Stimulation of septation in mitochondria by diamide,a thiol oxidising agent

Summary Diamide (10^–4M), a thiol oxidizing agent, rapidly promotes septation in isolated frog liver mitochondria and also in situ in liver slices. The effect is partially inhibited by dithioerythritol. DNP does not have this effect, and it is concluded that diamide does not promote septation via an uncoupling action. The septate mitochondria have a different appearance from typical dividing mitochondria previously described; it is suggested that (1) diamide may act by favouring the fusion of the internal membranes, and (2) -SH oxidation is of importance in mitochondria in ageing and in various pathological conditions. The ways in which septa may develop in mitochondria in the orthodox and condensed configuration are discussed.     

Attenuation of 4-nitroquinoline 1-oxide induced in vitro lipid peroxidation by green tea polyphenols

Lipid peroxidation is believed to play an important role in pathogenesis of diseases. 4-Nitroquiunoline 1-oxide (4-NQO) a potent oral carcinogen, widely used for induction of oral carcinogenesis, was found to induce lipid peroxidation in vivo and in vitro. Green tea contains high content of polyphenols, which are potent antioxidants. Thus green tea polyphenols (GP) can play a protective role in 4-NQO induced in vitro lipid peroxidation. 4-NQO at the concentration of 1.5 mM was found to induce lipid peroxidation in 5% liver homogenate in phosphate buffered saline and extent of lipid peroxidation at the different time intervals 0, 15, 30 and 45 min where studied by assessing parameters such as hydroxyl radical production (OH), thiobarbituric acid reactants (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). It was found that addition of 4-NQO caused an increase in OH and TBARS level and a decrease in activity of SOD, CAT and the levels of GSH. Simultaneous addition of GP 10 mg/ml significantly decreased lipid peroxidation and increased in antioxidant status. Thus, we conclude that GP, a potent antioxidant, was found to nullify 4-NQO induced lipid peroxidation in vitro and 4-NQO acts initially by causing oxidative stress and leads to carcinogenesis.     

Effect of DTNB on rat intestinal galactose transport in vivo

The effect of the non-penetrating reagent of -SH groups: acid 5,5'-dithiobis (2-nitrobenzoic), (DTNB), on 1 mM galactose absorption in rat intestine in vivo has been studied. DTNB inhibits sugar absorption in about 35%, which is due to an action on the mediated transport component, but without affecting the diffusional passive one. Consequently it does not modify galactose absorption in the presence of 0.5 mM phlorizin or that of the non-transportable sugar 2-deoxy-glucose. Galactose transport inhibition appears after a not longer than 5 min preexposure period and it remains constant at least up to 30 min. The inhibitory effect does not vary between 0.1 and 1 mM DTNB and it reverses completely with 0.5 mM dithioerythritol. Protection by excess of substrate has not been observed. Results show that DTNB affects sulfhydryl groups very probably located at the luminal side and related to the proteins of the cotransport system.     

Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide

In right-side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin-mediated K+ diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S-(2,4-Dinitrophenyl)glutathione and N-ethylmaleimide showed intermediate inhibitory effects.     

Natural and recombinant interferons provide different protection of human cells with impaired repair system (Marfan syndrome) against mutagens

It was shown that pretreatment of human cells with interferons (IF) of different origin has an unequal protective effect under the action of various mutagens with different activity. The protective effect of IF was estimated using the test of sister chromatid exchanges. Natural leucocyte alpha IF is highly effective in healthy human cells and in those of patients having Marfan syndrome. The latter are characterised by disorder in DNA repair under the action of 4-nitroquinoline-1-oxide (4-NQO), 8-methoxypsoralen and gamma-rays. Recombinant interferon (alpha 2) displayed no activity against gamma-rays in cells of healthy donors and patients with Marfan syndrome. Nor was it effective in the cells of patients in the experiments with 4-NQO. The absence of correlation between the ability of IF to protect the cells and their influence on the rate of cell proliferations was established.     

Synergism between cysteine and alkylating agents

Contrary to expectation, l-cysteine did not protect Escherichia coli from the lethal action of two monofunctional alkylating agents (nitrosomethylurethane and methylmethane sulfonate). The antibacterial action of these compounds was actually greatly enhanced by l-cysteine. This synergistic effect was also exhibited, to some extent, by d-cysteine but not by homocysteine, S-methylcysteine, or serine. The synergistic action between methylating agents and l-cysteine was not due to the formation of S-methylcysteine. l-Cysteine had no effect on the bacteriostatic action of ethylmethane sulfonate.     

A study of the growth inhibition of Lactobacillus casei by N-alkyl homologs of N-nitro-N-nitrosoguanidine.

N-methyl-(MNNG), N-ethyl-(ENNG), and N-propyl-(PNNG) derivatives of N'-nitro-N-nitrosoguanidine inhibited the growth of Lactobacillus casei in the order of potency, MNNG greater than ENNG greater than PNNG. L-Cysteine, gluathione, and dithioerythritol reversed the inhibition on a molar basis. The -SH compounds accelerated the loss of the N-nitroso group in vitro, yielding non-inhibitory N-alkyl nitroguanidines. The significance of the loss of the nitroso group and the size of the N-alkyl group is discussed.     

Effect of mercury ions on cysteine metabolism in Xenopus laevis tissues

The effect of mercury ions on the level of cysteine, glutathione, sulfane sulfur, and on the activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and γ-cystathionase in brain, heart muscle, liver, kidneys, testes and skeletal muscle of adult Xenopus laevis was investigated. Frogs of both sexes were exposed for 7 or 14 days to 1.353mgL(-1) (ppm) of mercury chloride (HgCl(2)) dissolved in water. The activity of the investigated enzymes participating in cysteine metabolism depends on cysteine in their active sites. Mercury ions can bind to -SH groups and, therefore, lower the activity of enzymes and change the level of sulfane sulfur, a product of l-cysteine desulfuration. The effect of mercury was found to depend on the time of exposure and the kind of tissue. In the liver, the main site of glutathione biosynthesis, the ratio of GSH to GSSG was essentially unchanged. The total glutathione level was decreased after 7 days of exposure to mercury, similarly as the activity of rhodanese. Sulfane sulfur levels were significantly increased after a shorter duration, while they decreased after a longer time of exposure. The kidney, brain and testes were able to enhance the level of GSH, probably thanks to high γ-glutamyltranspeptidase activity. These tissues showed an increased value of GSH/GSSG ratio during the shorter exposure to mercury. The activity of sulfurtransferases was decreased, especially after the longer exposure to mercury. In the heart and skeletal muscle, the level of GSH, sulfane sulfur, and the activity of the investigated sulfurtransferases was diminished after 14 days of exposure to Hg. It can be concluded that the main mechanism of toxic Hg activity is generation of reactive oxygen species in cells due to depleted GSH level, and a decreased sulfurtransferases activity either by blocking or oxidation of their -SH groups, what in consequence results in a diminished sulfane sulfur levels in tissues, especially the heart and testes.     

Synergistic DNA damaging effects of 4-nitroquinoline-1-oxide and non-effective concentrations of methyl methanesulfonate in human fibroblasts

DNA damage and DNA repair in human fibroblasts induced by the combination mixture of the genotoxic agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) were studied using the comet assay and the unscheduled DNA synthesis (UDS), respectively. Cells were simultaneously treated for 1h with the no observed effect concentration (noec) of MMS and increasing concentrations of 4-NQO or vice versa. Different results were obtained with the two types of mixtures. When the noec of 4-NQO was combined with increasing concentrations of MMS, no combination effects were observed. However, in experiments with increasing concentrations of 4-NQO and the noec of MMS, an increase in DNA damage and repair (and an enhancement of cytotoxicity) was demonstrated. Quantitative analysis of the effects by the isobologram method confirmed synergistic responses in both tests. We are proposing interactive actions between 4-NQO and MMS, whereby 4-NQO facilitates the attack of MMS on the DNA bases.     

Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols

1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.     

Purification and some properties of polyphenol oxidase from the yam tubers, Dioscorea bulbifera

In a comparison of the polyphenol oxidase activity of various species of yam tubers the greatest enzyme activity was found in D. bulbifera. The enzyme was purified from acetone powder extracts of this plant. Ammonium sulphate fractionation, followed by ion exchange chromatography and gel filtration gave 22-fold purification. The final product gave a single band on polyacrylamide disc gel electrophoresis. The purified enzyme showed activity towards catechol, pyrogallol and dl-β-3,4-dihydroxyphenylalanine (dl-DOPA) and had a MW 115000 ± 2000. It was characterized by response to various inhibitors. β-Mercaptoethanol, dithioerythritol, l-cysteine, sodium metabisulphite and KCN inhibited strongly.     

Insulin-like growth factor-I (IGF-I) receptor activation rescues UV-damaged cells through a p38 signaling pathway. Potential role of the IGF-I receptor in DNA repair

The activated insulin-like growth factor-I receptor (IGF-IR) is implicated in mitogenesis, transformation, and anti-apoptosis. To investigate the role of the IGF-IR in protection from UV-mimetic-induced DNA damage, 4-nitroquinoline N-oxide (4-NQO) was used. In this study we show that the activation of the IGF-IR is capable of rescuing NWTb3 cells overexpressing normal IGF-IRs from 4-NQO-induced DNA damage as demonstrated by cellular proliferation assays. This action was specific for the IGF-IR since cells expressing dominant negative IGF-IRs were not rescued from 4-NQO UV-mimetic treatment. DNA damage induced by 4-NQO in NWTb3 cells was significantly decreased after IGF-IR activation as measured by comet assay. IGF-I was also able to overcome the cell cycle arrest, observed after 4-NQO treatment, thereby enhancing the ability of NWTb3 cells to enter S phase. Interestingly, the p38 mitogen-activated protein kinase pathway was shown to represent the main signaling pathway involved in the IGF-IR-mediated rescue of UV-like damaged cells. The ability of the IGF-IR to induce DNA repair was also demonstrated by infecting NWTb3 cells with UV-irradiated adenovirus. Activation of the IGF-IR resulted in enhanced beta-galactosidase reporter gene activity demonstrating repair of the damaged DNA. This study indicates a direct role of the IGF system in the rescue of damaged cells via DNA repair.     

Selenium as a sulphydrylic group inductor in plants

We investigated the effect of selenium form and dose on the total glutathione and non-protein -SH group contents in the edible spinach (Spinacia oleracea L.) and ground tomato (Lycopersicon esculentum Mill.) plants. Our experiments were carried out in a hydroponic culture. Selenium was added to the culture medium in its selenite (Na2SeO3 x 5H2O) and selenate (Na2SeO4) forms. Regardless of the selenium form, we observed an increase in the non-protein thiol content. The non-protein -SH group content depended on the form and dose of selenium as well as on the organ and plant species. Regardless of the selenium form, a higher content of non-protein -SH groups were found in the spinach biomass than in the tomato biomass. Selenite contributed to a larger accumulation of non-protein -SH groups in the roots, whereas selenate contributed to their accumulation in the shoots     

Mutagenic activity of 4-nitroquinoline-N-oxide in upper aerodigestive tissue in lacZ mice (MutaMouse) and the effects of 1, 4-phenylenebis(methylene)selenocyanate

4-Nitroquinoline-N-oxide (4-NQO) was administered to lacZ mice at a concentration of 20 microg/ml in drinking water for 2 weeks, and the mutagenic fractions in a number of organs were assayed. The mutant fractions in tongue, esophagus and other pooled oral tissues were, respectively, 117+/-26, 73+/-15, and 48+/-15 mutants/10(5) plaque-forming units (pfu) (ca. 15-40xbackground). 4-NQO was not mutagenic in lung, liver or colon at conditions used here. We had previously demonstrated that the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), an established chemopreventive agent, greatly reduced carcinogenicity in 4-NQO in rat tongue, and we observed here that administration of p-XSC (10 ppm se) in the diet for 6 weeks (2 weeks before, during, and 2 weeks after 4-NQO) resulted in a 33% decrease in mutagenesis in oral tissue, a 17% decrease in esophagus, and a slight increase in tongue. Only the decrease in oral tissue reached statistical significance (p<0.04). The results reported here demonstrate that 4-NQO was extremely mutagenic in lacZ mouse tongue, with lower, but highly significant activities in esophagus and other pooled oral tissues. The high activity of 4-NQO in lacZ mouse tongue is consistent with the organ specificity of 4-NQO in the rat. Inhibition of 4-NQO-induced mutagenesis by p-XSC was observed mainly in pooled oral tissues, other than tongue. Possible reasons for the difference between inhibition of mutagenesis and carcinogenesis in tongue are discussed, as well as advantages and disadvantages of in vivo mutagenesis assays as surrogates for carcinogenicity assays in chemoprevention studies.     

人源性基因PLC-γ1中SH2-SH2-SH3 结构域的克隆、表达及纯化

利用RT-PCR技术将人源性PLC-γ1中的SH2-SH2-SH3 结构域基因扩增并克隆到载体pGEX-2T中,经热激反应转化到大肠杆菌BL21菌株内,在低温（33℃）下、利用IPTG诱导表达构建的重组基因的高效表达体系,利用谷胱甘肽琼脂糖（sepharose ）4B纯化得到高表达的重组蛋白。SDS-PAGE 和Western blot检测表明,PLC-γ1的SH2-SH2-SH3 结构域重组基因可在低温（33℃）条件下在E .coliBL21细胞中稳定完整表达,但是在较高温度下（37℃）会产生SH2-SH2-SH3 结构域不完全表达的现象。上述研究结果为人源性重组基因PLC-γ1中SH2-SH2-SH3 结构域的深入研究和广泛应用奠定了基础。     

Further studies on the role of sulfhydryl groups in the morphogenesis of the chick embryo

Summary The teratogenic effects on chick embryos of chloroacetophenone, a specific sulfhydryl reagent, are known to be reversible by cysteine and glutathione. Dithiothreitol (Cleland's reagent) and thiomalic acid cannot ameliorate the -SH block syndrome. While cysteine and glutathione can elicit differentiation of axial structures in the post-nodal pieces of the chick blastoderm, dithiothreitol and thiomalic acid cannot do so. This finding seems to suggest that the naturally occurring thiols, cysteine and glutathione, play an important role in the primary organizer action.This work was supported by a grant from the Indian Council of Medical Research. The author wishes to express his thanks to Miss N. Tripura Sundari for technical assistance. The Cleland's reagent used in this study was a gift from Dr. D. E. S. Truman of the University of Edinburgh.     

Toxicity of 4-nitroquinoline 1-oxide in Chinese hamster ovary cells: influence of cell density and of position in the cell cycle

Various factors influencing toxicity of 4-nitroquinoline 1-oxide (4-NQO) in Chinese hamster ovary cells were determined. Cell density during 4-NQO treatment and volume of treatment medium had a great effect on cell survival indicating that not the 4-NQO concentration per se, but the amount of 4-NQO per cell determines the toxic effect. When the cell-cycle response for 4-NQO-induced cell killing was measured in synchronous cells, a characteristic age response was seen in wild-type cells with greatly increased sensitivity in late G1 to early S and resistance increasing through the S-phase. In contrast, a UV-hypersensitive mutant, which is also more sensitive to 4-NQO showed only minor cell-cycle variations in its response to 4-NQO. Therefore, it appears that the cell-cycle pattern observed in the wild-type cells is associated with DNA repair.     

Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue Rat2 cell line.

This paper describes the spectrum of mutations induced by 4-nitroquinoline N-oxide (4-NQO) in the lacI target gene of the transgenic Big Blue Rat2 cell line. There are only a few report for the mutational spectrum of 4-NQO in a mammalian system although its biological and genetic effects have been well studied. Big Blue Rat2 cells were treated with 0.03125, 0.0625 or 0.125 microg/ml of 4-NQO, the highest concentration giving 85% survival. Our results indicated that the mutant frequency (MF) induced by 4-NQO was dose-dependent with increases from three- to seven-fold. The DNA sequence analysis of lacI mutants from the control and 4-NQO treatment groups revealed an obvious difference in the spectra of mutations. In spontaneous mutants, transition (60%) mutations, especially G:C-->A:T transition (45%), were most frequent. However, the major type of base substitution after treatment of 4-NQO was transversions (68.8%), especially G:C-->T:A (43.8%), while only 25% of mutants were transitions. These results are consistent with those produced by 4-NQO in other systems and the transgenic assay system will be a powerful tool to postulate more accurately the mechanism of chemical carcinogenesis involved.     

Inhibition by aldehydes as a possible further mechanism for glucose-6-phosphatase inactivation during CCl4-poisoning

Diffusable aldehydes are known to be produced during lipoperoxidative deterioration of unsaturated fatty acids. Malealdehyde (MLA) and 4-hydroxy-2,3-trans-penten-1-al (4-HPE) inhibit rat liver glucose-6-phosphatase activity in vitro. With MLA inhibition is significant at 0.25 mM concentration. With 4-HPE inhibition takes place at 0.5 mM. 1 mM MLA inhibited by about 89%, 6 mM 4-HPE by about 67%. Maximal inhibition is present as early as 5 min after addition of both aldehydes. Preincubation of aldehydes with 2 mM cysteine or glycine in the absence of microsomes almost completely prevents the inhibitory influence. Previous incubation of microsomes with 2 mM glutathione or 2 mM dithiothreitol or 2 mM cysteine affords a good protection towards the inhibitory action of the aldehydes; on the contrary, no protection is seen when microsomes are preincubated in the presence of either 2 mM glycine or asparagine. The total content of microsomes -SH groups is strongly decreased after incubation with 2mM malealdehyde.These results support the idea that the two aldehydes inhibit glucose-6-phosphatase mostly through interaction with protein -SH groups. The possibility that aldehydes derivated from the peroxidative decomposition of lipids may play a cooperative role in the inhibition of glucose-6-phosphatase occurring early after CCl₄-poisoning is discussed.