Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and Identification of Vesicular-Arbuscular Mycorrhiza-Stimulatory Compounds from Clover (Trifolium repens) Roots

Two isoflavonoids isolated from clover roots grown under phosphate stress were characterized as formononetin (7-hydroxy,4′-methoxy isoflavone) and biochanin A (5,7-dihydroxy,4′-methoxy isoflavone). At 5 ppm, these compounds stimulated hyphal growth in vitro and root colonization of an undescribed vesicular-arbuscular mycorrhiza, a Glomus sp. (INVAM-112). The permethylated products of the two compounds were inactive. These findings suggest that the isoflavonoids studied may act as signal molecules in vesicular-arbuscular mycorrhiza symbiosis.     

Biochemical mechanism for the inhibition of phenylalanine ammonia-lyase induction in the absence of oxygen in potato tuber tissue

Induction of phenylalanine ammonia-lyase (PAL) in excised potato parenchyma tissue in the presence of light displayed a rigid requirement for oxygen. A     

X-Pro peptides: Solution and solid-state conformation of benzyloxycarbonyl-(Aib-Pro)2methyl ester,a type I β-turn

The synthesis of the tetrapeptide benzyloxycarbonyl(α-aminoisobutyryl-L -prolyl)₂-methyl ester (Z-(Aib-Pro)₂-OMe) and an analysis of its conformation in solution and the solid state are reported. Stepwise synthesis using dicyclohexylcarbodiimide leads to racemization at Pro(2). Evidence for the presence of diastereomeric tetrapeptides is obtained from 270-MHz¹H-nmr and 67.89-MHz ¹³C-nmr. The all-L tetrapeptide is obtained by fractional crystallization from ethyl acetate. The NH of Aib(3) is shown to be involved in an intramo-lecular hydrogen bond by variable-temperature ¹H-nmr and the solvent dependence of NH chemical shifts. The results are consistent with a β-turn conformation with Aib(1) and Pro(2) at the corners stabilized by a 4 → 1 hydrogen bond. The molecule crystallizes in the space group P2₁2₁2₁, with a = 8.839, b = 14.938, and c = 22.015 Å. The structure has been refined to an R value of 0.051. The peptide backbone is all-trans, and a 4 → 1 hydrogen bond, between the CO group of the urethane moiety and Aib(3) NH, is observed. Aib(1) and Pro(2) occupy the corner positions of a type I β-turn with ? = ?55.4°, Ψ = ?31.3° for Aib(1) and ? = ?71.6°, Ψ = ?38° for Pro(2). The tertiary amide unit linking Pro(2) and Aib(3) is significantly distorted from planarity (Δω = 14.3°).     

A quantitating reagent for methanethiolation of protein sulfhydryl groups

p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH₃S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH₃S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups.     

Isolation and properties of a particulate fraction for the desaturation of palmitic acid from Alcaligenes faecalis.

A particulate fraction obtained from Alcaligenes faecalis could desaturate palmitic acid to palmitoleic acid. NADPH, ATP, CoA, Fe2+ and Mg2+ were essential cofactors for the reaction. The desaturation showed an absolute requirement for O2. Metal ions like Mn2+, Mo6+ and Cu2+ did not affect the desaturation, while Zn2+ was inhibitory. Sulfhydryl agents such as cysteine, glutathione and beta-mercaptoethanol had no effect, but SH-blocking agents like HgCl2 and p-hydroxymercuribenzoate inhibited the reaction. Azide and cyanide strongly inhibited the reaction while CO had no effect. The presence of a b-type cytochrome in the enzyme preparation was confirmed by the spectral studies on the reaction of enzyme with NADPH. Involvement of b-type cytochrome in the desaturation reaction was demonstrated by the reoxidation of b-type cytochrome initially reduced with NADPH, by the addition of palmitic acid and other cofactors. The pH optimum for the enzyme activity was 7.4. The optimum temperature for enzyme activity was 25 degrees C and maximum activity was obtained at the end of 45 min.     

Autocatalytic activation of the proenzyme form of the C1s subunit of the first component of complement.

The autocatalytic activation of the proenzyme form of the Cls subunit of the first component of complement is reported for the first time. Incubation of the purified proenzyme at 37° and pH 7.4 results in the evolution of esterolytic activity according to a second-order autocatalytic rate law. The lag phase portion of the sigmoidal activation curve can be shortened either by increasing the proenzyme concentration or by addition of the activated Cls subunit.     

Strategically Functionalized Adenosines: Agonists for Adenosine Receptors

Abstract

The syntheses of three classes of adenosine analogues involving cyclosubstitution at the 6-position and functionalization at the 2-position are reported. The target molecules synthesized are stable with respect to hydrolytic deamination by mammalian adenosine deaminase, and, because of major structural changes at the 2- and 6-positions, these compounds are expected to be poor phosphorylation substrates for the kinases. Adenosine receptor binding data reveal that several of the compounds synthesized show excellent A₁ receptor affinity and A₂/A₁ selectivity.