Chronic peritoneal inflammation by cyanate in rats.
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OBJECTIVE: During peritoneal dialysis, the peritoneum is exposed to waste products, including urea. Urea forms cyanate spontaneously at body temperature and pH, and cyanate carbamylates amino acids, peptides, and proteins. Cyanate may contribute to peritoneal injury with morphological changes in the peritoneum. To test this hypothesis, we injected cyanate into rats. METHODS: Experiments were performed in two groups of 7 rats each. In the cyanate group, each rat received 1 mL of 1.5 micromol/L potassium cyanate dissolved in 40 mmol/L sodium bicarbonate solution intraperitoneally each experiment day. In the control group, each rat received 1 mL of 1.5 micromol/L potassium bicarbonate instead of potassium cyanate. The rats in both groups were anesthetized and killed at the 85th day after the first injection. After formalin fixation, tissue samples from abdominal walls and livers were sliced, embedded in a standard manner, and stained with hematoxylin and eosin. RESULTS: Parietal peritoneum from rats in the cyanate group showed a mild increase in the number of fibroblasts, with collagen deposits, infiltration by mononuclear cells, vascular congestion, round-shaped transformation of mesothelial cells, widening of submesothelial spaces, and abundant denudation of mesothelial cells. The visceral peritoneum from rats in the cyanate group showed collagen deposits with fibroblastic proliferation. CONCLUSIONS: Cyanate can induce chronic inflammation in the peritoneum, and exposure of the peritoneum to cyanate may contribute to peritoneal injury in patients being treated with peritoneal dialysis. (+info)
Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III.
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Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing +info)
Carbamoylation of glomerular and tubular proteins in patients with kidney failure: a potential mechanism of ongoing renal damage.
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BACKGROUND: Cyanate formed spontaneously from urea carbamoylates non-protonated amino groups of protein, irreversibly altering function, charge and structure. Carbamoylated proteins in renal tissue have not been examined hitherto. OBJECTIVES: To identify homocitrulline (epsilon-amino-carbamoyl-lysine), a result of in vivo carbamoylation by urea-derived cyanate, from patients with renal disease or in newly transplanted kidneys by immunohistochemistry. To evaluate enzymatic activity of carbamoylated and non-carbamoylated matrix metalloproteinase-2 and correlate this with renal tissue carbamoylated in vivo. DESIGN: Anti-homocitrulline antibody is specific for homocitrulline and was used to identify carbamoylation of epsilon-amino-lysine in renal biopsies from patients with elevated BUN, with isolated proteinuria, and as controls, from normal donors at time of transplantation. Enzymatic activity of matrix metalloproteinase-2 carbamoylated in vitro was evaluated. RESULTS: Homocitrulline was present in glomerular basement membrane (8/10), mesangium (8/10), tubular epithelium and cytoplasm (7/10) and Bowman's capsule (1/10) in patients with elevated BUN. The discordant patterns of glomerular and tubular localization of homocitrulline versus immune complexes indicated that the carbamoylated proteins were not a component of immune deposits but were modified proteins in renal tissue. No homocitrulline was found in transplanted kidneys (14/15) or in proteinuric patients (2/2). Enzymatic activity of both human and rat matrix metalloproteinase-2 was strongly inhibited in a dose-dependent fashion when incubated with cyanate. CONCLUSIONS: In situ carbamoylation in proteins occurred in kidneys of patients with renal dysfunction but not in normal newly transplanted kidneys. Decreased enzymatic activity of carbamoylated enzymes may alter specific renal regulatory mechanisms. Carbamoylated proteins with altered function and charge may represent a previously underestimated mechanism in renal pathophysiology. (+info)
Deflavination of flavo-oxidases by nucleophilic reagents.
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Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked. (+info)
The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.
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1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor. (+info)
Development of immunoassays for biomonitoring of hexamethylene diisocyanate exposure.
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Hexamethylene diisocyanate (HDI) is used widely to manufacture polyurethanes for paints and coatings. It is an irritant and a chemical asthmagen. The U.S. Occupational Safety and Health Administration time-weighted average permissible exposure limit is 5 ppb and the ceiling limit is 20 ppb. We sought to develop a sensitive and specific immuno-bioassay to supplement workplace air monitoring and detect recent HDI exposure. For this, we produced rabbit antiserum to HDI-adducted keyhole limpet hemocyanin (HDI-KLH). The specificity of the antiserum was demonstrated by its reaction with a variety of HDI-conjugated proteins and the absence of reactions with conjugates of other diisocyanates, namely toluene diisocyanate and diphenyl methylene diisocyanate. Four immunoassays were developed and compared for their ability to detect decreasing quantities of HDI-adducted human serum albumin (HSA) containing 2 mol HDI adduct per mol HSA (HDI(2)-HSA) as determined by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. The sensitivities of some of the assays are within the range (0.82-45 nM) of current analytic methods. A Western analysis procedure has a sensitivity of 600 nM HDI adduct on HSA. ELISA inhibition assay, in which microtiter plates are coated with the HDI(2)-HSA antigen, has a sensitivity of 300 nM HDI adduct. An immunoblot assay has a sensitivity of 9 nM HDI adduct. The most sensitive bioassay (1.8 nM HDI adduct) is a three-antibody sandwich ELISA in which wells of microtiter plates are coated with the IgG fraction of the anti-HDI-KLH antisera. Compared with analytic methods for HDI biomonitoring, the immunoassays are faster and less costly and accommodate numerous samples simultaneously. The assays have the potential to affect industrial biomonitoring programs significantly. (+info)
Respiratory chain network in mitochondria of Candida parapsilosis: ADP/O appraisal of the multiple electron pathways.
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In this study we demonstrated that mitochondria of Candida parapsilosis contain a constitutive ubiquinol alternative oxidase (AOX) in addition to a classical respiratory chain (CRC) and a parallel respiratory chain (PAR) both terminating by two different cytochrome c oxidases. The C. parapsilosis AOX is characterized by a fungi-type regulation by GMP (as a stimulator) and linoleic acid (as an inhibitor). Inhibitor screening of the respiratory network by the ADP/O ratio and state 3 respiration determinations showed that (i) oxygen can be reduced by the three terminal oxidases through four paths implying one bypass between CRC and PAR and (ii) the sum of CRC, AOX and PAR capacities is higher than the overall respiration (no additivity) and that their engagement could be progressive according to the redox state of ubiquinone, i.e. first cytochrome pathway, then AOX and finally PAR. (+info)
Iron metabolism, sickle cell disease, and response to cyanate.
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In an attempt to understand the variability of the hematologic response to oral sodium cyanate, iron metabolism was studied in a group of 39 patients with sickel cell disease. Eleven of the 39 patients were found to have no stainable iron in the marrow despite the fact that patients with sickle cell disease are generally considered to have hemosiderosis. The mean per cent saturation and total iron-binding capacity were in the low-normal range in sickle cell patients whether or not stainable iron was present in the bone marrow aspirate. Serum ferritin concentrations, on the other hand, were found to be high in both groups (greater than 500 mu g/liter) when compared to controls (60 mu g/liter). The high serum ferritin levels denoted significant total-body iron deposition which may be unavailable for normal metabolic processes. One patient with no stainable iron in the bone marrow aspirate did respond to iron therapy alone with an increase in hemoglobin concentration. Serum ceruloplasmin levels were also found to be high in sickle cell disease patients. The ability to respond to oral cyanate therapy was correlated with the amount of stainable iron in the bone marrow aspirate. These studies emphasize the necessity of a reevaluation of iron metabolism in the pathophysiology and treatment of sickle cell disease. (+info)