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(1/972) N,N'-Diacetyl-L-cystine-the disulfide dimer of N-acetylcysteine-is a potent modulator of contact sensitivity/delayed type hypersensitivity reactions in rodents.

Oral N-acetyl-L-cysteine (NAC) is used clinically for treatment of chronic obstructive pulmonary disease. NAC is easily oxidized to its disulfide. We show here that N,N'-diacetyl-L-cystine (DiNAC) is a potent modulator of contact sensitivity (CS)/delayed type hypersensitivity (DTH) reactions in rodents. Oral treatment of BALB/c mice with 0.003 to 30 micromol/kg DiNAC leads to enhancement of a CS reaction to oxazolone; DiNAC is 100 to 1000 times more potent than NAC in this respect, indicating that it does not act as a prodrug of NAC. Structure-activity studies suggest that a stereochemically-defined disulfide element is needed for activity. The DiNAC-induced enhancement of the CS reaction is counteracted by simultaneous NAC-treatment; in contrast, the CS reaction is even more enhanced in animals treated with DiNAC together with the glutathione-depleting agent buthionine sulfoximine. These data suggest that DiNAC acts via redox processes. Immunohistochemically, ear specimens from oxazolone-sensitized and -challenged BALB/c mice treated with DiNAC display increased numbers of CD8(+) cells. DiNAC treatment augments the CS reaction also when fluorescein isothiocyanate is used as a sensitizer in BALB/c mice; this is a purported TH2 type of response. However, when dinitrofluorobenzene is used as a sensitizer, inducing a purported TH1 type of response, DiNAC treatment reduces the reaction. Treatment with DiNAC also reduces a DTH footpad-swelling reaction to methylated BSA. Collectively, these data indicate that DiNAC in vivo acts as a potent and effective immunomodulator that can either enhance or reduce the CS or DTH response depending on the experimental conditions.  (+info)

(2/972) The bspA locus of Lactobacillus fermentum BR11 encodes an L-cystine uptake system.

BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, from Lactobacillus reuteri. Compared with the database of Escherichia coli proteins, BspA is most similar to the L-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take up L-cystine. Uptake rates of L-glutamine, L-histidine, and L-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of L. fermentum BR11 with L-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed. L. fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.  (+info)

(3/972) Degradation of hepatic zinc-thionein after parenteral zinc administration.

A low-molecular-weight protein, zinc-thionein, a metallothionein, was implicated as having a regulatory function in zinc metabolism. The half-life (t 1/2) of hepatic zinc-thionein was determined by pulse-labelling with either L-[35S] cystine and/or 65Zn. In two experiments with L-[35S]cystine, the t 1/2 of zinc-thionein was 18h and 19h. Most of the soluble 35S-labelled hepatic proteins had a t 1/2 of 4 days. The t 1/2 of zinc-thionein calculated by using 65Zn was 20h. The close similarity between the calculated and measured t 1/2 values for zinc-thionein suggests that release of Zn2+ from zinc-thionein probably occurs simultaneously with degradation of the protein moiety.  (+info)

(4/972) Stimulation of cystine uptake by nitric oxide: regulation of endothelial cell glutathione levels.

Nitric oxide (NO) is known to produce some of its biological activity through modification of cellular thiols. Return of cellular thiols to their basal state requires the activity of the GSH redox cycle, suggesting important interactions between NO signaling and regulation of cellular redox status. Because continuous exposure to NO may lead to adaptive responses in cellular redox systems, we investigated the effects of NO on cellular GSH levels in vascular endothelial cells. Acute exposure (1 h) of cells to >1 mM S-nitroso-N-acetyl-penicillamine (SNAP) led to depletion of GSH. On the other hand, chronic exposure to lower concentrations of SNAP (+info)

(5/972) Elevated expression of liver gamma-cystathionase is required for the maintenance of lactation in rats.

Liver gamma-cystathionase activity increases in rats during lactation; its inhibition due to propargylglycine is followed by a significant decrease in lactation. This is reversible by N-acetylcysteine administration. To study the role of liver gamma-cystathionase and the intertissue flux of glutathione during lactation, we used lactating and virgin rats fed liquid diets. Virgin rats were divided into two groups as follows: one group was fed daily a diet containing the same amount of protein that was consumed the previous day by lactating rats (high protein diet-fed rats); the other virgin group was fed the normal liquid diet (control). The expression and activity of liver gamma-cystathionase were significantly greater in lactating rats and in high protein diet-fed virgin rats compared with control rats. The total glutathione [reduced glutathione (GSH) + oxidized glutathione (GSSG)] released per gram of liver did not differ in lactating rats or in high protein diet-fed rats, but it was significantly higher in these two groups than in control virgin rats. Liver size and the GSH + GSSG released by total liver were significantly higher in lactating rats than in high protein diet-fed virgin rats, and this difference was similar to the amount of glutathione taken up by the mammary gland (454.2 +/- 36.0 nmol/min). The uptake of total glutathione by the lactating mammary gland was much higher than the uptakes of free L-cysteine and L-cystine, which were negligible. These data suggest that the intertissue flux of glutathione is an important mechanism of L-cysteine delivery to the lactating mammary gland, which lacks gamma-cystathionase activity. This emphasizes the physiologic importance of the increased expression and activity of liver gamma-cystathionase during lactation.  (+info)

(6/972) 1H-NMR structural studies of a cystine-linked peptide containing residues 71-93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy.

The Ile-->Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using 1H-NMR spectroscopy. The peptide, containing residues 71-93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix-turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84-->Ser substitution it was found that the substitution does not greatly disrupt the overall three-dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix-turn motif is still present, but differences in chemical shifts and amide-exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.  (+info)

(7/972) PCR random mutagenesis into Escherichia coli serine acetyltransferase: isolation of the mutant enzymes that cause overproduction of L-cysteine and L-cystine due to the desensitization to feedback inhibition.

PCR random mutagenesis in the cysE gene encoding Escherichia coli serine acetyltransferase was employed to isolate the mutant enzymes that, due to a much less feedback inhibition by L-cysteine, cause overproduction of L-cysteine and L-cystine in the recombinant strains. The L-cysteine auxotrophic and non-utilizing E. coli strain was transformed with plasmids having the altered cysE genes. Then, several transformants overproducing L-cysteine were selected by detecting the halo formation of the L-cysteine auxotroph. The production test of amino acids and analysis of the catalytic property on the mutant enzymes suggest that the carboxy-terminal region of serine acetyltransferase plays an important role in the desensitization to feedback inhibition and the high level production of L-cysteine and L-cystine.  (+info)

(8/972) Differential reconstitution of mitochondrial respiratory chain activity and plasma redox state by cysteine and ornithine in a model of cancer cachexia.

The mechanism of wasting, as it occurs in malignant diseases and various etiologically unrelated conditions, is still poorly understood. We have, therefore, studied putative cause/effect relationships in a murine model of cancer cachexia, C57BL/6 mice bearing the fibrosarcoma MCA-105. The plasma of these mice showed decreased albumin and increased glutamate levels, which are typically found in practically all catabolic conditions. Skeletal muscles from tumor-bearing mice were found to have an abnormally low mitochondrial respiratory chain activity (mito.RCA) and significantly decreased glutathione (GSH) levels. The decrease in mito.RCA was correlated with an increase in the i.m. GSH disulfide/GSH ratio, the plasma cystine/thiol ratio, and the GSH disulfide/GSH ratio in the bile. This is indicative of a generalized shift in the redox state extending through different body fluids. Treatment of tumor-bearing mice with ornithine, a precursor of the radical scavenger spermine, reversed both the decrease in mito.RCA and the change in the redox state, whereas treatment with cysteine, a GSH precursor, normalized only the redox state. Treatment of normal mice with difluoromethyl-ornithine, a specific inhibitor of ornithine decarboxylase and spermine biosynthesis, inhibited the mito.RCA in the skeletal muscle tissue, thus illustrating the importance of the putrescine/spermine pathway in the maintenance of mito.RCA. Ornithine, cysteine, and N-acetyl-cysteine (NAC) also reconstituted the abnormally low concentrations of the GSH precursor glutamate in the skeletal muscle tissue of tumor-bearing mice. Higher doses, however, enhanced tumor growth and increased the plasma glucose level in normal mice. In the latter, cysteine and NAC also decreased i.m. catalase and GSH peroxidase activities. Taken together, our studies on the effects of ornithine, cysteine, and NAC illuminate some of the mechanistic pathways involved in cachexia and suggest targets for therapeutic intervention.  (+info)