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Yohimbine is a small indole alkaloid derived from the bark of the yohimbe tree with documented biological activity, including anti-inflammatory, erectile dysfunction relieving, and fat-burning properties. Hydrogen sulfide (H 2 S) and sulfane sulfur-containing compounds are regarded as important molecules in redox regulation and are involved in many physiological processes. Recently, their role in the pathophysiology of obesity and obesity-induced liver injury was also reported. The aim of the present study was to verify whether the mechanism of biological activity of yohimbine is related to reactive sulfur species formed during cysteine catabolism. We tested the effect of yohimbine at doses of 2 and 5 mg/kg/day administered for 30 days on aerobic and anaerobic catabolism of cysteine and oxidative processes in the liver of high-fat diet (HFD)-induced obese rats. Our study revealed that HFD resulted in a decrease in cysteine and sulfane sulfur levels in the liver, while sulfates were elevated. In the liver of obese rats, rhodanese expression was diminished while lipid peroxidation increased. Yohimbine did not influence sulfane sulfur and thiol levels in the liver of obese rats, however, this alkaloid at a dose of 5 mg decreased sulfates to the control level and induced expression of rhodanese. Moreover, it diminished hepatic lipid peroxidation. It can be concluded that HFD attenuates anaerobic and enhances aerobic cysteine catabolism and induces lipid peroxidation in the rat liver. Yohimbine at a dose of 5 mg/kg can alleviate oxidative stress and reduce elevated concentrations of sulfate probably by the induction of TST expression.

Background: Modulation of the host immune response by nematode parasites has been widely reported. Rhodaneses (thiosulfate: cyanide sulfurtransferases) are present in a wide range of organisms, such as archaea, bacteria, fungi, plants and animals. Previously, it was reported that a rhodanese homologue could be bound by goat peripheral blood mononuclear cells (PBMCs) in vivo., Methods: In the present study, we cloned and produced a recombinant rhodanese protein originating from Haemonchus contortus (rHCRD), a parasitic nematode of small ruminants. rHCRD was co-incubated with goat PBMCs to assess its immunomodulatory effects on proliferation, apoptosis and cytokine secretion., Results: We verified that the natural HCRD protein localized predominantly to the bowel wall and body surface of the parasite. We further demonstrated that serum produced by goats artificially infected with H. contortus successfully recognized rHCRD, which bound to goat PBMCs. rHCRD suppressed proliferation of goat PBMCs stimulated by concanavalin A but did not induce apoptosis in goat PBMCs. The production of TNF-α and IFN-γ decreased significantly, whereas secretion of IL-10 and TGF-β1 increased, in goat PBMCs after exposure to rHCRD. rHCRD also inhibited phagocytosis by goat monocytes. Moreover, rHCRD downregulated the expression of major histocompatibility complex (MHC)-II on goat monocytes in a dose-dependent manner, but did not alter MHC-I expression., Conclusions: These results propose a possible immunomodulatory target that may help illuminate the interactions between parasites and their hosts at the molecular level and reveal innovative protein species as candidate drug and vaccine targets.

We have analyzed a series of peptides derived from the C-terminus of alpha-synuclein for chaperone-like activity. Specifically, a cyclic peptide generated by introducing a disulfide bond was observed to increase chaperone-like activity. This is the first example of a disulfide-crosslinked peptide that exhibits activity against protein aggregation and activity loss.

3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S(4)O(6)(2-)) inhibited rat MST (k(i) = 3.3 min(-1), K(i) = 120.5 microM and k(i) = 2.5 min(-1), K(i) = 178.6 microM, respectively). The activity was completely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys(247), suggesting that Cys(247) is a target of the oxidants. A matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis revealed that hydrogen peroxide- and tetrathionate-inhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate (S(2)O(3)(-)), respectively. Treatment with dithiothreitol restored modified MST to the original mass. These findings suggested that there was no nearby cysteine with which to form a disulfide, and mild oxidation of MST resulted in formation of a sulfenate (SO(-)) at Cys(247), which exhibited exceptional stability and a lower redox potential than that of glutathione. Oxidative stress decreases MST activity so as to increase the amount of cysteine, a precursor of thioredoxin or glutathione, and furthermore, these cellular reductants restore the activity. Thus the redox state regulates MST activity at the enzymatic level, and on the other hand, MST controls redox to maintain cellular redox homeostasis.

The role of vascular smooth muscle inward rectifier K+ (K(IR)) channels in the mechanisms underlying vasodilation is still unclear. The hypothesis that K(IR) channels are involved in sodium nitroprusside (SNP)-induced dilation of rat-tail small arteries was tested. SNP relaxed tail small arteries with an EC50 of 2.6x10(-8) mol/L. Endothelium removal did not attenuate this effect. Vessel pretreatment with hydroxocobalamin, a nitric oxide (NO) scavenger, but not with rhodanese and sodium thiosulfate, inactivators of cyanide (CN), abolished the SNP effect. Vessel pretreatment with 10(-5) mol/L Ba2+, a specific blocker of K(IR) channels at micromolar concentrations, reduced the SNP effect. Low concentrations of K+ dilated the vessels; this effect was attenuated largely after pretreatment with 3x10(-5) mol/L Ba2+. In freshly isolated smooth muscle cells, a barium-sensitive current was observed at potentials negative to the potassium equilibrium potential. Application of 10(-4) mol/L SNP increased the barium-sensitive current 1.79+/-0.23-fold at -100 mV and hyperpolarized the membrane potential by 8.6+/-0.5 mV. In tissue from freshly dissected vessels, transcripts for K(IR) 2.1 and 2.2, but not for K(IR) 2.3 and 2.4, were found. However, only K(IR) 2.1 antibodies immunostained the tunica media of the vessel. These data suggest that vascular smooth muscle K(IR) 2.1 channels are involved in the SNP-induced dilation of rat-tail small arteries.

In the DnaK (Hsp70) molecular chaperone system of Escherichia coli, the substrate polypeptide is fed into the chaperone cycle by association with the fast-binding, ATP-liganded form of the DnaK. The substrate binding properties of DnaK are controlled by its two cochaperones DnaJ (Hsp40) and GrpE. DnaJ stimulates the hydrolysis of DnaK-bound ATP, and GrpE accelerates ADP/ATP exchange. DnaJ has been described as targeting the substrate to DnaK, a concept that has remained rather obscure. Based on binding experiments with peptides and polypeptides we propose here a novel mechanism for the targeting action of DnaJ: ATP.DnaK and DnaJ with its substrate-binding domain bind to different segments of one and the same polypeptide chain forming (ATP.DnaK)m.substrate.DnaJn complexes; in these ternary complexes efficient cis-interaction of the J-domain of DnaJ with DnaK is favored by their propinquity and triggers the hydrolysis of DnaK-bound ATP, converting DnaK to its ADP-liganded high affinity state and thus locking it onto the substrate polypeptide.

When the enzyme rhodanese was inactivated with hydrogen peroxide (H(2)O(2)), it underwent significant conformational changes, leading to an increased exposure of hydrophobic surfaces. Thus, this protein seemed to be an ideal substrate for GroEL, since GroEL uses hydrophobic interactions to bind to its substrate polypeptides. Here, we report on the facilitated reactivation (86%) of H(2)O(2)-inactivated rhodanese by GroEL alone. Reactivation by GroEL required a reductant and the enzyme substrate, but not GroES or ATP. Further, we found that GroEL interacted weakly and/or transiently with H(2)O(2)-inactivated rhodanese. A strong interaction with rhodanese was obtained when the enzyme was pre-incubated with urea, indicating that exposure of hydrophobic surfaces alone on oxidized rhodanese was not sufficient for the formation of a strong complex and that a more unfolded structure of rhodanese was required to interact strongly with GroEL. Unlike prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that GroEL can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein. Additionally, the mechanism for the GroEL-facilitated reactivation of rhodanese shown here appears to be different than that for the chaperonin-assisted folding of chemically unfolded polypeptides in which a nucleotide and sometimes GroES is required., ((c) 2002 Elsevier Science (USA).)

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degrees C compared with > or =60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45 degrees C and 37 degrees C for activase in the presence and absence of ATPgammaS, respectively, and at 65 degrees C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

In the last years, nitric oxide (NO) has emerged as an important intra- and intercellular transmitter involved in the control of the hypothalamic-pituitary axis, and NO synthase (NOS) has been identified in pituitary cells. To determine the role of NO in the control of GH secretion acting directly at the pituitary level, we have studied GH release by hemipituitaries incubated in the presence of different concentrations (10(-7)-10(-3) M) of sodium nitroprusside (SNP), a potent NO donor. We found that SNP (10(-4)-10(-3) M) stimulated GH release. This effect was mediated by the release of NO since it was abolished in the presence of hemoglobin, a scavenger of NO, but preserved in the presence of rhodanese + sodium thiosulfate (inactivators of cyanides generated from SNP). To analyze the participation of cyclic guanosine monophosphate (cGMP), the second messenger for a wide range of NO actions, in SNP-stimulated GH secretion, hemipituitaries were incubated in the presence of 8-bromo-cGMP (8-Br-cGMP; 10(-7)-10(-3) M). In addition, hemipituitaries were stimulated with SNP plus oxadiazoloquinoxaline (OQD) or LY 83,583 (inhibitors of guanylyl cyclases). We found that 8-Br-cGMP was ineffective in eliciting GH release, and that the stimulatory effect of SNP was maintained in presence of OQD and LY 83,583. Finally, to analyze calcium dependence, the SNP effect was studied in hemipituitaries incubated in free medium calcium, in the presence of nifedipine and verapamil (blockers of calcium channels) and after depletion of intracellular Ca(2+) stores with caffeine. We found that the SNP-induced GH secretion is also detected after incubation of hemipituitaries in free calcium medium, in the presence of nifedipine and verapamil, and after caffeine preincubation. We conclude that NO stimulates GH secretion in vitro through a specific calcium-cGMP-independent mechanism. Copyrightz1999S.KargerAG,Basel

The role of nitric oxide (NO) in glucose-induced insulin secretion was studied in pancreatic beta-cells, HIT-T15. A role for NO is suggested since glucose stimulated NO production in a concentration-dependent manner. NG-monomethyl-L-arginine, a potent inhibitor of nitric oxide synthase, significantly inhibited glucose-induced nitric oxide production as well as insulin release in HIT-T15. Furthermore, this inhibitory effect can be reversed by sodium nitroprusside (SNP), a well known NO donor. While SNP alone did not stimulate insulin release, it potentiated the secretory response of HIT-T15 cells to glucose by approximately two-fold. Potentiation by SNP appears to be mediated by NO, since (i) the potentiation was completely abolished by 10 microM hemoglobin, a scavenger of NO; and (ii) was not affected by rhodanese plus sodium thiosulphate. Neither hemoglobin alone nor the combination of rhodanese and sodium thiosulphate had any effect on glucose induced insulin release. These results are consistent with the hypothesis that glucose-induced formation of NO may potentiate the effect of glucose by a positive feedback mechanism., (Copyright 1998 Academic Press.)

There is increasing evidence that endogenously generated reactive oxygen (ROS) and reactive nitrogen (RNS) species at sites of inflammation and in tumors may be genotoxic. We have developed a murine tumor model (MN-11) in which mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus, arising both in vitro and in vivo, can be detected. In the present report, we describe an in vitro study of the ability of ROS and RNS to induce mutations in our model system. 137Cs radiation and radiomimetic drugs caused a dose-dependent increase in mutant frequency. At D0, radiation induced about 170 mutants per 10(5) viable cells, compared to 50 and 95 for streptonigrin and bleomycin, respectively. H2O2 induced a lower frequency of mutants, 20-30 per 10(5), for enzymatically generated or bolus, respectively. For the following treatments, mutant frequency at 50% survival is shown. Incubation with human granulocytes induced a low frequency of mutants (about 15 per 10(5)). RNS was tested using a series of NO-donating drugs. Spermine/NO. induced cytotoxicity but no mutants while S-nitroso-N-acetylpenicillamine induced a low level, 10 per 10(5). Both release nitrogen monoxide spontaneously, with a t1/2 < 3 h. Glyceryl trinitrate and sodium nitroprusside are two drugs that were slowly metabolized by MN-11 cells (> 12 h). Glyceryl trinitrate induced about 20 per 10(5) while nitroprusside induced 50 per 10(5). Our results indicate that RNS can readily induce mutations detectable in MN-11 cells. At equicytotoxic doses, the induced mutant frequency varied considerably for different drugs, suggesting that different states of nitrogen monoxide (such as NO+ or NO.) may be generated and these may vary in their mutagenic/cytotoxic potential.

A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation.

Previous studies reported that resealed erythrocytes containing rhodanese (CRBC) and NA2S2O3 rapidly metabolize cyanide to the less toxic thiocyanate both in vitro and in vivo. This provided a new conceptual approach to prevent and treat cyanide intoxication. Although the rhodanese-containing carrier cells with thiosulfate as the sulfur donor were efficacious, this approach has potential disadvantages, as thiosulfate has limited penetration of cell membrane and product inhibition of rhodanese can occur due to inorganic sulfite accumulation. In order to circumvent substrate limitation and product inhibition by sodium thiosulfate, organic thiosulfonates were explored. These thiosulfonates have higher lipid solubility than thiosulfate and therefore can replenish the depleted sulfur donor, as they can readily penetrate cell membranes. Also, product inhibition of rhodanese is less apt to occur. This change in sulfur donors should greatly enhance cyanide detoxication, replenish the sulfur donor, and minimize product inhibition of rhodanese. Present studies demonstrate the enhanced efficacy of exogenous organic thiosulfonates over sodium thiosulfate in the CRBC antidotal system to detoxify the lethal effects of cyanide either alone or in combinations with exogenously administered NaNO2. Murine carrier erythrocytes containing purified bovine liver rhodanese were administered intravenously into male Balb/C mice. Subsequently, butanethiosulfonate (BTS) or Na2S2O3 (ip), and NaNO2 (sc) were co-administered prior to KCN (sc). Potency ratios, derived from the LD50 values, were compared in groups of mice treated with CRBC-Na2S2O3 or CRBC-BTS either alone or in combination with NaNO2.(ABSTRACT TRUNCATED AT 250 WORDS)

We demonstrate that the previously observed stabilizing effect by the enzyme rhodanese of the oligomeric structure of Cpn60 is general and can be provided by six other proteins that can interact with Cpn60. All these substrate proteins, which include examples that are monomeric, as well as oligomeric polypeptides in their native states, were shown previously to be assisted in their refolding by the chaperonin. Strikingly, during the disassembly of Cpn60 in the presence of any of the substrate proteins, significant amounts of intermediates were detected. Furthermore, unfolded substrate proteins induce the reassembly of tetradecameric Cpn60 from monomers, and binding of each substrate protein stabilizes Cpn60 quaternary structure.

Rhodanese (thiosulfate cyanide sulfurtransferase; E.C. 2.8.1.1) is a mitochondrial enzyme that is unprocessed after import. We describe in vitro experiments showing that partially folded rhodanese can interact with lipid bilayers. The interaction was monitored by measuring the ability of rhodanese to disrupt small unilamellar vesicles composed of phosphatidylserine and to release 6-carboxyfluorescein that was trapped in the liposomes. Partially folded rhodanese, derived by dilution of urea-unfolded enzyme, efficiently induced liposome leakage. Native rhodanese had no effect on liposome integrity. Liposome disruption progressively decreased as rhodanese was given the opportunity to refold or aggregate before introduction of the liposomes. A synthetic 23 amino acid peptide representing the N-terminal sequence of rhodanese was very efficient at disrupting the liposomes. Shorter peptides chosen from within this sequence (residues 11-23 or residues 1-17) had no effect on liposome disruption. A peptide representing the tether region that connects the domains of the enzyme was also without effect. These results are consistent with the hypothesis that the N-terminal sequence of rhodanese is an uncleaved leader sequence, and can interact with membrane components that are involved in the mitochondrial uptake of this protein.

Previous studies have indicated that nitric oxide is involved in the lysis of pancreatic islet cells by inflammatory macrophages. Here we show that the incubation of islet cells with chemical NO-donors leads to cell lysis in a concentration and time dependent way. Islet cell death could be prevented by nicotinamide and 3-aminobenzamide, which are known to inhibit ADP-ribosylation, while several scavengers of oxygen radicals, N-acetylcysteine, dihydrolipoic acid, dimethylthiourea and citiolone, provided no protection.

The inactive 2Fe species of the Fe protein of the nitrogenase of Klebsiella pneumoniae was generated by treating oxidized Fe protein (Kp2) with MgATP and chelator. Incubation of the 2Fe species of Kp2 with the sulphurtransferase rhodanese in the presence of thiosulphate, ferric citrate and reduced lipoate reproducibly restored activity. The extent of restoration of activity depended on the molar ratio of 2Fe Kp2 to rhodanese and was time-dependent. Re-activation did not occur in the reaction mixture lacking rhodanese.