Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants.
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The responses to oxidative stress induced by chronic exercise (8-wk treadmill running) or acute exercise (treadmill running to exhaustion) were investigated in the brain, liver, heart, kidney, and muscles of rats. Various biomarkers of oxidative stress were measured, namely, lipid peroxidation [malondialdehyde (MDA)], protein oxidation (protein carbonyl levels and glutamine synthetase activity), oxidative DNA damage (8-hydroxy-2'-deoxyguanosine), and endogenous antioxidants (ascorbic acid, alpha-tocopherol, glutathione, ubiquinone, ubiquinol, and cysteine). The predominant changes are in MDA, ascorbic acid, glutathione, cysteine, and cystine. The mitochondrial fraction of brain and liver showed oxidative changes as assayed by MDA similar to those of the tissue homogenate. Our results show that the responses of the brain to oxidative stress by acute or chronic exercise are quite different from those in the liver, heart, fast muscle, and slow muscle; oxidative stress by acute or chronic exercise elicits different responses depending on the organ tissue type and its endogenous antioxidant levels. (+info)
Temperature-sensitive recA mutant of Escherichia coli K-12: deoxyribonucleic acid metabolism after ultraviolet irradiation.
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A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles. It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process. (+info)
Reactive half-cystine peptides of the secretory component of human exocrine immunoglobulin A.
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On the basis of previous work the two forms of human secretory component, namely that which is covalently bound as a part of the exocrine immunoglobulin A molecule and the free form, are probably different states of the same protein. From autoradiographs of trypic peptide maps of bound and free secretory components which were radioactively alkylated after partial reduction, it was concluded that the same half-cystines in each are sensitive to reduction. in the present work the easily reduced half-cystines of the bound and free secretory components have been studies in more detail. In each form there are two such half-cystines. In the case of bound secretory component they provide the linkage to the remainder of the exocrine immunoglobulin A molecule. Peptides from the sensitive half-cystines were isolated from tryptic-peptic digests of free secretory component and sequenced. By diagonal electrophoresis these two peptides were shown to be joined in an intrachain disulfide bridge. Therefore, it is proposed that the exocrine immunoglobulin A molecule becomes fully assembled when a single, reactive intrachain disulfide bridge in free secretory component rearranges to yeild two interchain bridges with dimeric serum-type immunoglobulin A. This process is thought to occur within the epithelial lining cells of mucous membranes. (+info)
Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.
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Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern. (+info)
Reduction of GC --> TA transversion mutation by overexpression of MutS in Escherichia coli K-12.
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Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants. (+info)
Derivatization of cysteine and cystine for fluorescence amino acid analysis with the o-phthaldialdehyde/2-mercaptoethanol reagent.
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Previous reports (Drescher, D.G., and Lee, K.S. (1978) Anal. Biochem. 84, 559-569; Lee, K.S., and Drescher, D.G. (1978) Int. J. Biochem. 9, 457-467) have shown that high performance liquid chromatographic analysis of amino acids with the o-phthaldialdehyde/2-mercaptoethanol reagent (OPA/2-ME) is one of the most sensitive procedures currently available for micro amino acid analysis. In the present paper, methods are presented for the modification of cysteine and cystine in proteins for micro amino acid analysis using OPA/2-ME. Cysteine and cystine, which both show low fluorescence with OPA/2-ME, are converted to cysteic acid with performic acid directly, or to S-3-sulfopropylcysteine with 1,3-propane sultone after reduction of cystine with tri-n-butylphosphine. Cysteic acid and S-3-sulfopropylcysteine form highly fluorescent adducts with OPA/2-ME. The formation of S-3-sulfopropylcysteine in proteins and the subsequent hydrolysis of the proteins with methanesulfonic acid are particularly useful for complete amino acid analysis at the picomole level using a single sample. (+info)
Arg133Cys mutation of Notch3 in two unrelated Japanese families with CADASIL.
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OBJECTIVE: More than 80 unrelated, but all Caucasian, patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), originating from various communities around the world, have been molecularly identified. To clarify the occurrence of CADASIL in Orientals, we investigated Japanese families presenting as CADASIL. METHODS: We performed the PCR-SSCP and sequence analyses using genomic DNA, isolated from venous blood of participants under informed consent. PATIENTS: We identified two unrelated Japanese families with CADASIL, including 5 affected members through 2 generations. RESULTS: Each of the affected individuals developed recurrent strokes without risk factors resulting in progressive dementia, pseudobulbar palsy, and gait disturbances which started after the fifth decade of life. Although affected individuals had no vascular risk factors, they showed various degrees of narrowing of retinal arteries. Their MRI/CTs showed characteristics of the disease; bilateral small infarcts in the thalamus, basal ganglia, brain stem, and deep white matter in addition to the findings of leukoaraiosis. On SPECT imaging, there was severe hypoperfusion in the cortex as well as in the white matter. Ultrastructural studies revealed an abnormal deposition of granular osmiophilic materials (GOM) within the basal lamina of pericytes in muscular capillaries. On PCR-SSCP and sequence analyses, a heterozygous Arg133Cys mutation was present, in the affected individuals, in the exon 4 of Notch3 gene which is the hot spot region for CADASIL mutations in Caucasian families. None of the non-affected members nor the 50 Japanese normal controls revealed this mutation. CONCLUSION: Thus, our results confirm that CADASIL is a geographically widespread disorder caused by a Notch3 mutation. (+info)
The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione.
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During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage. (+info)