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R K Sinha

Publications and source records attributed to R K Sinha.

At least 55 records · Page 3Linked to original sources
PubMed1984

Biosynthesis of peptidoglycan in Gaffkya homari: processing of nascent glycan by reactivated membranes.

Membranes from Gaffkya homari reactivated by freezing and thawing were used to study the processing events involved in the assembly of both sodium dodecyl sulfate (SDS)-insoluble peptidoglycan (PG) and SDS-soluble PG. The ability to reactivate membranes for the synthesis of these polymers provided an opportunity to monitor those events that are not influenced by wall-linked PG. In G. homari, processing for the formation of cross-links requires the selective actions of DD-carboxypeptidase, LD-carboxypeptidase, and NE-(DAla)-Lys transpeptidase. Time courses of cross-link formation, as measured by the amounts of amidated bisdisaccharide peptide dimer and nonamidated bisdisaccharide peptide dimer, showed a lack of correlation with those for the synthesis of SDS-insoluble PG. SDS-soluble PG, which is significantly cross-linked when synthesized in the absence of penicillin G, was a precursor of the SDS-insoluble PG. In the presence of penicillin G, un-cross-linked SDS-soluble PG was synthesized. This PG was also utilized and processed for the synthesis of cross-linked SDS-insoluble PG after removal of the beta-lactam. This protocol provided a method for separating stages in the synthesis and elongation of PG from those involved in processing. Cross-linkage in the various PG fractions ranged from 0 to 19% in SDS-soluble PG and from 2 to 24% in SDS-insoluble PG. Thus, the results indicated that there is no direct correlation between SDS insolubility and the degree of cross-linkage. Instead, they suggested that additional features may contribute to the insolubility of PG in SDS.

Acetylglucosamine↗
PubMed1981

Effect of penicillin G on release of peptidoglycan fragments by Neisseria gonorrhoeae: characterization of extracellular products.

The effect of penicillin G (penG) on the turnover and release of peptidoglycan (PG) by Neisseria gonorrhoeae RD5 was investigated. We previously showed that exponentially growing gonococci (labeled in the glycan moiety with glucosamine and muramic acid and in the peptide with diaminopimelic acid) turn over ca. 35% of their PG per generation. In current studies, addition of penG accelerated the rate of PG hydrolysis by more than twofold and resulted in a corresponding increase in the amount of soluble PG fragments found in the medium. This increase of soluble PG was accounted for mainly by the release of nonreducing (anhydro-muramyl-containing) disaccharide peptide dimers (molecular weight, about 2,000) and trimers that were composed of subunits, linked not by peptide cross-linking bonds, but probably only by glycosidic bonds. The enhanced release of these products suggested that penG, directly or indirectly, stimulates the activity of a glycan-splitting, gonococcal PG:PG-6 muramyl transferase (transglycosylase). PG monomers that were released in the presence of penG were identical, both qualitatively and quantitatively, to the monomers released as a result of turnover in the absence of penG and consisted of anhydro-muramyl-containing disaccharide tripeptides and tetrapeptides. PenG-treated bacteria consistently released slightly less free disaccharide and free peptides than did control cultures, implying a penG-associated depression in the activity of the gonococcal N-acetylmuramyl-L-alanine amidase. In addition to stimulating the release of PG fragments, penG was also associated with greatly enhanced release from cells of glucosamine-containing non-PG macromolecule(s).

Hydrolysis↗
PubMed1980

Variations in amino acids and sugars in different tissues of broad bean (Vicia faba L.) during the pathogenesis of Uromyces fabae (Pers.) de Bary.

In response to infection by the rust pathogen Uromyces fabae the different tissues of broad bean (Vicia faba L.) showed varied pattern of amino acid metabolism; some of them being exclusively present in a particular region only. In the inflorescence tissue, for example, tryptophan, glycine, aspartic acid, serine, proline, and arginine were present. In the stem, however, the amino acids, present during and after infection, were tryptophan, serine, glutamine, homoserine, and dl-alanine. Post-infectionally induced amino compounds, lysine, histidine, vomoserine, proline, tyrosine and dl-threonine, were found in the leaves; in the petiole serine and histidine were the only two such amino acids. Out of these amino acids only histidine and proline, with their specific presence and activity, encouraged uredospore differentiation. L-cysteine, too, by being actively utilized, served as promoter of uredosporulation. Asparagine and methionine showed moderate to heavy depletion during bean tissue infection. On the other hand, l-leucine/isoleucine, beta-alanine, valine, and glutamic acid showed moderate to pronounced increase during pathogenic establishment. Concomitant to uredospore differentiation there was a drastic lowering in the amount of sucrose in leaf and petiole tissue. The amount of glucose also declined during pathogenesis.

Amino Acids↗
PubMed1980

Extent of peptide cross-linking in the peptidoglycan of Neisseria gonorrhoeae.

The extent of peptide cross-linking in peptidoglycan (PG) isolated from various strains of Neisseria gonorrhoeae was examined. Purified PG, specifically labeled in the peptide moiety with [(3)H]diaminopimelic acid (DAP) and labeled in the glycan with [(14)C]glucosamine and [(14)C]muramic acid, was digested completely with Chalaropsis B muramidase. Gel filtration of the digest on connected columns of Sephadex G-50 and G-25 revealed four well-defined peaks corresponding to soluble PG fragments and containing a constant ratio of (3)H to (14)C. On the basis of (i) K(D) values, (ii) amino acid composition, (iii) free amino group analysis of [(3)H]DAP residues, (iv) borohydride reduction, (v) the beta-elimination reaction, (vi) high-voltage electrophoresis, and (vii) paper chromatography in various solvents, the PG fragments were identified as un-cross-linked disaccharide peptide monomer, typical of chemotype I PG, and the corresponding peptide cross-linked dimers, trimers, and tetramers. The percent cross-linking of PG basically reflects the percentage of DAP residues that are involved in peptide cross-linking bonds. This value was estimated from the distribution of labeled fragments that resulted from the enzymatic digestion of PG and was confirmed by the analysis of free amino groups in [(3)H]DAP of intact PG. Although there were subtle, strain- and medium-dependent differences in percent cross-linking, these values varied only over a relatively narrow range (36 to 44%). The percent cross-linking of PG in the prototype strain, RD(5), grown in a standard gonococcal medium (LGCB(+)) was 41.0 +/- 2.0%. This is a relatively high degree of peptide cross-linking for a gram-negative bacterium. We also confirmed previous observations that the extent of PG cross-linking among isogenic gonococci was higher in strains, e.g., FA140 and FA136, carrying loci that govern increased resistance to multiple drugs.

Amino Acids↗
PubMed1980

Release of soluble peptidoglycan from growing conococci: demonstration of anhydro-muramyl-containing fragments.

Previous analysis of soluble peptidoglycan (PG) fragments released by exponentially growing gonococci implicated the combined action of both hexosaminidase and amidase activities in PG turnover. Current studies further characterized PG fragments which were labeled in the glycan with D-glucosamine and in the peptide moiety with meso-diaminopimelic acid of L- and D-alanine. Labeled PG fragments were isolated by gel filtration and characterized on the bases of (i) KD values, (ii) free amino group analysis using fluorodinitrobenzene, (iii) borohydride reduction, (iv) alkali-catalyzed beta-elimination, (v) paper chromatography in various solvents, (vi) electrophoretic mobility at various pH values, (vii) digestibility by Charonia lampas glycosidases, and (viii) content of labeled D- and L-alanine. A set of well-characterized PG fragments was used as standards. The monomer fraction (the major extracellular product) was found to contain two components. Most (about 80%) appeared to be N-acetylglucosaminyl-beta-1 leads to 4-1,6-anhydro-N-acetylmuramyl-L-ala-D-glu-meso-diaminopimelic acid; the remainder was the corresponding disaccharide tetrapeptide containing a C-terminal D-alanine. An unusual feature of these products was the presence of the anhydro-muramyl (non-reducing) ends, reflecting the activity of a gonococcal transglycosylase, and the near absence of products containing detectable reducing ends. Otherwise, the structures of the monomer fragments were typical of those expected for a gram-negative bacterium (chemotype I). The corresponding peptide-cross-linked dimer and the free disaccharide also contained nonreducing ends, exclusively. Free peptides (products of amidase activity) consisted of both tripeptide and tetrapeptide. In summary, all gonococci examined appear to possess an unusual transglycosylase activity which contributes to the release of soluble PG fragments containing nonreducing, anhydro-muramyl ends. The release of these fragments in vivo might be a unique aspect of gonococci-host interactions.

Amino Acids↗