Vargaftig (1985) Biochim. Biophys. Acta 833, 111-118]. We report here that human platelets can produce PAF on thrombin stimulation. This production is marginal and transient, reaching a maximum at 10 min and decreasing thereafter. In contrast, 10-12 times more PAF is produced when platelets are treated with PMSF and stimulated with thrombin. Under these conditions, the maximum formation is observed at 30 min and no decline occurs for up to 60 min after stimulation. In addition, these platelets (treated with PMSF and stimulated with thrombin) incorporate exogenous labelled acetate in the 2-position of PAF, probably by an acetyltransferase-dependent mechanism. Production of PAF by human platelets during physiological stimulation can be demonstrated when PAF degradation is suppressed by the acetyl-hydrolase inhibitor PMSF.
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Mari, M., T. Cembali, E. Baraldi, and L. Casalini. "Peracetic Acid and Chlorine Dioxide for Postharvest Control of Monilinia laxa in Stone Fruits." Plant Disease 83, no. 8 (August 1999): 773–76. http://dx.doi.org/10.1094/pdis.1999.83.8.773.
Full textAbstract:
The effects of different concentrations of peracetic acid (PAA; 62.5, 125, 250, 500, and 1,000 μg/ml)and chlorine dioxide (ClO2; 12.5, 25, 50, 100, and 200 μg/ml) on germination of Monilinia laxa conidia were tested. Conidia germination was related to the concentration of chemical product used, as well as duration of treatment. Complete inhibition of germination was observed with PAA at 500 μg/ml after 5 min of contact with conidia and with ClO2 at 50 μg/ml after 1 min of contact with conidia. The results of in vitro tests were confirmed by inoculation of fruits with treated conidia. The PAA treatment also was effective 1 h after pathogen inoculation but only on plums, for which a 1,000 μg/ml treatment significantly reduced decay incidence by 50%. In a semi-commercial test, pathogen conidia dipped for 20 min in PAA at 250 μg/ml or ClO2 at 10 μg/ml or for 5 min in PAA at 250 μg/ml were completely inhibited, and no brown rot was observed in inoculated wounded nectarines and plums.
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May, Carl J., Gavin I. Welsh, Musleeha Chesor, Phillipa J. Lait, Lauren P. Schewitz-Bowers, Richard W. J. Lee, and Moin A. Saleem. "Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation." American Journal of Physiology-Renal Physiology 317, no. 4 (October 1, 2019): F913—F921. http://dx.doi.org/10.1152/ajprenal.00093.2019.
Full textAbstract:
The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 in podocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS.
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Ritchie, Helen, Alec Jamieson, and Nuala A. Booth. "Regulation, Location and Activity of Plasminogen Activator Inhibitor 2 (PAI-2) in Peripheral Blood Monocytes, Macrophages and Foam Cells." Thrombosis and Haemostasis 77, no. 06 (1997): 1168–73. http://dx.doi.org/10.1055/s-0038-1656132.
Full textAbstract:
SummaryMonocytes, macrophages and foam cells are central to atherogenesis. We have examined the potential ability of monocytes, macrophages and foam cells to affect the stability of deposited fibrin, characteristic of the atherosclerotic plaque, by their production of plasminogen activators and their inhibitors. Monocytes respond to thrombin and LPS by up-regulation of PAI-2 synthesis, and PAI-2 is their major product among the plasminogen activators/inhibitors. In contrast, macrophages and foam cells, while they did produce PAI-2, did not respond to thrombin and LPS by an increase in its synthesis. All PAI-2 produced by macrophages and foam cells was accumulated intracellularly, whereas monocytes also secreted PAI-2. Secreted PAI-2 was active as an inhibitor of u-PA, whereas intracellular PAI-2 required detergent treatment to generate activity. Thus monocytes, but not macrophages or foam cells, produce and secrete active PAI-2, thus potentially affecting fibrin stability in the local environment.
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Schaefer, Ulrich, Takuji Machida, Sandra Vorlova, Sidney Strickland, and Roberto Levi. "The plasminogen activator system modulates sympathetic nerve function." Journal of Experimental Medicine 203, no. 9 (August 28, 2006): 2191–200. http://dx.doi.org/10.1084/jem.20060077.
Full textAbstract:
Sympathetic neurons synthesize and release tissue plasminogen activator (t-PA). We investigated whether t-PA modulates sympathetic activity. t-PA inhibition markedly reduced contraction of the guinea pig vas deferens to electrical field stimulation (EFS) and norepinephrine (NE) exocytosis from cardiac synaptosomes. Recombinant t-PA (rt-PA) induced exocytotic and carrier-mediated NE release from cardiac synaptosomes and cultured neuroblastoma cells; this was a plasmin-independent effect but was potentiated by a fibrinogen cleavage product. Notably, hearts from t-PA–null mice released much less NE upon EFS than their wild-type (WT) controls (i.e., a 76.5% decrease; P < 0.01), whereas hearts from plasminogen activator inhibitor-1 (PAI-1)–null mice released much more NE (i.e., a 275% increase; P < 0.05). Furthermore, vasa deferentia from t-PA–null mice were hyporesponsive to EFS (P < 0.0001) but were normalized by the addition of rt-PA. In contrast, vasa from PAI-1–null mice were much more responsive (P < 0.05). Coronary NE overflow from hearts subjected to ischemia/reperfusion was much smaller in t-PA–null than in WT control mice (P < 0.01). Furthermore, reperfusion arrhythmias were significantly reduced (P < 0.05) in t-PA–null hearts. Thus, t-PA enhances NE release from sympathetic nerves and contributes to cardiac arrhythmias in ischemia/reperfusion. Because the risk of arrhythmias and sudden cardiac death is increased in hyperadrenergic conditions, targeting the NE-releasing effect of t-PA may have valuable therapeutic potential.
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Ritchie, Helen, and Nuala Booth. "The Distribution of the Secreted and Intracellular Forms of Plasminogen Activator Inhibitor 2 (PAI-2) in Human Peripheral Blood Monocytes Is Modulated by Serum." Thrombosis and Haemostasis 79, no. 04 (1998): 813–17. http://dx.doi.org/10.1055/s-0037-1615070.
Full textAbstract:
SummaryPlasminogen activator inhibitor 2 (PAI-2) is produced by activated monocytes in two forms, intracellular and secreted. We have studied the distribution of these two forms in unstimulated human peripheral blood monocytes and after stimulation by thrombin. Fetal calf serum (FCS) in the culture medium was absolutely necessary for accumulation of intracellular PAI-2; but not for synthesis and secretion. Even at a concentration as low as 0.1%, FCS restored accumulation of intra-cellular PAI-2. Increasing concentrations of FCS resulted in an increase in the ratio of intracellular to secreted PAI-2. The factor that promoted accumulation of intracellular PAI-2 was not a platelet product. Failure of monocytes to accumulate PAI-2 did not reflect leakage due to cell death, as assessed by LDH in culture supernatants. We propose that accumulation of intracellular PAI-2 is not simply due to poor secretion, but is an active process that is modulated by factor(s) found in serum.
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Bricmont, P. A., and T. G. Cooper. "A gene product needed for induction of allantoin system genes in Saccharomyces cerevisiae but not for their transcriptional activation." Molecular and Cellular Biology 9, no. 9 (September 1989): 3869–77. http://dx.doi.org/10.1128/mcb.9.9.3869.
Full textAbstract:
The allantoin-degradative pathway of Saccharomyces cerevisiae consists of several genes whose expression is highly induced by the presence of allophanic acid. Induced expression requires a functional DAL81 gene product. Analysis of these genes has demonstrated the presence of three cis-acting elements in the upstream regions: (i) an upstream activation sequence (UAS) required for transcriptional activation in an inducer-independent fashion, (ii) an upstream repression sequence (URS) that mediates inhibition of this transcriptional activation, and (iii) an upstream induction sequence (UIS) needed for a response to inducer. The UIS element mediates inhibition of URS-mediated function when inducer is present. We cloned and characterized the DAL81 gene and identified the element with which it was associated. The gene was found to encode a rare 3.2-kilobase-pair mRNA. The amount of DAL81-specific RNA responded neither to induction nor to nitrogen catabolite repression. Deletion of the DAL81 gene resulted in loss of induction but did not significantly affect basal level expression of the DAL7 and DUR1,2 genes or the UAS and URS functions present in plasmid constructions. These data suggest that (i) transcriptional activation of the DAL genes and their responses to inducer are mediated by different factors and cis-acting sequences and (ii) the UIS functions only when a wild-type DAL81 gene product is available.
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Bricmont, P. A., and T. G. Cooper. "A gene product needed for induction of allantoin system genes in Saccharomyces cerevisiae but not for their transcriptional activation." Molecular and Cellular Biology 9, no. 9 (September 1989): 3869–77. http://dx.doi.org/10.1128/mcb.9.9.3869-3877.1989.
Full textAbstract:
The allantoin-degradative pathway of Saccharomyces cerevisiae consists of several genes whose expression is highly induced by the presence of allophanic acid. Induced expression requires a functional DAL81 gene product. Analysis of these genes has demonstrated the presence of three cis-acting elements in the upstream regions: (i) an upstream activation sequence (UAS) required for transcriptional activation in an inducer-independent fashion, (ii) an upstream repression sequence (URS) that mediates inhibition of this transcriptional activation, and (iii) an upstream induction sequence (UIS) needed for a response to inducer. The UIS element mediates inhibition of URS-mediated function when inducer is present. We cloned and characterized the DAL81 gene and identified the element with which it was associated. The gene was found to encode a rare 3.2-kilobase-pair mRNA. The amount of DAL81-specific RNA responded neither to induction nor to nitrogen catabolite repression. Deletion of the DAL81 gene resulted in loss of induction but did not significantly affect basal level expression of the DAL7 and DUR1,2 genes or the UAS and URS functions present in plasmid constructions. These data suggest that (i) transcriptional activation of the DAL genes and their responses to inducer are mediated by different factors and cis-acting sequences and (ii) the UIS functions only when a wild-type DAL81 gene product is available.
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Canfield, A. E., A. M. Schor, D. J. Loskutoff, S. L. Schor, and M. E. Grant. "Plasminogen activator inhibitor-type I is a major biosynthetic product of retinal microvascular endothelial cells and pericytes in culture." Biochemical Journal 259, no. 2 (April 15, 1989): 529–35. http://dx.doi.org/10.1042/bj2590529.
Full textAbstract:
Previous studies have shown that a glycoprotein of Mr 47,000 (designated Gp47) is a major biosynthetic product of retinal endothelial cells in vitro (Canfield, Schor, West, Schor & Grant (1987) Biochem. J. 246, 121-129). We now present data indicating that (a) an identical protein is secreted by bovine retinal pericytes, (b) this protein is plasminogen activator inhibitor-type I (PAI-1), as revealed by immunoprecipitation with specific antibodies and reverse fibrin zymography, and (c) retinal endothelial cells and pericytes synthesize different species of matrix macromolecules, that is: type IV collagen is the major collagen secreted by endothelial cells, whereas pericytes produce predominantly type I collagen; fibronectin and thrombospondin are synthesized by both cell types. Our studies also indicate that PAI-1 is produced, albeit at considerably lower levels, by large vessel vascular cells (aortic endothelial and smooth muscle cells) and human skin fibroblasts. PAI-1 produced by human skin fibroblasts appears to be a distinct molecular species compared to its bovine counterpart as assessed by its slower mobility on SDS/polyacrylamide-gel electrophoresis. The potential significance of elevated PAI-1 production by retinal endothelial cells and pericytes, as well as their distinctive patterns of matrix biosynthesis, is discussed in terms of the involvement of these cells in the maintenance and remodelling of microvessel basement membrane.
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Mikulová, Mária Bodnár, Dáša Kružlicová, Daniel Pecher, Claudiu T. Supuran, and Peter Mikuš. "Synthetic Strategies and Computational Inhibition Activity Study for Triazinyl-Substituted Benzenesulfonamide Conjugates with Polar and Hydrophobic Amino Acids as Inhibitors of Carbonic Anhydrases." International Journal of Molecular Sciences 21, no. 10 (May 22, 2020): 3661. http://dx.doi.org/10.3390/ijms21103661.
Full textAbstract:
Various sulfonamide derivatives are intensively studied as anticancer agents owing to their inhibitory activity against human tumor-associated carbonic anhydrase isoforms. In this work, different synthetic procedures for the series of 1,3,5-triazinyl-aminobenzenesulfonamide conjugates with amino acids, possessing polar uncharged, negatively charged, and hydrophobic side chain, were studied and optimized with respect to the yield/purity of the synthesis/product as well as the time of synthetic reaction. These procedures were compared to each other via characteristic HPLC-ESI-DAD/QTOF/MS analytical product profiles, and their benefits as well as limitations were discussed. For new sulfonamide derivatives, incorporating s-triazine with a symmetric pair of polar and some less-polar proteinogenic amino acids, inhibition constants (KIs) against four human carboanhydrases (hCAs), namely cytosolic hCA I, II, transmembrane hCA IV, and the tumor-associated, membrane-bound hCA IX isoforms, were computationally predicted applying various methods of the advanced statistical analysis. Quantitative structure-activity relationship (QSAR) analysis indicated an impressive KI ratio (hCA II/hCA IX) 139.1 and hCA IX inhibition constant very similar to acetazolamide (KI = 29.6 nM) for the sulfonamide derivative disubstituted with Gln. The derivatives disubstituted with Ser, Thr, and Ala showed even lower KIs (8.7, 13.1, and 8.4 nM, respectively).
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Shimada, Toshio, Taeko Hirose, Itsuro Matsumoto, and Tadaomi Aikawa. "Platelet-activating factor acts on cortisol secretion by perfused guinea-pig adrenals via calcium-/phospholipid-dependent mechanisms." Journal of Endocrinology 184, no. 2 (February 2005): 381–91. http://dx.doi.org/10.1677/joe.1.05937.
Full textAbstract:
Bilateral adrenals of the guinea pig were perfused in situ with an artificial medium equilibrated with 95% O2/5% CO2. Platelet-activating factor (PAF) induced biphasic cortisol responses, which reached a maximum at 10 nM PAF and declined at 100 nM. The effect of the PAF receptor antagonists CV-3988 and CV-6209 on PAF-stimulated cortisol secretion was examined. Prior exposure of adrenal glands to 10 μM CV-3988 or a simultaneous incubation with 10 μM CV-6209 abolished the cortisol response to 10 nM PAF. Lyso-PAF (a PAF precursor and breakdown product) did not affect cortisol secretion. Concentrations of 5–12.5 μM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C (PKC) inhibitor, abolished subsequent cortisol secretion in response to 10 nM PAF. N-[2-(Methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-8), a protein kinase A inhibitor, was less effective. A calcium ionophore (A23187) at 3.3 and 10 μM increased cortisol secretion, but the activator of PKC, l-α-1-oleoyl-2-acetyl-sn-3-glycerol (OAG), at 50 μM had no effect. When infused simultaneously, OAG (50 μM) and A23187 (3.3 μM) stimulated cortisol secretion synergistically. The secretory response of cortisol to repeated infusions of adrenocortico-trophin (100 pg/ml) or forskolin (10 μM) was essentially reproducible. By contrast, cortisol secretion in response to repeated infusions of PAF (10 nM) or OAG plus A23187 was not reproducible and the second response was diminished compared with the first. Our findings suggest that PAF plays a role in the regulation of steroidogenesis via a mechanism mediated by the PAF receptor and PKC.
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Srinivasan, Padmanabhan, Edwin C. Thrower, Fred S. Gorelick, and Hamid M. Said. "Inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone." American Journal of Physiology-Gastrointestinal and Liver Physiology 310, no. 10 (May 15, 2016): G874—G883. http://dx.doi.org/10.1152/ajpgi.00461.2015.
Full textAbstract:
Thiamin is essential for normal metabolism in pancreatic acinar cells (PAC) and is obtained from their microenvironment through specific plasma-membrane transporters, converted to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria through the mitochondrial TPP (MTPP) transporter (MTPPT; product of SLC25A19 gene). TPP is essential for normal mitochondrial function. We examined the effect of long-term/chronic exposure of PAC in vitro (pancreatic acinar 266-6 cells) and in vivo (wild-type or transgenic mice carrying the SLC25A19 promoter) of the cigarette smoke toxin, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), on the MTPP uptake process. Our in vitro and in vivo findings demonstrate that NNK negatively affects MTPP uptake and reduced expression of MTPPT protein, MTPPT mRNA, and heterogenous nuclear RNA, as well as SLC25A19 promoter activity. The effect of NNK on Slc25a19 transcription was neither mediated by changes in expression of transcriptional factor NFY-1 (known to drive SLC25A19 transcription), nor due to changes in methylation profile of the Slc25a19 promoter. Rather, it appears to be due to changes in histone modifications that involve significant decreases in histone H3K4-trimethylation and H3K9-acetylation (activation markers). The effect of NNK on MTPPT function is mediated through the nonneuronal α7-nicotinic acetylcholine receptor (α7-nAChR), as indicated by both in vitro (using the nAChR antagonist mecamylamine) and in vivo (using an α7-nAchR −/− mouse model) studies. These findings demonstrate that chronic exposure of PAC to NNK negatively impacts PAC MTPP uptake. This effect appears to be exerted at the level of Slc25a19 transcription, involve epigenetic mechanism(s), and is mediated through the α7-nAchR.
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Srinivasan, Padmanabhan, Svetlana Nabokina, and Hamid M. Said. "Chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse 266-6 cell line and primary cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 309, no. 9 (November 1, 2015): G750—G758. http://dx.doi.org/10.1152/ajpgi.00226.2015.
Full textAbstract:
Thiamin is essential for normal metabolic activity of all mammalian cells, including those of the pancreas. Cells obtain thiamin from their surroundings and enzymatically convert it into thiamin pyrophosphate (TPP) in the cytoplasm; TPP is then taken up by mitochondria via a specific carrier the mitochondrial TPP transporter (MTPPT; product of the SLC25A19 gene). Chronic alcohol exposure negatively impacts the health of pancreatic acinar cells (PAC), but its effect on physiological/molecular parameters of MTPPT is not known. We addressed this issue using mouse pancreatic acinar tumor cell line 266-6 and primary PAC of wild-type and transgenic mice carrying the SLC25A19 promoter that were fed alcohol chronically. Chronic alcohol exposure of 266-6 cells (but not to its nonoxidative metabolites ethyl palmitate and ethyl oleate) led to a significant inhibition in mitochondrial TPP uptake, which was associated with a decreased expression of MTPPT protein, mRNA, and activity of the SLC25A19 promoter. Similarly, chronic alcohol feeding of mice led to a significant inhibition in expression of MTPPT protein, mRNA, heterogeneous nuclear RNA, as well as in activity of SLC25A19 promoter in PAC. While chronic alcohol exposure did not affect DNA methylation of the Slc25a19 promoter, a significant decrease in histone H3 euchromatin markers and an increase in H3 heterochromatin marker were observed. These findings show, for the first time, that chronic alcohol exposure negatively impacts pancreatic MTPPT, and that this effect is exerted, at least in part, at the level of Slc25a19 transcription and appears to involve epigenetic mechanism(s).
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Wu, Xue, Jia-Li Liang, Yu-Hong Liu, Jia-Zhen Wu, Qiong-Hui Huang, Yu-Cui Li, and Qing-Feng Xie. "Comparison of anti-inflammatory effect between β-patchoulene epoxide and β-patchoulene in LPS-stimulated RAW264.7 macrophages." European Journal of Inflammation 16 (January 1, 2018): 205873921878507. http://dx.doi.org/10.1177/2058739218785075.
Full textAbstract:
β-patchoulene (β-PAE) is one of the essential tricyclic sesquiterpenes of patchouli oil while β-patchoulene epoxide (β-PAO) is the oxidative product of β-PAE which can only be found in the oil with long storage period. Our previous researches demonstrated that both β-PAE and β-PAO exert potent anti-inflammatory activity in vivo, but which one is more valuable still remains uncertain. Therefore, this study adopts the model of LPS-stimulated RAW264.7 macrophages to compare β-PAO with β-PAE on the anti-inflammatory activity. According to our results, β-PAO was superior to β-PAE on anti-inflammation as evidence by lowering the protein and mRNA expressions of several pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interleukin-1β (IL-1β), and monocyte chemotactic protein-1 (MCP-1). β-PAO was also better than β-PAE in reducing the productions of nitric oxide (NO) and prostaglandin E2 (PGE2) through inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 signaling pathway. The results above provided experimental basis for the conclusion that β-PAO was more potent than β-PAE in anti-inflammatory activity in vitro.
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Zentella, A., F. M. Weis, D. A. Ralph, M. Laiho, and J. Massagué. "Early gene responses to transforming growth factor-beta in cells lacking growth-suppressive RB function." Molecular and Cellular Biology 11, no. 10 (October 1991): 4952–58. http://dx.doi.org/10.1128/mcb.11.10.4952.
Full textAbstract:
The growth-suppressive function of the retinoblastoma susceptibility gene product, RB, has been implicated in the mediation of growth inhibition and negative regulation of certain proliferation related genes by transforming growth factor-beta 1 (TGF-beta 1). Early gene responses to TGF-beta 1 were examined in order to determine their dependence on the cell cycle and on the growth-suppressive function of RB. TGF-beta 1, which rapidly elevates the steady-state level of junB and PAI-1 mRNAs and decreases that of c-myc mRNA, induces these responses in S-phase populations of Mv1Lu lung epithelial cells containing RB in a phosphorylated state. Since in this state RB is presumed to lack growth-suppressive activity, the response to TGF-beta 1 was also examined in DU145 human prostate carcinoma cells whose mutant RB product lacks growth-suppressive function. In these cells, TGF-beta 1 also decreases c-myc expression at the transcription initiation level. These results suggests that the c-myc, junB, and PAI-1 responses to TGF-beta 1 are not restricted to the G1 phase of the cell cycle and that down-regulation of c-myc expression by TGF-beta 1 can occur through a mechanism independent from the growth-suppressive function of RB.
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Zentella, A., F. M. Weis, D. A. Ralph, M. Laiho, and J. Massagué. "Early gene responses to transforming growth factor-beta in cells lacking growth-suppressive RB function." Molecular and Cellular Biology 11, no. 10 (October 1991): 4952–58. http://dx.doi.org/10.1128/mcb.11.10.4952-4958.1991.
Full textAbstract:
The growth-suppressive function of the retinoblastoma susceptibility gene product, RB, has been implicated in the mediation of growth inhibition and negative regulation of certain proliferation related genes by transforming growth factor-beta 1 (TGF-beta 1). Early gene responses to TGF-beta 1 were examined in order to determine their dependence on the cell cycle and on the growth-suppressive function of RB. TGF-beta 1, which rapidly elevates the steady-state level of junB and PAI-1 mRNAs and decreases that of c-myc mRNA, induces these responses in S-phase populations of Mv1Lu lung epithelial cells containing RB in a phosphorylated state. Since in this state RB is presumed to lack growth-suppressive activity, the response to TGF-beta 1 was also examined in DU145 human prostate carcinoma cells whose mutant RB product lacks growth-suppressive function. In these cells, TGF-beta 1 also decreases c-myc expression at the transcription initiation level. These results suggests that the c-myc, junB, and PAI-1 responses to TGF-beta 1 are not restricted to the G1 phase of the cell cycle and that down-regulation of c-myc expression by TGF-beta 1 can occur through a mechanism independent from the growth-suppressive function of RB.
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Christ, G., D. Seiffert, P. Hufnagl, A. Gessl, J. Wojta, and BR Binder. "Type 1 plasminogen activator inhibitor synthesis of endothelial cells is downregulated by smooth muscle cells." Blood 81, no. 5 (March 1, 1993): 1277–83. http://dx.doi.org/10.1182/blood.v81.5.1277.1277.
Full textAbstract:
Abstract Plasminogen activator inhibitor type 1 (PAI-1), the physiologic inhibitor of both tissue-type plasminogen activator (tPA) and urokinase- type plasminogen activator (uPA), is a major biosynthetic product of endothelial cells in vitro; endothelial cells in vivo, in contrast, do not appear to produce significant amounts of PAI-1 as made evident by in situ-hybridization studies in normal mice. This suggests that the high rate of PAI-1 synthesis of endothelial cells in vitro might be a result of the culture conditions. When human umbilical vein endothelial cells (HUVEC) were grown on human amniotic membranes, resembling the natural growth support instead of coated plastic, their morphology was changed from the cobblestone-like appearance on plastic to an in vivo like flagstone pattern. However, this morphological change had no significant effect on the synthesis and secretion of PAI-1. When smooth muscle cell (SMC) conditioned media (CM) were added to HUVEC cultures, PAI-1 antigen secretion of HUVEC was reduced by 40% to 60% as measured by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation experiments using 36S-methionine metabolically labeled HUVEC and Northern blot analysis of HUVEC PAI-1 mRNA indicate that this reduction was attributable to decreased PAI-1 synthesis and reduced steady-state levels of both the 3.2 kb and 2.2 kb form of PAI-1 mRNA. This effect was dose-dependent and observed under serum-containing as well as serum- free conditions, in the absence or presence of endothelial cell growth supplement (ECGS, 0 to 100 micrograms/mL) and attributable to a nondialyzable factor. Our data suggest that the high level of PAI-1 biosynthesis of endothelial cells in vitro may be attributable to the lack of a soluble factor produced by SMC, which controls and suppresses PAI-1 biosynthesis of endothelial cells in vivo.
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Christ, G., D. Seiffert, P. Hufnagl, A. Gessl, J. Wojta, and BR Binder. "Type 1 plasminogen activator inhibitor synthesis of endothelial cells is downregulated by smooth muscle cells." Blood 81, no. 5 (March 1, 1993): 1277–83. http://dx.doi.org/10.1182/blood.v81.5.1277.bloodjournal8151277.
Full textAbstract:
Plasminogen activator inhibitor type 1 (PAI-1), the physiologic inhibitor of both tissue-type plasminogen activator (tPA) and urokinase- type plasminogen activator (uPA), is a major biosynthetic product of endothelial cells in vitro; endothelial cells in vivo, in contrast, do not appear to produce significant amounts of PAI-1 as made evident by in situ-hybridization studies in normal mice. This suggests that the high rate of PAI-1 synthesis of endothelial cells in vitro might be a result of the culture conditions. When human umbilical vein endothelial cells (HUVEC) were grown on human amniotic membranes, resembling the natural growth support instead of coated plastic, their morphology was changed from the cobblestone-like appearance on plastic to an in vivo like flagstone pattern. However, this morphological change had no significant effect on the synthesis and secretion of PAI-1. When smooth muscle cell (SMC) conditioned media (CM) were added to HUVEC cultures, PAI-1 antigen secretion of HUVEC was reduced by 40% to 60% as measured by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation experiments using 36S-methionine metabolically labeled HUVEC and Northern blot analysis of HUVEC PAI-1 mRNA indicate that this reduction was attributable to decreased PAI-1 synthesis and reduced steady-state levels of both the 3.2 kb and 2.2 kb form of PAI-1 mRNA. This effect was dose-dependent and observed under serum-containing as well as serum- free conditions, in the absence or presence of endothelial cell growth supplement (ECGS, 0 to 100 micrograms/mL) and attributable to a nondialyzable factor. Our data suggest that the high level of PAI-1 biosynthesis of endothelial cells in vitro may be attributable to the lack of a soluble factor produced by SMC, which controls and suppresses PAI-1 biosynthesis of endothelial cells in vivo.
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Reinke, Ashley A., Shih-Hon Li, Mark Warnock, Maxim E. Shaydakov, Naga Sandhya Guntaka, Enming J. Su, Jose A. Diaz, Cory D. Emal, and Daniel A. Lawrence. "Dual-reporter high-throughput screen for small-molecule in vivo inhibitors of plasminogen activator inhibitor type-1 yields a clinical lead candidate." Journal of Biological Chemistry 294, no. 5 (December 3, 2018): 1464–77. http://dx.doi.org/10.1074/jbc.ra118.004885.
Full textAbstract:
Plasminogen activator inhibitor type-1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, arterial and venous thrombosis, and chronic fibrotic diseases. These associations have made PAI-1 an attractive pharmaceutical target. However, the complexity of the serpin inhibitory mechanism, the inherent metastability of serpins, and the high-affinity association of PAI-1 with vitronectin in vivo have made it difficult to identify pharmacologically effective small-molecule inhibitors. Moreover, the majority of current small-molecule PAI-1 inhibitors are poor pharmaceutical candidates. To this end and to find leads that can be efficiently applied to in vivo settings, we developed a dual-reporter high-throughput screen (HTS) that reduced the rate of nonspecific and promiscuous hits and identified leads that inhibit human PAI-1 in the high-protein environments present in vivo. Using this system, we screened >152,000 pure compounds and 27,000 natural product extracts (NPEs), reducing the apparent hit rate by almost 10-fold compared with previous screening approaches. Furthermore, screening in a high-protein environment permitted the identification of compounds that retained activity in both ex vivo plasma and in vivo. Following lead identification, subsequent medicinal chemistry and structure–activity relationship (SAR) studies identified a lead clinical candidate, MDI-2268, having excellent pharmacokinetics, potent activity against vitronectin-bound PAI-1 in vivo, and efficacy in a murine model of venous thrombosis. This rigorous HTS approach eliminates promiscuous candidate leads, significantly accelerates the process of identifying PAI-1 inhibitors that can be rapidly deployed in vivo, and has enabled identification of a potent lead compound.
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Dakin, Catherine L., Caroline J. Small, Adrian J. Park, Asha Seth, Mohammad A. Ghatei, and Stephen R. Bloom. "Repeated ICV administration of oxyntomodulin causes a greater reduction in body weight gain than in pair-fed rats." American Journal of Physiology-Endocrinology and Metabolism 283, no. 6 (December 1, 2002): E1173—E1177. http://dx.doi.org/10.1152/ajpendo.00233.2002.
Full textAbstract:
Oxyntomodulin (OXM) is a product of proglucagon processing in the intestine and the central nervous system. We reported that intracerebroventricular (ICV) and intranuclear administration of OXM caused an inhibition of food intake in rats (Dakin CL, Gunn I, Small CJ, Edwards CM, Hay DL, Smith DM, Ghatei MA, and Bloom SR. Endocrinology 142: 4244–4250, 2001). In this study, we investigated the effect of twice-daily ICV administration of OXM, 1 nmol, for 7 days. A pair-fed control was included. These animals were restricted to the food intake of the OXM group but injected twice daily with saline. OXM-treated animals gained significantly less weight than either control group ( day 8: OXM, 12.2 ± 1.9 g vs. pair fed, 21.0 ± 2.1 g; P < 0.005). OXM treatment caused a reduction in epididymal white adipose tissue (OXM, 1.13 ± 0.03 g vs. pair fed, 1.29 ± 0.04 g; P < 0.05) and interscapular brown adipose tissue (OXM, 0.15 ± 0.01 g vs. pair fed, 0.18 ± 0.01 g; P < 0.05) and increased core temperature compared with saline control, suggestive of enhanced energy expenditure. The food restriction-induced suppression in plasma TSH, seen in the pair-fed group, was prevented by OXM, potentially via increased release of hypothalamic TRH. In summary, ICV OXM causes reduced body weight gain and body adiposity following chronic administration.