Method of electrophoretic determination of S-methyl methionine in plants]

A method to determine S-methyl methionine (vitamin U) in plants has been devised. The method includes S-methyl methionine extraction extract purification on cationite KU-2 in the NH4+-form, S-methyl methionine eluation with 2n water solution of NH4OH from the cation exchange resin, eluate evaporation in vacuo, dissolution of the residue in a minimal amount of water acidified with hydrochloric acid, electrophoretic separation of S-methyl methionine from basic amino acids, ninhydrine development of the electrophoregram, methanol eluation of the spot corresponding to S-methyl methionine, and colorimetry of the solution at 506 mmu. Relative error is +/-2.5%.     

Synthesis of beta-(S-methyl)thioaspartic acid and derivatives

Beta-(S-Methyl)thioaspartic acid occurs as a posttranslational modification at position 88 in Escherichia coli ribosomal protein S12, a position that is a mutational hotspot resulting in both antibiotic-resistant and antibiotic-sensitive phenotypes. Critical to research designed to determine the biological function of beta-(S-methyl)thioaspartic acid will be the availability of synthetic beta-(S-methyl)thioaspartic acid as well as derivatives designed for peptide incorporation. We report here the synthesis of beta-(S-methyl)thioaspartic acid and derivatives. The installation of the beta-methylthio moiety into the aspartic acid structure was accomplished by electrophilic sulfenylation of N-protected-l-aspartic acid derivatives with 2,4-dinitrophenyl methyl disulfide. Following this key transformation, we were able to prepare protected beta-(S-methyl)thioaspartic acid derivative suitable for peptide coupling.     

Reactions of [PtCl(dien)]Cl with glutathione,oxidized glutathione and S-methyl glutathione. Formation of an S-bridged dinuclear unit

The reaction of the monofunctional platinum compound [PtCl(dien)]Cl with the tripeptide glutathione (GSH), oxidized glutathione (GSSG) and S-methyl glutathione (GS-Me) has been investigated by ¹H, ¹³C and ¹⁹⁵Pt magnetic resonance spectroscopy and by potentiometric titrations. It appears that platinum binds with a high degree of specificity to the GSH sulfhydryl group. The reaction of platinum with GSH proceeds in two steps. In the first step only one platinum binds to the sulfur atom and, in the second step, another [Pt(dien)]²⁺ unit binds to [Pt(dien)GS]⁺ forming an S-bridged dinuclear unit [{Pt(dien)}₂GS]³⁺. The rate of the first binding step is pH-dependent, whereas the rate of the second step is not. At pH < 7 the rate of the first binding step is slow compared to the rate of the second binding step. At pH > 10, on the other hand, the rate of the first binding step is faster than the rate of the second binding step. Consequently, at pH < 7 one can only isolate the [{Pt(dien)}₂GS]³⁺ complex. In the presence of free GSH, at pH > 7, one [Pt(dien)]²⁺ unit of [{Pt(dien)}₂GS]³⁺ dissociates forming [Pt(dien)GS]⁺. The mechanism of the pH-dependent rate of the first platinum binding step and the ligand-exchange reaction are discussed. GSSG reacts with [Pt(dien)]²⁺, also forming the S-bridged dinuclear unit [{Pt(dien)}₂GS]³⁺, probably through a redox disproportionation reaction with a catalytic function of [PtCl(dien)]Cl. GS-Me reacts with [Pt(dien)]²⁺ forming the S-coordinated [Pt(dien)GS-Me]²⁺. [Pt(dien)GS-Me]²⁺ exists as a pair of diastereomers due to different configurations about sulfur. The rate of the inversion of configuration at the coordinated sulfur atom is slow on the NMR time-scale.     

Synthesis and biological evaluation of 5R- and 5S-methyl substituted D- and L-configuration 1,3-dioxolane nucleoside analogs

1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemotherapy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxymethyl)-5-methyldioxolane. Dioxolanes of alpha-D-, beta-D-, alpha-L-, and beta-L-configuration were prepared, that included 5-methyl derivatives of both 5R and 5S configuration. Molecular mechanics calculations indicate that the 5S and 5R diastereoisomeric 1,3-dioxolanes possess distinct conformational bias, suggesting that methyl substitution may alter the conformational preference of 1,3-dioxolanes. The ability of the 1,3-dioxolanes to inhibit HCV RNA replication was evaluated in a cell-based, subgenomic replicon assay. In addition, activity against vaccinia and HIV was evaluated in cell-based assays. The 2-hydroxymethyl-5-methyl-1,3-dioxolanes were found to be inactive.     

Novel applications of modification of thiol enzymes and redox-regulated proteins using S-methyl methanethiosulfonate (MMTS)

S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of natural thiol-disulfide states of redox-sensitive proteins and proteins which have undergone S-nitrosylation. The reagent can also be employed as an inhibitor of enzymatic activity, since nucleophilic cysteine thiolates are commonly present at active sites of various enzymes. The advantage of using MMTS for this purpose is the reversibility of the formation of methylthio mixed disulfides, compared to irreversible alkylation using conventional agents. Additional benefits include good accessibility of MMTS to buried protein cysteines due to its small size and the simplicity of the protection and deprotection procedures. In this study we report examples of MMTS application in experiments involving oxidoreductase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), redox-regulated protein (recoverin) and cysteine protease (triticain-α). We demonstrate that on the one hand MMTS can modify functional cysteines in the thiol enzyme GAPDH, thereby preventing thiol oxidation and reversibly inhibiting the enzyme, while on the other hand it can protect the redox-sensitive thiol group of recoverin from oxidation and such modification produces no impact on the activity of the protein. Furthermore, using the example of the papain-like enzyme triticain-α, we report a novel application of MMTS as a protector of the primary structure of active cysteine protease during long-term purification and refolding procedures. Based on the data, we propose new lines of MMTS employment in research, pharmaceuticals and biotechnology for reversible switching off of undesirable activity and antioxidant protection of proteins with functional thiol groups.     

S-甲基异硫脲对大鼠门脉高压症食管静脉曲张的影响

目的观察诱导型一氧化氮合酶抑制剂SMT对大鼠门脉高压症食管静脉曲张的影响。方法健康雄性SD大鼠60只随机分为5组,假手术组、模型组、低剂量组、中剂量组和高剂量组。假手术组仅分离门静脉、左肾上腺静脉关腹,其余组门脉缩窄两步法加左肾上腺静脉结扎,建立门脉高压症食管静脉曲张模型。假手术组与模型组手术后给予腹腔注射生理盐水,其余3组手术后给予腹腔注射不同浓度SMT。手术后21 d,检测大鼠门脉血中TNOS、iNOS的活性及NO的浓度,免疫组化CD34标记食管血管内皮,测量每组大鼠食管横切面黏膜下血管的数目、面积。结果模型组大鼠门脉血中TNOS活性与iNOS活性以及NO浓度和食管黏膜下血管数目,血管平均截面积,血管总面积均较假手术组显著升高（P〈0.01）。中、高剂量组大鼠门脉血中TNOS活性与iNOS活性以及NO浓度和食管黏膜下血管的数目、血管平均面积、血管总面积较模型组均显著下调（P≤0.01）。结论大鼠门脉高压食管静脉曲张的发病机制中有NO参与,门脉缩窄型门脉高压食管静脉曲张病中NO主要由iNOS生成,SMT对大鼠门脉高压食管静脉曲张可能具有一定保护作用。     

S-methyl glutathione synthesis is catalyzed by the cheR methyltransferase in Escherichia coli.

The cheR methyltransferase, known to be necessary for the methyl esterification of receptors involved in chemotaxis, is shown to be essential to the synthesis of S-methyl glutathione from glutathione and S-adenosylmethionine in intact Escherichia coli. S-Methyl glutathione is not, however, found to be essential for chemotaxis. It is suggested that the synthesis of S-methyl glutathione may be due to a "parasitic" reaction of glutathione with S-adenosylmethionine bound to the methyltransferase.     

Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.     

Control of postharvest black rot caused by Alternaria alternata in strawberries by the combination of Cryptococcus laurentii and Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester

The biological control activity of Cryptococcus laurentii alone or in combination with Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) against postharvest black rot caused by Alternaria alternata in strawberries was investigated. As a stand-alone treatment, C. laurentii significantly reduced the incidence and lesion diameter of black rot in strawberries at 20 °C. The incidence and lesion diameter in strawberries treated with BTH alone was not significantly different from that in the control. C. laurentii in combination with BTH (0.1 g L⁻¹) was more effective than C. laurentii alone or BTH alone. BTH only slightly increased the population of C. laurentii in strawberry wounds and nutrient yeast dextrose broth (NYDB) and had little inhibition effect on the growth of A. alternate in potato dextrose agar (PDA). The enzyme analysis results showed that BTH significantly increased the activity of defense enzymes, including polyphenol oxidase (PPO), peroxidase (POD), and catalase (CAT) in strawberries treated with C. laurentii in combination with BTH. All these results indicated that the action mode of BTH enhancing the biocontrol efficacy of C. laurentii against A. alternata may involve in its ability to induce defense enzymes including PPO, POD and CAT in strawberries rather than its direct effect on C. laurentii or A. alternata.     

Aptitude of cheese bacteria for volatile S-methyl thioester synthesis. II. Comparison of coryneform bacteria, Micrococcaceae and some lactic acid bacteria starters

Various strains of coryneform bacteria, Micrococcaceae and commercial starters of Lactococcus lactis and Leuconostoc were compared for their aptitude to form S-methyl thioesters. Resting cells were incubated with methanethiol alone at pH 7 and in conjunction with a mixture of straight, branched and hydroxy short-chain fatty acids up to C₆ at pH 7 and 5. Results showed that all the strains synthesized at least S-methyl thioacetate, with strains that were low and high producers in each group. This is the only thioester formed in small amount by Leuconostoc. Brevibacterium linens (six strains) and Micrococcaceae (five strains) were able to form branched-chain thioesters especially from their intracellular fatty acids at neutral pH, and straight-chain thioesters mostly from exogenous fatty acids at acid pH. Coryneform bacteria other than B. linens (four strains) and L. lactis (four starters) synthesized thioesters up to S-methyl thiobutyrate from endogenous or exogenous fatty acids but not branched-chain ones, except for one starter which formed a very little thioisovalerate. Some particular effects of pH and added fatty acids revealed differences between species or strains in their specific enzymatic systems. Received: 7 April 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997     

Control of potato cyst-nematode, Heterodera rostochiensis, in sandy loam, by Du Pont 1410 (S-methyl l-(dimethylcarbamoyl)-N-[(methylcarbamoyl) oxy] thioformimidate) applied to the soil at planting time

When applied to heavily infested sandy loam soil at planting time, as little as 5 ppm Du Pont 1410 (5-methyl I-(dimethylcarbamoyl)-N-[(methylcar-bamoyl) oxy] thioformimidate) in pots, or 2–5 ppm in field plots, effectively controlled potato cyst-nematode, Heterodera rostochiensis Woll., and greatly increased the growth and yield of susceptible potatoes. Dipping the shoots of potted King Edward potatoes once in aqeuous solution containing 2000 ppm did not control potato cyst-nematode. Nematode control was not increased when 2 or 4 kg a.i./ha was sprayed on the foliage of young Pentland Crown potatoes growing in soil already treated with the nematicide.     

A comparative quantum chemical study of methyl acetate and S-methyl thioacetate. Toward an understanding of the biochemical reactivity of esters of coenzyme A

The electronic structures of methyl acetate and S-methyl thioacetate and the corresponding anions have been investigated using the INDO-MO method. Equilibrium geometries, gas-phase anion proton affinities and barriers to internal rotation have been computed. Analysis of the effect of the d-type functions on sulfur on the static and dynamic properties of the thioester and its anion reveal no role for (p-d) pi conjugative effects. The results of this work indicate that the unique properties of thioester, and hence esters of coenzyme A, may be attributed to the lack of resonance, rather than to a sulfur d-orbital expansion.     

Benzo-thiadiazole-7-carbothioic Acid S-methyl Ester does not protect Melon Fruits against Fusarium pallidoroseum Infection but Induces Defence Responses in Melon Seedlings

The present study investigated the potential of benzo-thiadiazole-7-carbothioic acid S -methyl ester (BTH) to protect postharvest melons var. 'Orange Flesh' from the fruit rot caused by Fusarium pallidoroseum . It was noticed that melon fruits immersed in BTH and postinoculated with the fungus presented the same pattern of disease incidence/severity and activity of the defence-related enzymes superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, phenylalanine ammonia-lyase, and β-1,3-glucanase of controls, indicating that BTH was ineffective in protecting melons from the fruit rot disease. However, the preflowering application of BTH in melon seedlings induced stunted growth, probably related to enhanced lignification which is related to the plant cell wall reinforcement and increase of resistance against invading pathogens, and alterations of the activity of the studied defence-related enzymes in comparison with controls, suggesting that this strategy could probably be effective for the control of the postharvest rot of melon fruits through activation of systemic resistance.     

Antidiabetic and antioxidant effects of S-methyl cysteine sulfoxide isolated from onions (Allium cepa Linn) as compared to standard drugs in alloxan diabetic rats

Antidiabetic and antoxidant effects of S-methyl cysteine sulfoxide (SMCS) isolated from A. cepa and two standard drugs, glibenclamide and insulin were studied and compared in alloxan diabetic rats after using each of them for treatment for two months. These drugs ameliorated the diabetic condition significantly, viz. maintenance of body weight and control of blood sugar in rats. Further they lowered the levels of malondialdehyde, hydroperoxide and conjugated dienes in tissues exhibiting antioxidant effect on lipid peroxidation in experimental diabetes. This is achieved by their stimulating effects on glucose utilization and the antioxidant enzymes, viz. superoxide dismutase and catalase. The probable mechanism of action of SMCS and glibenclamide may be partly dependent on the stimulation of insulin secretions and partly due to their individual actions. In the amelioration of diabetes the standard drugs showed a better action, but as an antioxidant SMCS proved to be a better one.     

S-methyl thioesters are produced from fatty acids and branched-chain amino acids by brevibacteria: focus on L-leucine catabolic pathway and identification of acyl-CoA intermediates

Despite their importance as potent odors that contribute to the aroma of numerous cheeses, S-methyl thioesters formation pathways have not been fully established yet. In a first part of our work, we demonstrated that Brevibacterium antiquum and Brevibacterium aurantiacum could produce S-methyl thioesters using short-chain fatty acids or branched-chain amino acids as precursors. Then, we focused our work on l-leucine catabolism using liquid chromatography tandem mass spectrometry and gas chromatography-mass spectrometry analyses coupled with tracing experiments. For the first time, several acyl–CoAs intermediates of the l-leucine to thioesters conversion pathway were identified. S-methyl thioisovalerate was produced from l-leucine, indicating that this amino acid was initially transaminated. Quite interestingly, data also showed that other S-methyl thioesters, e.g., S-methyl thioacetate or S-methyl thioisobutyrate, were produced from l-leucine. Enzymatic and tracing experiments allowed for postulating catabolic pathways leading to S-methyl thioesters biosynthesis.     

Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.     

Primary structure of the glycan chains of normal C 1 esterase inhibitor (C 1-INH) after NMR analysis at 400 MHz

The primary structural analysis of O- and N-linked carbohydrate chains of the C-1-esterase inhibitor purified from normal serum was carried out by 400-MHz 1H-NMR spectroscopy. C-1-esterase inhibitor protein of a molecular weight of 116,000 daltons contains 24 O-glycans: NeuAc (alpha 2-3) Gal (beta 1-3) GalNAc, 4 N-glycans: NeuAc (alpha 2-6) Gal (beta 1-4) (GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-6) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc and 2 N-glycans: NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc. 30% of the N-glycans are fucosylated.     

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.     

Structural study of cell wall mannan of a Candida albicans (serotype A) strain.

The structure of the mannan of Candida albicans NIH A-207 strain (serotype A) was investigated by adopting mild acetolysis followed by enzymolysis with an Arthrobacter GJM-1 exo-alpha-mannosidase. The resultant oligosaccharides, from pentaose to octaose (where manp = D-mannopyranose), were identified as manp beta (1----2)manp alpha (1----2)manp alpha (1----2)manp alpha (1----2)manp, manp beta (1----2)manp beta (1----2)manp alpha (1----2)manp alpha (1----2)- manp alpha (1----2)manp, manp beta (1----2)manp beta (1----2)manp beta (1----2)manp alpha (1----2)manp alpha (1----2)manp alpha (1----2)manp and manp beta (1----2)manp beta (1----2)manp beta (1----2)manp beta (1----2)manp alpha (1----2)manp alpha (1----2)manp alpha (1----2)manp, respectively. Analyses of alpha-linked oligosaccharides obtained by acetolysis under conventional conditions gave the same oligosaccharides, from biose to heptaose, as those obtained from the mannans of C. albicans NIH B-792 (serotype B) and J-1012 (serotype A, formerly serotype C).     

Cyclopeptides from Sagina japonica (Caryophyllaceae)

Two new cyclic peptides, named sajaponicin C (1) and sajaponicin D (2), were isolated from the whole plants of Sagina japonica (Caryophyllaceae). Their structures were determined as cyclo(Pro(2)-Leu(2)-Tyr-Leu(1)-Phe(1)-Pro(3)-Phe(2)-Pro(1)) (1) and cyclo(Pro(1)-Pro(2)-Pro(3)-Pro(4)-Phe(1)-Gly-Thr-Ser-Phe(2)-Ile-Tyr) (2) on the basis of spectroscopic data, especially by two-dimensional (2D) NMR techniques.