Calpain mediates progressive plasma membrane permeability and proteolysis of cytoskeleton-associated paxillin, talin, and vinculin during renal cell death.
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The goal of the present study was to determine the role of calpain in changes in plasma membrane permeability and cytoskeleton-associated paxillin, vinculin, talin, and alpha-actinin levels during acute renal cell death. The mitochondrial inhibitor antimycin A or hypoxia produced graded plasma membrane permeability in renal proximal tubules (RPTs), first allowing propidium iodide (PI, molecular mass 668 Da) influx and then lactate dehydrogenase (LDH, molecular mass 130 kDa) release. Cytoskeleton-associated paxillin levels decreased concomitantly with PI influx and before LDH release, whereas cytoskeleton-associated talin and vinculin levels decreased concomitantly with LDH release. Cytoskeleton-associated alpha-actinin levels did not change during antimycin A exposure or hypoxia. Purified micro-calpain cleaved paxillin, talin, vinculin, but not alpha-actinin. The dissimilar calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) or chloroacetic acid N'-[6,7-dichloro-4-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl] hydrazide (SJA7029) preserved cytoskeleton-associated paxillin, talin, and vinculin levels and prevented PI influx and LDH release in antimycin A-exposed or hypoxic RPTs. These results suggest that calpain mediates increased plasma membrane permeability and hydrolysis of cytoskeleton-associated paxillin, vinculin, and talin during renal cell death. (+info)
Effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c1 in the cytochrome bc1 complex.
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Famoxadone is a new cytochrome bc(1) Q(o) site inhibitor that immobilizes the iron-sulfur protein (ISP) in the b conformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c(1) were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c(1) was found to be 16,000 s(-1) in bovine cyt bc(1). Binding famoxadone decreased this rate constant to 1,480 s(-1), consistent with a decrease in mobility of the ISP. Reverse electron transfer from cyt c(1) to 2Fe-2S was found to be biphasic in bovine cyt bc(1) with rate constants of 90,000 and 7,300 s(-1). In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s(-1). It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc(1) mutants involving residues at the interface between the Rieske protein and cyt c(1) and/or cyt b. (+info)
Effects of the AT1-receptor antagonist eprosartan on the progression of left ventricular dysfunction in dogs with heart failure.
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1. We examined the effects of eprosartan, an AT(1) receptor antagonist, on the progression of left ventricular (LV) dysfunction and remodelling in dogs with heart failure (HF) produced by intracoronary microembolizations (LV ejection fraction, EF 30 to 40%). 2. Dogs were randomized to 3 months of oral therapy with low-dose eprosartan (600 mg once daily, n=8), high-dose eprosartan (1200 mg once daily, n=8), or placebo (n=8). 3. In the placebo group, LV end-diastolic (EDV) and end-systolic (ESV) volumes increased after 3 months (68+/-7 vs 82+/-9 ml, P<0.004, 43+/-1 vs 58+/-7 ml, P<0.003, respectively), and EF decreased (37+/-1 vs 29+/-1%, P<0.001). In dogs treated with low-dose eprosartan, EF, EDV, and ESV remained unchanged over the course of therapy, whereas in dogs treated with high-dose eprosartan, EF increased (38+/-1 vs 42+/-1%, P<0.004) and ESV decreased (41+/-1 vs 37+/-1 ml, P<0.006), Eprosartan also decreased interstitial fibrosis and cardiomyocyte hypertrophy. 4. We conclude that eprosartan prevents progressive LV dysfunction and attenuates progressive LV remodelling in dogs with moderate HF and may be useful in treating patients with chronic HF. (+info)
Effects of isosorbide mononitrate and AII inhibition on pulse wave reflection in hypertension.
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The aortic pulse wave contour in isolated systolic hypertension often shows a prominent reflection peak, which combines with the incident wave arising from cardiac ejection so as to widen pulse pressure. We investigated the effects of an extended-release nitrate preparation and of 2 angiotensin II (AII) inhibitors (an AII receptor antagonist and an ACE inhibitor) on the aortic pulse wave contour and systemic blood pressure in hypertensive subjects with high augmentation index caused by exaggerated pulse wave reflection. Two double-blind, randomized, placebo-controlled crossover studies were carried out in a total of 16 elderly patients with systolic hypertension resistant to conventional antihypertensive therapy. In 1 study, pharmacodynamic responses to single doses of placebo, isosorbide mononitrate, eprosartan, and captopril were determined; in the other, single-dose isosorbide mononitrate and placebo were compared in subjects treated with AII inhibitors at baseline. Blood pressure was measured by sphygmomanometry and pulse wave components by applanation tonometry at the radial artery. All 3 agents were shown to decrease brachial systolic blood pressure, aortic systolic blood pressure, and aortic pulse pressure. Qualitative effects on the aortic pulse wave contour differed: augmentation index was not significantly altered by either captopril or eprosartan but was decreased (P<0.0001) by approximately 50% of the placebo value with isosorbide mononitrate in both study groups. We propose that isosorbide mononitrate corrected the magnified wave reflection in systolic hypertension of these elderly patients by an effect that was distinct from that exercised by either acute or chronic AII inhibition. (+info)
Thermal stability of immobilized enzymes circular dichroism, fluorescence and kinetic measurements of alpha-chymotrypsin attached to soluble carriers.
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The temperature-induced unfolding of alpha-chymotrypsin and of chymotrypsin covalently bound to two soluble transparent carriers, dextran and a copolymer of maleic acid anhydride and acrylic acid, has been studied by tryptophan fluorescence emission, circular dichroic and kinetic measurements. It has been shown that the structural and functional properties of the enzyme when bound to the anionic copolymer are strongly influenced by electrostatic interactions. A number of reference experiments with anionic polyelectrolytes and the hydrogenated monomers of the copolymer suggest that these changes are brought about by the cooperative ion pair formation between protein and polyanionic matrix. (+info)
Biocompatibility of poly2methoxyethylacrylate coating for cardiopulmonary bypass.
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The systemic inflammatory response to cardiopulmonary bypass (CPB) may contribute to the development of postoperative complications. Heparin-coated circuits and poly2methoxyethylacrylate (PMEA)-coated circuits have been developed to reduce the risk of such complications. We compared the biocompatibility of these circuits. Twelve patients scheduled to undergo elective coronary artery bypass grafting (CABG) with CPB were assigned to CPB with a PMEA-coated circuit (PMEA-coated group, n=6) or a heparin-coated circuit (heparin-coated group, n=6). The plasma concentrations of the following inflammatory markers were measured before CPB and just after, 4 hours after, and 24 hours after the termination of CPB: cytokines (interleukin [IL]-6, IL-8, IL-10), complement factor (C3a), polymorphonuclear elastase (PMNE), and coagulofibrinolytic factors (thrombin-antithrombin III complex [TAT], D-dimer). Postoperative clinical response was evaluated on the basis of respiratory index, blood loss, and the postoperative and preoperative body-weight percent ratio. There were no significant differences between the groups in the plasma concentrations of IL-6, IL-10, C3a, PMNE, TAT, or D-dimer. Plasma IL-8 concentrations were below the assay detection limits at all time points in both groups. Clinical variables did not differ significantly between the groups. In conclusion, PMEA-coated CPB circuits are as biocompatible as heparin-coated CPB circuits and prevent postoperative organ dysfunction in patients undergoing elective CABG with CPB. (+info)
Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5.
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The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs. Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates. To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5. In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form. MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not. Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5. In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively. Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP. Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps. These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low. (+info)
Mucoadhesive vaginal tablets as veterinary delivery system for the controlled release of an antimicrobial drug, acriflavine.
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The aim of the study was the development of mucoadhesive vaginal tablets designed for the local controlled release of acriflavine, an antimicrobial drug used as a model. The tablets were prepared using drug-loaded chitosan microspheres and additional excipients (methylcellulose, sodium alginate, sodium carboxymethylcellulose, or Carbopol 974). The microspheres were prepared by a spray-drying method, using the drug to polymer weight ratios 1:1 and 1:2 and were characterized in terms of morphology, encapsulation efficiency, and in vitro release behavior, as MIC (Minimum Inhibitory Concentration), MBC (Minimum Bacterial Concentration), and killing time (KT). The tablets were prepared by direct compression, characterized by in vitro drug release and in vitro mucoadhesive tests. The microparticles have sizes of 4 to 12 microm; the mean encapsulation yields are about 90%. Acriflavine, encapsulated into the polymer, maintains its antibacterial activity; killing time of the encapsulated drug is similar to that of the free drug. In vitro release profiles of tablets show differences depending on the excipient used. In particular Carbopol 974, which is highly cross-linked, is able to determine a drug-controlled release from the matrix tablets for more than 8 hours. The in vitro adhesion tests, carried out on the same formulation, show a good adhesive behavior. The formulation containing microspheres with drug to polymer weight ratios of 1:1 and Carbopol 974 is characterized by the best release behavior and shows good mucoadhesive properties. These preliminary data indicate that this formulation can be proposed as a mucoadhesive vaginal delivery system for the controlled release of acriflavine. (+info)