Synthesis, chemical identification, antioxidant capacities and immunological evaluation studies of a novel silver(I) carbocysteine complex.

A new silver-carbocysteine (Ccy-Ag) complex [Ag2(Ccy)2(H2O)2] has been synthesized and characterized by using a combination of FTIR, Raman, molar conductivity, (1)H NMR, electronic spectra, thermal analyses, X-ray powder diffraction (XRD) and scanning electron microscopy (SEM). The infrared spectrum of Ccy-Ag complex in comparison with carbocysteine ligand prove that Ccy behaves as monobasic bidentate chelate to the silver metal ions via the deprotonated carboxylate O atom. The assessments of Ccy and its complexation with Ag(+) in treating COPD, evaluating immune activities through measuring IL-8, TGF-ß1, VEGF and TNF-α, antioxidant activities of (Ccy-Ag) complex by measuring SOD, MDA and GPX and bronchial asthma were discussed.

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction.

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N (ε)-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.

Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis.

All cloned sialyltransferases from vertebrates are classified into four subfamilies and are characterized as having type II transmembrane topology. The catalytic domain has highly conserved motifs known as sialylmotifs. Besides sialylmotifs, each family has several unique conserved cysteine (Cys) residues mainly in the catalytic domain. The number and loci of conserved amino acids, however, differ with each subfamily, suggesting that the conserved Cys-residues and/or disulphide linkages they make may contribute to linkage specificity. Using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF)-mass spectrometry, the present study performed disulphide linkage analysis on soluble mouse ST6Gal-I, which has six Cys-residues. Results confirmed that there were no free Cys-residues, and all six residues contributed to disulphide linkage formation, C(139)-C(403), C(181)-C(332) and C(350)-C(361). Study of single amino acid-substituted mutants revealed that the disulphide linkage C(181)-C(332) was necessary for molecular expression of the enzyme, and that the disulphide linkage C(350)-C(361) was necessary for enzyme activity. The remaining disulphide linkage C(139)-C(403) was not necessary for enzyme expression or for activity, including substrate specificity. Crystallographic study of pig ST3Gal I has recently been reported. Interestingly, the loci of disulphide linkages in ST6Gal-I differ from those in ST3Gal I, suggesting that the linkage specificity of sialyltransferase may results from significant structural differences, including the loci of disulphide linkages.

Carbocisteine can scavenge reactive oxygen species in vitro.

BACKGROUND AND OBJECTIVE: Reactive oxygen species (ROS) play an important role in the pathogenesis of various respiratory diseases. Carbocisteine, a mucoregulatory drug, is used in the treatment of several disease states but little information is available about its scavenger effects on ROS. The present study was designed to examine the scavenger effects of carbocisteine on ROS. METHODS: The oxidation-reduction potential of carbocisteine was measured, and its scavenger effects on hypochlorous acid (HOCl), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH(*)) and peroxynitrite (ONOO(-)) were examined in cell-free conditions. The effects of carbocisteine on ROS generated from rat neutrophils, intracellular oxidative stress and release of inflammatory cytokines (IL-8 and IL-6) from IL-1 beta-induced airway epithelial cells, NCI-H292 cells, were investigated. RESULTS: Carbocisteine provided a reducing stage and showed scavenger effects on H(2)O(2), HOCl, OH(*) and ONOO(-) in cell-free conditions. Carbocisteine inhibited ROS generation from rat neutrophils, intracellular oxidative stress and release of IL-8 and IL-6 from NCI-H292 cells. N-acetyl-L-cysteine, a radical scavenger, also inhibited these events related to ROS as well as carbocisteine. CONCLUSIONS: These results suggest that carbocisteine could exert anti-inflammatory and anti-oxidant effects through directly scavenging ROS in addition to its previously known mucoregulatory effect.

The suppression of enhanced bitterness intensity of macrolide dry syrup mixed with an acidic powder.

The aim of the present study was to identify a medicine which strongly enhanced the bitterness of clarithromycin dry syrup (CAMD) when administered concomitantly and to develop a method to suppress this enhanced bitterness. The bitterness enhancement was evaluated not only by gustatory sensation tests but also using pH and taste sensor measurements of the mixed sample. A remarkable bitterness enhancement was found when CAMD was mixed with the acidic powder L-carbocysteine. The acidic pH (pH 3.40) of the suspension made from these two preparations, seemed to be due to enhanced release of clarithromycin caused by the dissolution of the alkaline polymer film-coating. Several methods for preventing this bitterness enhancement were investigated. Neither increasing the volume of water taken with the mixture, nor changing the ratio of CAMD:L-carbocysteine in the mixture, were effective in reducing the bitterness intensity of the CAMD/L-carbocysteine mixture. The best way to achieve taste masking was to first administer CAMD mixed with chocolate jelly, which has a neutral pH, followed by the L-carbocysteine suspension. Similar results were obtained for the bitterness suppression of azithromycin fine granules with L-carbocysteine. The chocolate jelly will be useful for taste masking of bitter macrolide drug formulations, when they need to be administered together with acidic drug formulations.

Fluorimetric determination of some sulfur containing compounds through complex formation with terbium (Tb+3) and uranium (U+3).

Two simple, sensitive and specific fluorimetric methods have been developed for the determination of some sulphur containing compounds namely, Acetylcysteine (Ac), Carbocisteine (Cc) and Thioctic acid (Th) using terbium Tb+3 and uranium U+3 ions as fluorescent probes. The proposed methods involve the formation of a ternary complex with Tb+3 in presence of Tris-buffer method (I) and a binary complex with aqueous uranyl acetate solution method (II). The fluorescence quenching of Tb+3 at 510, 488 and 540 nm (lambda(ex) 250, 241 and 268 nm) and of uranyl acetate at 512 nm (lambda(ex) 240 nm) due to the complex formation was quantitatively measured for Ac, Cc and Th, respectively. The reaction conditions and the fluorescence spectral properties of the complexes have been investigated. Under the described conditions, the proposed methods were applicable over the concentration range (0.2-2.5 microg ml(-1)), (1-4 microg ml(-1)) and (0.5-3.5 microg ml(-1)) with mean percentage recoveries 99.74+/-0.36, 99.70+/-0.52 and 99.43+/-0.23 for method (I) and (0.5-6 microg ml(-1)), (0.5-5 microg ml(-1)), and (1-6 microg ml(-1)) with mean percentage recoveries 99.38+/-0.20, 99.82+/-0.28 and 99.93+/-0.32 for method (II), for the three cited drugs, respectively. The proposed methods were successfully applied for the determination of the studied compounds in bulk powders and in pharmaceutical formulations, as well as in presence of their related substances. The results obtained were found to be in agree statistically with those obtained by official and reported ones. The two methods were validated according to USP guidelines and also assessed by applying the standard addition technique.

Evidence for the formation of adducts and S-(carboxymethyl)cysteine on reaction of alpha-dicarbonyl compounds with thiol groups on amino acids, peptides, and proteins.

Nonenzymatic covalent adduction of glucose, or aldehydes derived from glucose or oxidation reactions, to proteins (glycation) has been proposed as a key factor in the vascular complications of diabetes. In conditions of chronic glucose elevation, alpha-dicarbonyl compounds, including glyoxal and methylglyoxal, are also present at elevated levels. These carbonyls react rapidly with nucleophilic groups on Lys and Arg side chains and the N-terminal amino group, to give poorly defined products, often called advanced glycation endproducts. These are present at elevated levels in tissue samples from people with diabetes and have been linked with disease development. As the thiol group of Cys is a powerful nucleophile, we hypothesized that adduction should occur rapidly and efficiently at Cys residues. It is shown here that Cys residues react with dicarbonyl compounds to give thiol-aldehyde adducts, which have been detected by electrospray ionization mass spectrometry. This process is accompanied by loss of the thiol group and formation of stable products. In the case of glyoxal, these reactions give S-(carboxymethyl)cysteine. The percentage conversion of thiol lost to product is substrate-dependent and < or = 32%. S-(Carboxymethyl)cysteine has been quantified by HPLC on thiol-containing, protected amino acids, peptides, and proteins, after exposure to glyoxal. The yield of this product has been shown to increase in a time- and dose-dependent manner with higher glyoxal concentrations and to also be formed on extended incubation of serum albumin with glucose. This novel, stable, advanced glycation endproduct is a potential marker of glycation.

Phenylalanine hydroxylase: possible involvement in the S-oxidation of S-carboxymethyl-l-cysteine.

Activated phenylalanine 4-monooxygenase, phenylalanine hydroxylase (PAH), is known to be involved in the S-oxidation of a number of sulfide compounds. One of these compounds, S-carboxymethyl-l-cysteine (SCMC), is currently used for the treatment of chronic obstructive pulmonary disease and otitis media with effusion as a mucolytic agent, and the S-oxides are the major metabolites found in urine. However, the enzyme catalyzing the S-oxidation of SCMC has yet to be identified. Here we report on the role of nonactivated phenylalanine 4-monooxygenase activity in rat liver cytosol in the S-oxidation of SCMC. Linearity of the enzyme assays was seen for both time (0-16 min) and cytosolic protein concentration (0.1-0.5mg/ml). The calculated K(m) and V(max) values for the formation of SCMC (S) S-oxide were 3.92+/-0.15 mM and 1.10+/-0.12 nmol SCMC (S) S-oxide formed/mg protein/min, respectively. The calculated K(m) and V(max) values for the formation of SCMC (R) S-oxide were 9.18+/-1.13 mM and 0.46+/-0.11 nmol SCMC (R) S-oxide formed/mg protein/min, respectively. These results indicate that in the female Wistar rat, nonactivated PAH showed a stereospecific preference for the formation of the (S) S-oxide metabolite of SCMC against the (R) S-oxide metabolite of SCMC.

The effect of cysteine analogues on the excretion of urinary sulphate in the rat following cysteine administration.

A major pathway for the production of sulphate within the mammalian body is known to be via the oxidative degradation of the sulphur moiety within the amino acid, L-cysteine. The ability of two structurally similar sulphur-containing drugs, the anti-rheumatic agent, D-penicillamine, and the mucoactive compound, S-carboxymethyl-L-cysteine, to interfere with this sulphate production was investigated. Co-administration to the male rat of D-penicillamine (p.o.) and S-carboxymethyl-L-cysteine (p.o.) with [35S]-L-cysteine (i.p.) led to a significant decrease in the subsequent urinary elimination of inorganic sulphate whilst having no measurable effect on organic sulphate excretion. The co-administration of L-valine, an amino acid not containing sulphur, had no effect. It is not known where, within the complex sequence of events surrounding the degradation of cysteine to sulphate, that D-penicillamine or S-carboxymethyl-L-cysteine may interact.

Spectrophotometric determination of penicillamine and carbocisteine based on formation of metal complexes.

A simple spectrophotometric method was developed for the determination of penicillamine and carbocisteine. The method depends on complexation of penicillamine with Ni, Co and Pb ions in acetate buffer pH of 6.3, 6.5 and 5.3, respectively, and carbocisteine with Cu and Ni ions in borate buffer pH of 6.7; 1-70 microg/ml of these drugs could be determined by measuring the absorbance of each complex at its specific lambdamax. The results obtained are in good agreement with those obtained using the official methods. The proposed method was successfully applied for the determination of these compounds in their dosage forms. Also, the molar ratio and stability constant of the metal complexes were calculated and a proposal of the reaction pathway was postulated.

Transfection of fluorescent probed antisense compounds: L-cysteine derivatives of nucleic acid bases.

Antisense with L-cysteine derivative (CAS) can recognize DNA and forms the complementary duplex with DNA. So the properties of CAS in vitro and in vivo were examined in this study. CAS was resistant to proteinase K and stabilized RNA against RNase HI. Moreover using fluorescent CAS, the localization was observed by fluorescence microscope and confocal microscope. As a result, CASs were accumulated inside the nucleus in NG108-15.

Study about the inhibition of L-cysteine derivatives of nucleic acid bases in protein production.

Isopoly (S-carboxymethyl-L-cysteine) derivatives of nucleic acids bases were prepared as antisense compounds. In past study, we investigated the properties of these compounds in vitro, and revealed that these compounds in vivo regulated the cell death presumably due to the inhibition of protein production. In this study, western and northern blots were carried out in order to reveal the mechanism of this inhibition for N-methyl-D-aspartate receptor in neuroblastoma x glioma hybrid NG108-15 cell line. In addition, we investigated the resistance of these compounds against cell extract and the metabolism. In conclusion, we proved that these compounds inhibited the protein production by antisense mechanism.

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase beta subunit determined by 13C-NMR.

Glutamic acid-190 in the beta subunit of F1-ATPase from thermophilic Bacillus PS-3 (TF1) was reported to be essential for the ATPase activity. The mutant TF1beta subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with 13C-labeled monoiodoacetic acid. The pKa value of the carboxymethylene group at the 190 position was determined as 5.6 +/- 0.4 by 13C-NMR. On the basis of this value, the pKa of the carboxylate of Glu-190 of the TF1beta subunit was estimated to be 6.8 +/- 0.5. The unusually high pKa could play a role in the catalytic mechanism of F1-ATPase.

Kinetics of the reaction of S-carboxymethyl-L-cysteine with palladium(II) chloride.

The influence of the acidity and the Cl- concentration on the kinetics of the complex formation between s-carboxymethyl-L-cysteine and Pd(II) chloride was studied with a stopped-flow technique in the pH range from 1.5 to 5.0 at 25 degrees C. The reaction mechanism involving Pd(H2O)4-nCln, LH-, LH2 and LH3+ species was proposed. Fairly good agreement between the computed and experimentally determined rate constants was found.

High-performance liquid chromatographic determination of hypotaurine and taurine after conversion to 4-dimethylaminoazobenzene-4'-sulfonyl derivatives and its application to the urine of cysteine-administered rats.

A method for the determination of urinary hypotaurine and taurine for the purpose of studying hypotaurine-taurine status in mammals is described. Hypotaurine and taurine were converted into 4-dimethylaminoazobenzene-4'-sulfonyl (dabsyl) derivatives under conditions minimizing hypotaurine oxidation. The dabsylhypotaurine and dabsyltaurine formed were determined by reversed-phase HPLC. Average excretions of taurine and hypotaurine in rat urine were 270.5 and 2.5 mumol/kg body mass per day, respectively. After intraperitoneal injection of 5 mmol of L-cysteine/kg body mass, the increased excretions of taurine and hypotaurine corresponded to 22.1 and 2.5%, respectively, of L-cysteine administered, indicating that hypotaurine production exceeded the capacity of hypotaurine oxidation in vivo.

Drug residue formation from ronidazole, a 5-nitroimidazole. VIII. Identification of the 2-methylene position as a site of protein alkylation.

Ronidazole protein-bound adducts were generated by the in vitro anaerobic incubation of [2-methylene-14C]ronidazole with microsomes from the livers of male rats. Acid hydrolysis of the protein adducts yielded an imidazole ring fragment bearing the radiolabel and an amino acid residue derived from the proteins. This fragment has been identified as carboxymethylcysteine by co-chromatography of the amino acid and its dansyl derivative with known standards under a variety of conditions. The carboxymethylcysteine was estimated to represent at least 15% of the radioactivity derived from the protein-bound adducts and provides unequivocal evidence that nucleophilic attack by protein cysteine thiols occurred at the 2-methylene position of ronidazole.