Viruses occurring in white clover (Trifolium repens L.) from permanent pastures in Britain

The uptake of captan from aqueous solution by conidia of Neurospora crassa can be markedly reduced by pretreatment of the cells with thiol reagents such as iodoacetic acid (IOA). The nature of this reaction has been investigated and it is shown that IOA largely reacts with the soluble thiol pool of the spores. Captan, however, reacts with both soluble and insoluble thiols and it is suggested that whilst the former is a detoxication process the latter may provide the key to its toxic action. Using glutathione as a model soluble thiol the molar ratio and pH dependence of the captan detoxication process has been determined and compared with the cellular reactions. Assay of ³⁵S-labelled captan has shown that captan is almost completely decomposed by the spores and that one-third of the accumulated captan is converted to sulphur compounds fixed to insoluble cell entities.     

Resonance Raman spectroscopic and kinetic consequences of a nitrogen ... sulphur enzyme-substrate contact in a series of dithioacylpapains.

The resonance Raman (RR) spectroscopic, conformational, and kinetic properties of six dithioacylpapain intermediates have been examined. Five of the intermediates are of the form N-(methyloxycarbonyl)-X-glycine-C(= S)S-papain, where X is L-phenyl-alanine, D-phenylalanine, glycine, L-phenylglycine, or D-phenylglycine. The sixth intermediate is N-phenylacetyl-glycine-C(= S)S-papain. Throughout the series there is an approximately 50-fold variation in kcat, the rate constant for deacylation, and a 1750-fold variation in kcat/KM. Existing RR spectra structure correlations allow us to define the torsional angles in the NH-CH2-C(= S)-S-CH2-CH fragment of the functioning intermediates. The values of these angles for each bound substrate appear to be very similar, with the substrates assuming a B-type conformer such that the nitrogen atom of the P1 glycine residue is cis to the thiol sulphur atom of cysteine-25. For each intermediate, the C(= S)S-CH2CH torsional angle is approximately -90 degrees, whereas for the SCH2-CH torisonal angle the cysteine-25 thiol sulphur (S) and cysteine-25 C alpha hydrogen (H) atoms are approximately trans. The three acyl-enzymes with the lowest catalytic rate constants, viz. N-(methyloxycarbonyl)-glycine-glycine-, N-(methyloxycarbonyl)-L-phenylglycine-glycine-, or N-(phenylacetyl)-glycine-dithioacylpapains, have atypical RR spectra in that they show a feature of medium intensity in the 1,085-cm-1 region. This band is sensitive to NH to ND exchange of the P1 glycine residues' (-NH-) function and, thus, the corresponding mode involves an excursion of the NH hydrogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

HMSS2: An advanced tool for the analysis of sulphur metabolism,including organosulphur compound transformation,in genome and metagenome assemblies

The global sulphur cycle has implications for human health, climate change, biogeochemistry and bioremediation. The organosulphur compounds that participate in this cycle not only represent a vast reservoir of sulphur but are also used by prokaryotes as sources of energy and/or carbon. Closely linked to the inorganic sulphur cycle, it involves the interaction of prokaryotes, eukaryotes and chemical processes. However, ecological and evolutionary studies of the conversion of organic sulphur compounds are hampered by the poor conservation of the relevant pathways and their variation even within strains of the same species. In addition, several proteins involved in the conversion of sulphonated compounds are related to proteins involved in sulphur dissimilation or turnover of other compounds. Therefore, the enzymes involved in the metabolism of organic sulphur compounds are usually not correctly annotated in public databases. To address this challenge, we have developed HMSS2, a profiled Hidden Markov Model-based tool for rapid annotation and synteny analysis of organic and inorganic sulphur cycle proteins in prokaryotic genomes. Compared to its previous version (HMS-S-S), HMSS2 includes several new features. HMM-based annotation is now supported by nonhomology criteria and covers the metabolic pathways of important organosulphur compounds, including dimethylsulphoniopropionate, taurine, isethionate, and sulphoquinovose. In addition, the calculation speed has been increased by a factor of four and the available output formats have been extended to include iTol compatible data sets, and customized sequence FASTA files.     

X-ray absorption spectroscopy of xanthine oxidase. The molybdenum centres of the functional and the desulpho forms.

X-ray absorption spectra have been recorded for the molybdenum K-edge region of xanthine oxidase. Both the absorption edge and the extended fine structure (e.x.a.f.s.) regions were investigated. Spectra were obtained for samples of the desulpho enzyme as well as for mixtures of this with the active enzyme. The spectrum of the pure active form was then obtained by difference. The desulpho enzyme shows a pronounced step in the absorption edge, of a type previously associated terminal oxygen ligands. In the active enzyme this step has decreased markedly. Satisfactory simulations of the e.x.a.f.s. spectrum of the desulpho enzyme could be obtained by assuming the molybdenum to be bonded to two terminal oxygen atoms (Mo = O about .175 nm), two sulphur atoms (presumably from cysteine residues, Mo-S about .0250 nm) and one sulphur atom (presumably from a methionine residue, Mo-S about 0.290 nm). E.x.a.f.s. of the active enzyme differed appreciably from this. In keeping with earlier proposals [Gutteridge, Tanner & Bray (1978) Biochem. J. 175, 887-897], the spectrum of the active enzyme could be simulated if a sulphur atom at about 0.225 nm (i.e. presumably a terminal sulphur atom) replaced one of the terminal oxygen atoms of the desulpho from, with small changes in the other bond distances. Validity of the interpretative procedures, which involved phase shift and amplitude calculations ab initio, was demonstrated by using low molecular weight compounds of known structure.     

Impact of pedospheric and atmospheric sulphur nutrition on sulphur metabolism of Allium cepa L., a species with a potential sink capacity for secondary sulphur compounds

Onion (Allium cepa L.) was able to use atmospheric H(2)S as sole sulphur source for growth. The foliarly absorbed H(2)S was rapidly metabolized into water-soluble, non-protein thiol compounds, including cysteine, and subsequently into other sulphur compounds in the shoots. In H(2)S-exposed plants, the accumulation of sulphur compounds in the shoots was nearly linear with the concentration (0.15-0.6 microl l(-1)) and duration of the exposure. Exposure of onion to H(2)S for up to 1 week did not affect the sulphur content of the roots. Secondary sulphur compounds formed a sink for the foliarly absorbed sulphide, and the sulphur accumulation upon H(2)S exposure could, for a great part, be ascribed to enhancement of the content of gamma-glutamyl peptides and/or alliins. Furthermore, there was a substantial increase in the sulphate content in the shoots upon H(2)S exposure. The accumulation of sulphate originated both from the pedosphere and from the oxidation of absorbed atmospheric sulphide, and/or from the degradation of accumulated secondary sulphur compounds. From studies on the interaction between atmospheric and pedospheric sulphur nutrition it was evident that H(2)S exposure did not result in a down-regulation of the sulphate uptake by the roots.     

A mutation in the cytosolic O-acetylserine (thiol) lyase induces a genome-dependent early leaf death phenotype in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol) lyase (OAS-TL) catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified.     

Gas chromatographic detection of sulphur compounds as methyl esters after growth of anaerobic bacteria in broth media

The use of a highly polar capillary column permits gas chromatographic analysis of organic acids as methyl esters. This method has found use in the study of end products of anaerobic bacteria. In addition, it also permits the detection of nonvolatile sulphur compounds which are neglected on the usual packed columns. These compounds have been identified by gas chromatography-mass spectrometry as methyl esters of 3-(methylthio)-propanoic acid and 4-(methylthio)-butanoic acid. When certain species were grown in a medium supplemented with 0·4% (w/v) DL-methionine, the relative amounts of both acids increased significantly. These nonvolatile sulphur compounds may serve as markers for specific bacteria.     

Inhibition of alcohol dehydrogenases by thiol compounds

2-Mercaptoethanol is a strong inhibitor of LADH. The inhibitory effect is likely due to the binding of the SH group to the enzymatic zinc ion. Various thiol compounds do not inhibit YADH and it is suggested that the zinc atoms involved in the catalytic mechanism of LADH and YADH may have different structural arrangements and that these zinc atoms in YADH may not be blocked by thiol compounds. Thiol compounds also quench the enhanced fluorescence of LADH-NADH in a pH-dependent manner. At pH 9.2, the binding of coenzyme to LADH is replaced by 2-mercaptoethanol, whilst at pH 7.3, it further quenches the fluorescence of NADH-LADH. This quenching of fluorescence is likely attributed to a conformational change and energy transfer upon binding of 2-mercaptoethanol to the LADH-NADH complex. Complete reversal of the inhibitory effect of thiol compounds on LADH can be obtained by dialysis.     

Thiol reactive nitroimidazoles: radiosensitization studies in vitro and in vivo

Using Chinese hamster V79 cells in vitro a study has been made of the radiosensitizing properties of 4- or 5-nitroimidazoles substituted in the 2, 5 or 4 position with various halo, sulphur ether, sulphonamide, sulphonate, ether or nitro groups. Values of E17 (the one-electron reduction potential measured versus the normal hydrogen electrode at pH7) vary in the range -178 to -565 mV. All the compounds, with one exception, are more efficient radiosensitizers than would be predicted from their redox potentials, and the factor, C1.6/C1.6, by which a compound is more efficient has been calculated. The second-order rate constants, k2, for reaction of these nitroimidazoles with glutathione and/or dithiothreitol were determined. Within each class of nitroimidazole there is a trend for k2 to increase with increasing redox potential. However, there is no clear trend between k2 and C1.6/C1.6. The concentration required to cause a 50 per cent depletion of intracellular glutathione was determined for selected compounds, as was the ability of glutathione-S-transferase to catalyse reaction with thiols. These observations suggested that the relative thiol reactivity measured under chemically controlled conditions does not necessarily indicate thiol reactivity intracellularly. Studies using the MT tumour in mice showed that the high levels of radiosensitization seen in vitro could not be duplicated in vivo. This was attributed to thiol reactivity, resulting in low metabolic stability and rapid depletion of sensitizer in vivo.     

Evaluation of benzofuroxan as a chromophoric oxidizing agent for thiol groups by using its reactions with papain, ficin, bromelain and low-molecular-weight thiols.

1. Benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) was evaluated as a specific chromophoric oxidizing agent for thiol groups. 2. Aliphatic thiol groups both in low-molecular-weight molecules and in the enzymes papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4) readily reduce benzofuroxan to o-benzoquinone dixime; potential competing reactions of amino groups are negligibly slow. 3. The fate of the thiol depends on its structure: a mechanism is proposed in which the thiol and benzofuroxan form an adduct which, if steric factors permit, reacts with another molecule of thiol to form a disulphide; when the thiol is located in the active site of a thiol proteinase and steric factors preclude enzyme dinner formation, the adduct reacts instead with water or HO- to form a sulphenic acid; attack on the sulphur atom of the adduct by either a sulphur or oxygen nucleophile releases o-benzoquinone dioxine. 4. Benzofuroxan contains n o proton-binding sites with pKa values in the range 3-10 and probably none in the range 0-14; o-benzoquinone dioxine undergoes a one-proton ionization with pKa=6.75.5. o-benzoquinone dioxime absorbs strongly at wavelengths greater than 410nm, where absorption by benzofuroxan, proteins and simple thiol compounds is negligible; 416 nm is an isosbestic point (epsilon 416 = 5110 litre. mol-1-cm-1); epsilon430=3740+[1460/(1+[H+]/Ka)] where pKa=6.75. 6. The possibility of acid-base catalysis of the oxidation by active-centre histidine residues of the thiol proteinases is discussed.     

Novel oxorhenium and oxotechnetium MO(NS)(S)2 complexes in the development of 5-HT1A receptor imaging agents

The [NS][S](2) mixed-ligand system was applied to synthesize oxorhenium and oxotechnetium complexes of the general formula MO(o-CH(3)OC(6)H(4)N(CH(2)CH(2))(2)NCH(2)CH(2)S)(p-CH(3)C(6)H(4)S)(2) (M=Re in 1, M=(99)Tc in 2, and M=(99m)Tc in 3). The bidentate [NS] ligand includes the 1-(2-methoxyphenyl)piperazine moiety which is a fragment of the true 5-HT(1A) antagonist WAY 100635. The oxorhenium complex 1 was prepared by a ligand exchange reaction using ReOCl(3)(PPh(3))(2) as precursor while [Bu(4)N][(99)TcOCl(4)] and (99)Tc-gluconate were used as precursors in the synthesis of the oxotechnetium-99 complex 2. Both complexes were characterized by elemental analysis and spectroscopic methods. Crystallographic analysis of 1 showed that the rhenium coordination geometry is trigonal bipyramidal. The basal plane of the trigonal bipyramid is defined by the oxo group and two sulphur atoms, one belonging to the [NS] ligand and the other to an aromatic thiol, while the apical positions are occupied by the nitrogen of the [NS] ligand and the sulphur of the second aromatic thiol. The oxotechnetium-99 complex 2 has almost identical unit cell parameters to those of the oxorhenium complex 1 indicating, in combination with the other analytical data, that the complexes are isostructural. The binding affinity of the oxorhenium complex 1 for the 5-HT(1A) receptor subtype was determined in rat brain hippocampal preparations (IC(50)=106 nM). The oxotechnetium-99m complex 3 was prepared by a ligand exchange reaction using (99m)Tc-glucoheptonate as the precursor. Its structure was established by comparative HPLC studies using the oxotechnetium-99 complex 2 as a reference. Complex 3 was administered by intravenous injection in rats. At 2 min post injection, 0.153% of the injected dose per gram of tissue was measured in rat brain.     

固氮酶铁钼辅因子FeMo-co在水与非水体系内重组活力与催化活力的比较研究

 本文提出一种测定FeMo-co催化活力的反应体系,用此反应体系,在测定FeMo-co催化活力的过程中,FeMo-co与变种UW45抽提液的重组活性始终保持不变。讨论了水含量、还原剂对FeMo-co催化活力和重组活力的影响。     

In vitro-refolding of a single-chain Fv fragment in the presence of heteroaromatic thiols

The aim of the present work was to explore the use of heteroaromatic thiol compounds, namely derivatives of pyridine and pyrimidine, as redox reagents for the in vitro-refolding of a recombinantly expressed single-chain Fv fragment (scFvOx). The mixed disulfide of scFvOx with glutathione was used as a starting material, while reduced glutathione, 4-mercaptopyridine, 2-mercaptopyrimidine, 2-mercaptopyridine N-oxide, and the mercaptobenzene derivative thiosalicylic acid, respectively, served as catalysts for the formation of native disulfide bonds during renaturation. In contrast to thiosalicylic acid, and despite their significantly lower thiol pKa values, none of the heteroaromatic thiol compounds accelerated the apparent kinetics of in vitro-refolding compared to the naturally occurring peptide glutathione. However, significantly improved renaturation yields were observed in the presence of 4-mercaptopyridine and 2-mercaptopyrimidine, demonstrating the usefulness of aromatic thiol compounds as reagents for the in vitro-refolding of antibody fragments.     

Cytotoxic mechanism of selenomethionine in yeast

Although selenium is an essential element, its excessive uptake is detrimental to living organisms. The significance of selenium for living organisms has been exploited for various purposes. However, the molecular basis of selenium toxicity is not completely understood. Here, we applied a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach to analysis of yeast cells treated with selenomethionine. The data indicated that intracellular thiol compounds are significantly decreased, and diselenide and selenosulfide compounds are increased in selenomethionine-treated cells. The growth defect induced by selenomethionine was recovered by extracellular addition of cysteine and by genetic modification of yeast cells that have an additional de novo synthetic pathway for cysteine. Because cysteine is an intermediate of thiol compounds, these results suggested that the loss of a reduced form of thiol compounds due to selenomethionine causes a growth defect of yeast cells.     

EXAFS study of zinc coordination in bacitracin A.

Bacitracin is a dodecapeptide antibiotic produced by Bacillus sp. The antibacterial activity depends upon the peptide binding a divalent metal. Hitherto, the exact coordination of the cation has not been established. In particular the role played by the sulphur and nitrogen atoms of the thiazoline ring of bacitracin A has not been clear. Here the coordination of Zn2+ by bacitracin A has been studied using extended X-ray absorption fine structure. The experimental data are consistent with a model in which zinc is coordinated by one oxygen and three nitrogen atoms with the sulphur atom of the thiazoline ring not being directly involved in the zinc coordination.     

Antigenotoxic effect of allicin against SCEs induced by methyl methanesulphonate in cultured mammalian cells

Allicin, one of the sulphur compounds of garlic (Allium sativum), possesses antioxidant and thiol disulphide exchange activity and is also shown to cause a variety of activities potentially useful for human health. In this investigation, the effect of 1,5,10 and 20 microM of allicin was determined for inhibiting the rate of SCE induced by 60 microM of MMS. Cultured human lymphocytes from two female donors were used for the experiment. The levels of SCEs were lowered by allicin suggesting its antigenotoxic activity in mammalian cells in vitro.     

Adequate S supply protects barley plants from adverse effects of salinity stress by increasing thiol contents

As the salt-affected areas are expected to increase substantially in subsequent years, the impact of salinity on plant growth and yield is likely to increase. One of the first consequences of plant exposure to high saline concentrations is the formation of reactive oxygen species (ROS). In order to allow adjustment of the cellular redox state, plant antioxidative system has to be activated. This system involves several enzymes and compounds, as the sulphur-containing metabolite glutathione (GSH). Therefore, our aim was to determine whether adequate sulphur nutrition might alleviate the adverse effects of salt stress on barley plants grown in the presence of different sulphate application rate and exposed to 100 mM NaCl, by studying differences in growth parameters, lipid peroxidation, sulphate and thiol accumulation and sulphur assimilation pathway. In salt-treated plants, an adequate sulphur supply allows adequate GSH synthesis (high-thiol concentration) thus avoiding the effects of ROS on photosynthetic functions (no effect on both chlorophyll and protein content), whereas in S-deficient plants, salt stress leads to excess ROS production that induces stress and plants showed reduction of photosynthetic efficiency (loss of chlorophyll and protein contents). As thiol levels are more abundant in S-sufficient plants than in those S-deficient, one might expect that S-sufficient plants are more able to remove the harmful effects of high salinity. The comparison of malondialdehyde levels between +S and ?S salt-treated plants strongly supports this idea. In conclusion, we found that plant sulphur nutritional status plays a key role in the metabolic modifications necessary to cope with salt stress.     

The distribution of sulphur in the mud of Lake Victoria

Summary Re-examination of mud from Lake Victoria showed the presence of pyrite. It has been claimed that these muds contain abnormally large amounts of organic sulphur, but pyritic and organic sulphur would not be distinguished by the method that was used. The organic sulphur content of the mud is therefore much less than previously reported, and there seems to be no reason to assume the presence of unusual organic sulphur compounds.     

Seasonal and spatial variation of reduced sulphur compounds in mistletoes (Viscum album) and the xylem sap of its hosts (Populus × euramericana and Abies alba)

In the present field study with adult trees inhabited by Viscum album , the question was addressed as to whether European mistletoes are able to remove reduced sulphur from the xylem sap of its hosts. For this purpose the reduced sulphur composition and content of the xylem sap of Viscum album and the corresponding hosts Populus  ×  euramericana and Abies alba were analysed. The xylem sap of Viscum was enriched in reduced sulphur compared to the hosts but still reflected the higher reduced sulphur content of Populus compared to Abies . Despite similar xylem sap composition of the hosts with glutathione as the dominating thiol, Viscum on Populus contained predominantly cysteine, Viscum on Abies predominantly glutathione in its xylem sap. These findings suggest selective and different removal of reduced sulphur from these hosts. Still the amount of reduced sulphur removed was too small to result in changes of the concentration of thiols in the xylem sap of the hosts that are statistically significant, probably due to the high variability encountered under field conditions. Despite the differences in the reduced sulphur composition and contents of the xylem sap between Viscum on Populus and Viscum on Abies , total thiol content as well as thiol composition of Viscum leaves on the two hosts were similar throughout the seasons. The seasonal pattern in the thiol composition and contents of Viscum leaves showed high levels in spring and autumn and low levels in summer. The significance of these seasonal changes is discussed.     

Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway

A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O-acetyl-L-serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2-methyl-2,4-thiazolidinedicarboxylic acid. N-acetyl-L-serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK'-'lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.