Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide / Níveis de glutationa e capacidade antioxidante total em células de Candida sp. expostas a estresse oxidativo causado por peróxido de hidrogênio

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

INTRODUÇÃO: A capacidade de suportar o estresse oxidativo imposto por fagócitos parece ser crítica para que espécies de Candida causem candidíase invasiva. MÉTODOS: Para melhor caracterizar a resposta ao estresse oxidativo (REO) de oito Candida sp. clinicamente relevantes, um componente vital do balanço redox intracelular, a glutationa, foi mensurada pelo método de reconversão DTNB-GSSG redutase e a capacidade antioxidante total (CAT) foi mensurada por um método modificado baseado na descoloração do ABTS*+. Ambos os métodos foram utilizados em extratos celulares das espécies de Candida tratadas ou não com peróxido de hidrogênio (0,5mM). RESULTADOS: O estresse oxidativo induzido pelo peróxido de hidrogênio claramente reduziu os níveis intracelulares de glutationa. Esta diminuição foi mais intensa em C. albicans e os níveis de glutationa em células não tratadas foram também maiores nesta espécie. A capacidade antioxidante total demonstrou variação intraespecífica na capacidade antioxidante. CONCLUSÕES: Os níveis de glutationa não se correlacionaram com a capacidade antioxidante total mensurada, apesar desta ser a defesa antioxidante intracelular não-enzimática mais importante. Os resultados indicam que a medição isolada da CAT não fornece um quadro claro da habilidade de certa espécie de Candida responder ao estresse oxidativo.

Site-directed mutagenesis and sulfhydryl PEGylation of lysostaphin / 生物工程学报

The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133-154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni(2+)-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.

Expression of macrophage inflammatory protein 1 alpha in the endothelial cells exposed to diamide / 华中科技大学学报（医学）（英德文版）

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.

Expression of macrophage inflammatory protein 1 alpha in the endothelial cells exposed to diamide / 华中科技大学学报（医学）（英德文版）

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.

Identification of Cross-linked 46 KDa Protein in Experimentally Induced Silicotic Nodule in Rat Lung / 대한산업의학회지

OBJECTIVES: This study was conducted in order to understand the cellular events associated with silica-induced pathogenesis of the rat lung. METHODS: Silicosis was induced by an intratracheal instillation of 50 mg of silica (SiO2, 0.15 - 10 micrometer) suspended in 500 microliter of a sterile saline solution in Sprague-Dawley rats weighing 200g. Silicotic nodules were excised from the rat lungs 4 weeks after silica instillation, then boiled for 4 days at 110 degrees in solution containing 2% SDS, 10 M urea and 40 mM DTT. The insoluble cellular encapsulates were electrophoresed on 4-12 % gradient SDS-PAGE, and the amino acid composition was analyzed. Affinity chromatographies of the homogenate supernatants of the control lung, silicotic nodule, and normal rat plasma were performed using rabbit IgG, anti-rat, cross-linked protein from the silicotic nodule. The amounts of N epsilon-(gamma-glutamyl) lysine cross-linked in the control lungs and silicotic nodules were determined using HPLC analysis. RESULTS: The remaining cross-linked protein was insoluble in the 10 M urea and 40 mM sulfhydryl reagents even under prolonged boiling conditions. The encapsulate revealed the retention of silica particles within the protein whose amino acid composition showed a high percentage of alanine, leucine and glycine. A 46 KDa protein was identified as a cross-linked protein in the silicotic nodule by affinity chromatography. The level of N epsilon-(gamma-glutamyl) lysine dipeptide in the nodule digest was prominently increased compared with that in the control lung. CONCLUSIONS: Transglutaminase (TGase)-catalyzed cross-linking appears to be involved in the silicotic nodule formation, and the 46 KDa protein may be cross-linked to itself and other extracellular matrix proteins during fibrosis and the formation of eventually insoluble nodule.

Protective effect of N-acetylcysteine against gamma ray induced damages in rats--biochemical evaluations.

The effect of N-acetylcysteine (NAC) (Ig/kg body weight in saline for 7 days) against the damages induced by gamma ray was studied. Whole body exposure of rats to gamma-rays (3.5 Gy) caused increases in lipid peroxides (P < 0.01). Reduced glutathione (GSH) (P < 0.01) and total sulphydryl groups (TSH) (P < 0.05), were found to be increased probably to counteract the damages produced by the lipid peroxides. The plasma antioxidant vitamins E, C and A were reduced. The activities of antioxidant enzymes, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were enhanced, which might be to eliminate the superoxide radical and H2O2 and accompanied by a fall in glutathione-s-transferase (GST) and glutathione reductase (GR) activity. The excessive production of free radicals and lipid peroxides might have caused the leakage of cytosolic enzymes such as aminotransferases (AST and ALT), lactate dehydrogenase (LDH), creatine kinase (CK) and phosphatases. Membrane damage is quite evident from histological studies undertaken in the intestinal tissue, which is susceptible to radiation damage. Intragastric pretreatment of NAC (1g/kg body weight in saline for 7 days) prevented the radiation induced damage to an appreciable extent. From the results it may be concluded that NAC is effective in protecting from the damages caused by gamma-ray radiations and its prospects as an adjuvant to radiotherapy should be considered.

Diamide and cyclosporin A enhanced arsenic trioxide-induced apoptosis in NB4 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of mitochondrial membrane permeability transition pore (MPT)-opened agent diamide and MPT-closed agent cyclosporin A on arsenic trioxide (As(2)O(3))-induced apoptosis in acute promyelocytic leukemia (APL) cell line NB4.</p><p><b>METHODS</b>NB4 cells were treated with As(2)O(3) alone or in combination with diamide or cyclosporin A in different concentrations. Cell apoptosis was assessed by the morphological observation, Annexin-V assay, distribution of cellular DNA contents and genomic DNA electrophoresis. The mitochondrial transmembrane potentials (DeltaPsim) were detected by flow cytometry according to the intensity of rhodamine 123 uptake in cells.</p><p><b>RESULTS</b>Both diamide and cyclosporin A significantly enhanced As(2)O(3)-induced apoptosis in NB4 cells. The DeltaPsim collapse induced by As(2)O(3) was also enforced by combined treatment with diamide or cyclospo-rin A. 1 micromol/L As(2)O(3) alone treatment for 72 hours led to DeltaPsim disruption in 27.9% of cells, while combined treatment of As(2)O(3) and diamide or cyclosporin A increased DeltaPsim disruption cells to 59.7% and 42.2%, respectively.</p><p><b>CONCLUSIONS</b>As(2)O(3)-induced DeltaPsim disruption possibly involves with thiol oxidation or crosslink of important components especially ANT-related molecules.</p>

Electron microscopic changes in spermatozoa of albino rats, induced by dithiothreitol [DDT], a protective reagent for - SH group

Disulfide bonds formation plays an important role in many chemical reactions. The maturity of the heads and tails of spermatozoa are highly dependent on such bonds. Prevention of these chemical bonds would certainly affect such maturity. This has been achieved through the administration of dithiothreitol [DTT], a chemical compound which prevents the formation of such chemical bonds by maintaining the - SH group of the amino acid cysteine. Thirty adult male rats were employed, divided to 10 control received saline and Tris buffer and 20 experimental groups received 0.2 ml of 9 mM solution of dithiothreitol in 0.05 M Tris buffer, pH 8,0 injected subcutaneously daily for thirty days. All animals were killed after 30 days except ten experimental animals that retained for 15 more days to recover. The testes were dissected out and processed for preliminary examination with light microscopy and for electron microscopy. Due to the limited magnification of the light microscope, no major changes were visible. The electron microscope did show variable changes, specifically there have been many deformities in the nuclei, and disruption of the normal configuration and arrangement of the miotchondria; decondensation of the nuclear chromatin in some samples has also been detected. This would emphasize that DTT when maintained the - SH group from being oxidized, had interfered with the integrity of the spermatozoa, which consequently affected their maturity. Depending upon this result, one could speculate that compound of this nature and this mode of action could be considered when a male antifertility agent, is thought of

Effect of chloride and diamide on angiotensin converting enzyme from sheep testis and epididymis.

Effect of chloride and diamide on testicular and epididymal angiotensin converting enzyme (ACE) activity was investigated using Hip-His-Leu as substrate in sheep. The chloride ions functioned as ACE activators, however, there was no linear correlation between the two. The optimum chloride concentrations were 500 mM for epididymal ACE and 900-1100 mM for testicular ACE. Further, optimum chloride concentration increased ACE activity of testis and epididymis 25.40- folds and 12.84- folds respectively of the activities at physiological chloride concentration. The differences found in the effect of chloride on testicular and epididymal ACE activity suggest dissimilar three dimensional structure of ACE in these tissues. Increased testicular and epididymal ACE activity on diamide pretreatment indicates that tissue oxidation may affect ACE activity.

Radioprotection: mechanisms and radioprotective agents including honeybee venom

Since 1949, a great deal of research has been carried out on the radioprotective action of chemical substances. These substances have shown to reduce mortality when administered to animals prior to exposure to a lethal dose of radiation. This fact is of considerable importance since it permits reduction of radiation-induced damage and provides prophylactic treatment for the damaging effects produced by radiotherapy. The following radiprotection mechanisms were proposed: free radical scavenger, repair by hydrogen donation to target molecules, formation of mixed disulfides, delay of cellular division and induction of hypoxia in the tissues. Radiprotective agents have been divided into four major groups: the thiol compounds, other sulfur compounds, pharmacological agents (anesthetic drugs, analgesics, tranquilizers, etc.) and other radioprotective agents (WR-1065, WR-2721, vitamins C and E, glutathione, etc). Several studies revealed the radioprotective action of Apis mellifera honeybee venom as well as that of its components mellitin and histamine. Radioprotective activity of bee venom involves mainly the stimulation of the hematopoietic system. In addition, release of histamine and reduction in oxygen tension also contribute to the radioprotective action of bee venom.

Purification and characterization of neutral invertase from chickpea nodules.

Neutral invertase from nodules of chickpea (Cicer arietinum L.) was isolated and purified by ammonium sulphate fractionation, gel filtration and DEAE-cellulose column chromatography. The purified enzyme was stable between 0 to 40 degrees C beyond which it was irreversibly denatured. Optimum temperature and pH of the enzyme were 37 degrees C and 7.0, respectively. K(m) for sucrose was 14.2 mM and Vmax was 4.8 mumole hr-1. The enzyme was inhibited by several metal ions. From the temperature effect on K(m) and Vmax values, the energy of activation (Ea), enthalpy change (delta H) and entropy change (delta S) of the enzyme were calculated to be 147 kJmol-1, -4.10 kJmol-1 and -2.33 JK-1mol-1, respectively. By employing photo-oxidation and chemical modification and by studying the effect of pH on K(m) and Vmax, the involvement of sulphydryl-, imidazole- and alpha-amino groups in the active site of the enzyme has been indicated.

Glutatión S-transferasas en no-vertebrados y en mamíferos: su importancia en la detoxificación de insecticidas / The Glutathione S-transferases (GST) of non-vertebrate organisms and mammals

Las Glutatión S-transferasas (GST) de organismos no-vertebrados no han sido estudiadas con la misma intensidad que las de mamíferos. El interés en las GST en no-vertebrados radica en su importancia como protección bioquímica de los organismos expuestos a compuestos químicos ambientales. En efecto, se ha observado que niveles elevados de GST podrían estar asociados con la tolerancia a pesticidas. La intención de esta actualización es revisar el nivel de conocimiento actual sobre estas enzimas en no-vertebrados, comparándolas con las de mamíferos. Evaluar la contribución de estos estudios al conocimiento del rol de las glutinatión transferasas en general, e intentar discernir la dirección de las futuras investigaciones en este campo

Isatin (2,3-dioxoindole): a competitive inhibitor of Na(+)-dependent lysine uptake in rat instestine.

Isatin (2,3-dioxoindole) competitively inhibited (27-40%) Na(+)-dependent L-lysine uptake in rat intestine. The value of Kt was increased from 3.04 mM in control to 5.88 mM in presence of 10 mM isatin. Effect of isatin on the Na(+)-independent amino acid uptake was insignificant (12-18%). The inhibitory constant (Ki) was 2.8 mM under these conditions. The observed inhibition was unaffected by -SH group reacting agents. Isatin (1-10 mM) inhibited Na+, K(+)-ATPase activity in intestine in vitro, the maximum inhibition (66%) being at 10 mM isatin concentration. But the drug had no effect on enzyme activity under in vivo conditions.

Biochemical studies on citrullus vulgaris urease: stability and reactivation by some reducing agents

Watermelon [Citrullus vulgaris cv. "Giza 1"] urease was inhibited by the divalent metal ions. Mg2+, Ca2+, Ba2+ [10 mM] and Mn2+ [0.02 mM] partially inhibited the enzyme with 15.9, 28.8, 8.0 and 12.0% inhibition, respectively. The heavy metal ions inhibited the enzyme in the order of Hg2+ > Cd2+ > Zn2+ > Cu2+ > Co2+ > Ni2+. Thiols as reducing agents at low concentrations ranging from 0.0001 to 0.01 mM caused activation of the enzyme and preserved its activity. The enzyme was stored in 0.01 mM dithiothreitol and 0.1% penicillin- streptomycin. It was stable and free of bacterial contamination for 6 moths. The specification of this enzyme meets the prerequisites needed for preparation of diagnostic urea kit

Inactivation of glyceraldehyde-3-phosphate dehydrogenase with SH-reagents and its relationship to the protein quaternary structure.

Inactivation of mung bean glyceraldehyde-3-phosphate dehydrogenase (GPDH) with excess iodoacetate or N-ethylmaleimide exhibits pseudo-first order kinetics at pH 7.3 and 8.6 in the absence and presence of NAD+, suggesting that all the reactive SH groups (four per tetrameric GPDH molecule) have equivalent reactivity towards these reagents. This is similar to the D2-symmetry conformation proposed on the basis of thermal inactivation data [Malhotra and Srinivasan, Arch. Biochem. Biophys. 236, 775-781 (1985)]. With p-chloromercury benzoate (p-CMB), the inactivation of GPDH is very fast and its kinetics can be monitored at low reagent concentration only. Keeping a high molar p-CMB: enzyme ratio (= 47), the kinetics were found to be biphasic, with half of the activity being lost in a fast and the remaining in a slow phase, characteristic of C2-symmetry conformation and half site reactivity. The p-CMB inactivation could be largely reversed on the addition of excess cysteine. A comparison of these data with literature reports on this and other GPDHs reveals that all reagents having large non-polar moieties exhibit half site reactivity with this enzyme.

Studies on the utilization of inorganic pyrophosphate by different metabolic systems in yeast mitochondria

A mitochondrial pyrophosphatase (PPase) from yeast cells (Saccharomyces cerevisiae) was studied and characterized. The hydrolytic activity towards inorganic pyrophosphate (PPi) was inhibited by different SH-reagents and increased in the presence of uncouplers, indicating a possible involvement of this enzyme in energy-linked processes. This view was also supported by the observation that these mitochondria were able to hydrolyze PPi, generating an electrical membrane potential (delta psi) of the same magnitude as that obtained with ATP. Both ATP and PPi inhibited the pyruvate dehydrogenase complex and it was demonstrated that PPi can be used as substrate by mitochondrial kinases leading to the same pattern of protein phosphorylation as when ATP is used

Effect of SH reagents on rod photoresponse of isolated frog retina.

Several SH reagents, N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), p-chloromercuribenzene sulphonate (PCMBS) and monoiodoacetic acid (MIAA) changed the wave form and the peak of the amplitude of the photoresponse remarkably. The effects of amino group modifying reagents, ethyl acetimidate (EA) and isethinyl acetimidate (ITA) on photoresponse were very slight. The possibility of a SH protein as cGMP-sensitive cation channel protein is discussed.

Impact of surface modifications of Acanthocheilonema viteae microfilariae on cell adhesion.

Exposure of A. viteae microfilariae to various lectins reduced their capacity to react with the peritoneal exudate cells of the host, Mastomys natalensis. Sugars corresponding to these lectins with the exception of N-acetyl glucosamine, did not affect the adhesion per se. They however, protected the parasite against the adverse effect of lectins. Neuraminidase and chitinase also suppressed adhesion capacity of the microfilariae. Except sodium dodecylsulphate which enhanced cell attachment, other surfactants inhibited this reaction considerably. The results indicate that antibody dependent adhesion of the microfilariae with the macrophages involves surface moieties of the parasite, where N-acetylglucosamine acts as the principal sugar residue. Participation of -SH groups also is inferred from the observations that p-chloromercuribenzoate and dithiobis-(2-nitrobenzoic acid) inhibited cell attachment and dithiothreitol provided protection against these agents.