Cysteamine prevents and reverses the inhibition of pyruvate kinase activity caused by cystine in rat heart.
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Cystinosis is a disorder associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending on the involved tissues. Accumulation of cystine in myocardium may lead to heart failure. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that thiolic enzymes like pyruvate kinase (PK) may be altered by disulfides like cystine, the main objective of the present study was to investigate the effect of cystine on PK activity in the heart of developing rats. We performed kinetic studies and investigated the effects of reduced glutathione (GSH), a biologically occurring thiol groups protector, and cysteamine, the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits PK activity by oxidation of the sulfhydryl groups of the enzyme. Although there is no definite proof of cystine within cytoplasm, there is indirect proof t it is able to escape lysosomes and come in contact with PK. Considering that cysteamine is used in patients with cystinosis because it causes parenchymal organ cystine depletion, the present data provide a possible new effect for this drug. (+info)
Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress.
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Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers. (+info)
Amperometric hydrogen peroxide biosensor based on horseradish peroxidase-labeled nano-Au colloids immobilized on poly(2,6-pyridinedicarboxylic acid) layer by cysteamine.
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A sensitive hydrogen peroxidase (H2O2) amperometric sensor based on horseradish peroxidase (HRP)-labeled nano-Au colloids has been proposed. Nano-Au colloids were immobilized by the thiol group of cysteamine, which was associated with the carboxyl groups of poly(2,6-pyridinedicarboxylic acid) (PPDA). With the aid of the hydroquinone, the sensor displayed excellent electrocatalytical response to the reduction of H2O2. Compared with the non-Au-colloid modified electrode, i.e., PPDA/HRP, the Au-colloid modified electrode exhibited better performance characteristics, including stability, reproducibility, sensitivity and accuracy. The biosensor shows a linear response to H2O2 in the range of 3.0 x 10(-7) - 2 x 10(-3) M. The detection limit was 1.0 x 10(-7) M. (+info)
Different inhibition characteristics of intracellular transglutaminase activity by cystamine and cysteamine.
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The treatment of cystamine, a transglutaminase(TGase) inhibitor, has beneficial effects in several diseases including CAG-expansion disorders and cataract. We compared the inhibition characteristics of cystamine with those of cysteamine, a reduced form of cystamine expect-ed to be present inside cells. Cystamine is a more potent inhibitor for TGase than cysteamine with different kinetics pattern in a non-reducing condition. By contrast, under reducing conditions, the inhibitory effect of cystamine was comparable with that of cysteamine. How-ever, cystamine inhibited intracellular TGase activity more strongly than cysteamine despite of cytoplasmic reducing environment, suggest-ing that cystamine itself inhibits in situ TGase activity by forming mixed disulfides. (+info)
The Corynebacterium glutamicum aecD gene encodes a C-S lyase with alpha, beta-elimination activity that degrades aminoethylcysteine.
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S-(beta-Aminoethyl)-cysteine (AEC) resistance was achieved in Corynebacterium glutamicum by cloning a chromosomal 1.5-kb EcoRV-BglII DNA fragment on a multicopy plasmid. DNA sequence analysis of the 1.5-kb DNA fragment revealed an open reading frame (ORF326) which represents the AEC resistance gene, designated aecD. The aecD gene directs the synthesis of a 36-kDa protein which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aecD gene is a nonessential gene and mediates AEC resistance only in an amplified state. C. glutamicum strains harboring an amplified aecD gene can utilize AEC as an alternative nitrogen source, indicating that the AEC resistance mechanism is due to AEC degradation. Since the AEC degradation products analyzed by high-pressure liquid chromatography were found to be pyruvate and aminoethanethiol (cysteamine), it was concluded that the aecD gene encodes a C-S lyase with alpha, beta-elimination activity. Besides AEC, the C-S lyase was also able to use cysteine, cystine, and cystathionine as substrates. (+info)
Cysteamine enhances the procoagulant activity of Factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine).
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We have recently identified the molecular defect responsible for cross-reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East Hartford[FVIII-EH]) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose- and time-dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII-EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function. (+info)
Factor VIII-East Hartford (arginine 1689 to cysteine) has procoagulant activity when separated from von Willebrand factor.
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Factor VIII East Hartford (FVIII-EH) procoagulant activity is reduced because the substitution of cysteine for arginine 1689 abolishes an essential Factor VIII light chain thrombin cleavage site. Incubation of FVIII-EH plasma with penicillamine or DTT causes a five- to sixfold increase in FVIII-EH VIII:C, at 80 and 1 mM, respectively. While there is no FVIII-EH light chain cleavage when thrombin is added in the presence of penicillamine or DTT, these reducing agents disrupt the FVIII-vWf complex. For example, the addition of 5 mM DTT to normal or FVIII-EH plasma causes a 50% reduction in Factor VIII binding to vWf. These observations suggested that DTT increases FVIII-EH VIII:C by partial dissociation of FVIII-EH from vWf. This was verified by showing that vWf-free FVIII-EH had VIII:C activity of 21 U/dl, while the starting plasma level was 2.5 U/dl. Removal of other FVIII-EH plasma proteins by agarose gel filtration had no effect on VIII:C activity. The demonstration that this mutant Factor VIII has cofactor function when separated from vWf indicates that the dissociation of Factor VIII from vWf is an essential effect of Factor VIII light chain cleavage at arginine-1689. (+info)
Enhanced ghrelin secretion in rats with cysteamine-induced duodenal ulcers.
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Ghrelin, produced and secreted by the A-like cells of the stomach, stimulates growth hormone secretion, gastric motility, and food intake. Cysteamine inhibits the release of somatostatin and induces the formation of duodenal ulcers in rats. The present study was conducted to investigate the dynamics of ghrelin secretion in rats treated with cysteamine. Male Wistar rats (7 wk old) were administered three doses of cysteamine (400 mg/kg) orally; at 50 h after the first dose, duodenal ulcers were induced, and the plasma level of somatostatin and gastric density of somatostatin-immunoreactive cells were significantly reduced. The plasma total and active ghrelin levels were significantly higher in the cysteamine-treated rats than in the control rats, whereas the gastric ghrelin levels, number of gastric ghrelin-immunoreactive cells, and preproghrelin mRNA expression levels were significantly lower. Even at the time points of 2 and 10 h after the first dose of cysteamine, at which time no significant ulcer formation or antral neutrophil accumulation was yet noted, a significant increase in the plasma ghrelin level and decrease in the gastric ghrelin level were observed. Furthermore, although lansoprazole treatment attenuated the duodenal ulceration induced by cysteamine, the increase in the plasma level of ghrelin could still be demonstrated. Because an inverse correlation was found between the plasma ghrelin and somatostatin levels, the inhibition of somatostatin secretion may be associated with the increased ghrelin secretion. In conclusion, an increase in the plasma ghrelin level precedes the formation of duodenal ulcers in rats treated with cysteamine. (+info)