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Biomedical subjects

A Datta

Publications and source records attributed to A Datta.

At least 109 records · Page 6Linked to original sources
PubMed1992

Molecular cloning of a gene encoding a seed-specific protein with nutritionally balanced amino acid composition from Amaranthus.

An albumin with a well-balanced amino acid composition and high levels of the essential amino acids was purified to homogeneity from the mature seeds of Amaranthus hypochondriacus. The amino acid composition of this protein is comparable to the World Health Organization recommended values for a highly nutritional protein. The protein is a 35-kDa monomer with four isoforms that can be separated by chromatofocusing. Antibodies raised against one of the isoforms, AmA1, cross-reacted with the other three isoforms. Affinity-purified AmA1 antibodies were used to isolate cDNA clones from a developing-seed expression library. The six immunopositive recombinants obtained were found to be related. The cDNA of the largest clone (1.2 kilobases) has a single major open reading frame corresponding to a 304-amino acid polypeptide. The clone was confirmed by hybrid-selected translation and immunoprecipitation. The size of the immunoprecipitated product was identical to the mature protein. Analysis of RNA and protein in developing seeds showed that AmA1 is synthesized during early embryogenesis, reaching a maximum by midmaturation. No RNA was detected in 1-day-old seedlings although the protein showed delayed breakdown on germination. Expression of the AmA1 gene was found to be seed-specific, as no protein or RNA was detected in other plant tissues.

Amino Acid Sequence↗
PubMed1992

N-acetyl-D-glucosamine-specific lectin purified from Vibrio cholerae 01.

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.

Acetylglucosamine↗
PubMed1992

The complex CD44 transcriptional unit; alternative splicing of three internal exons generates the epithelial form of CD44.

We have utilized the polymerase chain reaction (PCR) to isolate a 3.5 kilobase pair (kb) genomic fragment that encodes the additional extracellular domain unique to the epithelial isoform of CD44 (CD44E). Nucleotide sequence was determined for this complete region and sequence comparison to our previously determined CD44R1 and CD44R2 cDNA sequences revealed the R region to be comprised of three exons of 102 bp, 90 bp, and 204 bp. Northern blot analysis of CD44 expressing cell lines confirmed the presence of CD44R1 transcripts and indicates that the epithelial domain may be inserted through alternative splicing into all CD44 transcript classes. Southern blot analysis of the CD44E genomic fragments is consistent with a single copy per human haploid genome. The data presented here further supports our model of the human CD44 transcriptional unit as a single gene complex that utilizes an invariant 5' initiation site, alternative internal and 3' end splicing, and multiple poly (A) sites to generate through RNA processing a diverse number of human CD44 isoforms.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of a novel protease from solid substrate cultures of Phanerochaete chrysosporium.

The white-rot basidiomycete Phanerochaete chrysosporium is mostly known for its extracellular ligninolytic enzymes. In this paper, the purification and characterization of a novel extracellular protease secreted by this fungus in solid substrate cultures under ligninolytic conditions are described. The purification steps included extraction of enzymes from the wood substrate, concanavalin A-Sepharose chromatography, anion-exchange chromatography (Mono Q), and size-exclusion chromatography (Superose 12). The purified protein migrates with Mr = 40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has an isoelectric point of 5.6 and a sharp pH optimum at 4.0. The protease is totally inhibited by Hg2+, p-hydroxymercuribenzoic acid, and N-bromosuccinimide, but is insensitive to phenylmethanesulfonyl fluoride, pepstatin A, and EDTA. Its amino acid composition and NH2-terminal sequence have also been determined. The sequence data and the binding of the protease to concanavalin A indicate that the protease is a glycoprotein. The protease differs in its physicochemical properties and its response to inhibitors from other extracellular proteases previously found in another strain of P. chrysosporium. The data suggest that it has properties of both aspartate-type and thiol-type proteases.

Amino Acid Sequence↗
PubMed1992

Adhesion of Shigella dysenteriae type 1 and Shigella flexneri to guinea-pig colonic epithelial cells in vitro.

Adhesion of bacteria to guinea-pig colonic epithelial cells in vitro was inhibited by fucose with all the four strains tested (two of Shigella dysenteriae type 1 and two of S. flexneri). N-acetyl neuraminic acid and N-acetyl mannosamine also caused inhibition, suggesting a multiplicity of receptors on the epithelial cell. Congo-red binding of the strains correlated with their adhesive ability, whereas haemagglutination of rabbit erythrocytes by the bacteria did not.

Animals↗
PubMed1992

Indian policies on urbanisation and urban development.

"This article begins by examining the rationale of [India's] national urbanisation policy goals, before moving on to analyse some of the specific urban development schemes.... This is followed by a discussion of the modifications which need to be made to India's urbanisation goals...." The policy's three main objectives are to achieve "a) rural-urban mix in regional development; b) distribution of urban size-classes; and c) rural-urban distribution of public services."

Asia↗
PubMed1991

Oxalate decarboxylase from Collybia velutipes. Purification, characterization, and cDNA cloning.

The oxalate-degrading enzyme oxalate decarboxylase (EC 4.1.1.2), which is inducible by oxalic acid, was purified to homogeneity from a crude extract of Collybia velutipes, a basidiomycetous fungus. Two forms of the enzyme were resolved on chromatofocusing. The two isozymes were shown to be related by amino acid composition, peptide mapping, and immunological cross-reactivity. Peak A, eluting at pH 3.3, was used for further study; the Km was found to be 4.5 mM, and the Vmax was 166 mumol/min/mg. The subunit molecular mass of the glycosylated enzyme was 64 kDa, whereas the mass of the deglycosylated protein was 55 kDa. The enzyme showed an acidic pl, was very stable over a wide pH range, and was moderately thermostable. The cDNA encoding the enzyme was obtained by immunoscreening a lambda gt11 expression library. In vitro translation of hybrid-selected mRNA gave a 55-kDa protein. Genomic Southern hybridization indicated the oxalate decarboxylase is encoded by a single gene. The cDNA probe hybridized to a single 1.5-kilobase pair species of mRNA. The mRNA was shown to be induced by oxalic acid. A temporal relationship between enzyme activity and mRNA levels was observed, thus suggesting that the expression of oxalate decarboxylase is regulated at the transcriptional level.

Amino Acids↗
PubMed1991

Overexpression of the actin gene is associated with the morphogenesis of Candida albicans.

A progressive increase in the synthesis of actin mRNA was observed by Northern blot analysis, when cells were induced to form germ tubes at 37 degrees C by N-acetyl-D-glucosamine. Presence of trifluoperazine, a calmodulin inhibitor, or incubation of cells at 25 degrees C, or by replacing N-acetyl-D-glucosamine with glucose which inhibited germ tube formation lowered this synthesis. Furthermore, in vitro translation of total RNA revealed an increase in the synthesis of actin (45 kDa) during germ tube formation. These results suggest for the first time that the expression of actin gene is regulated during morphogenesis of C. albicans.

Acetylglucosamine↗
PubMed1991

Role of nutritional status of the cell in pH regulated dimorphism of Candida albicans.

Stationary phase cells of Candida albicans are under the control of glucose repression, as indicated by the inhibition of germ tube formation by glucose. This 'glucose effect' was absent in starved cells which were derived from similar stationary phase cells. Moreover, starved cells required glucose for germ tube formation, suggesting that it was depletion of energy reserves which was the main factor overriding the 'glucose repression machinery' during starvation. High concentration of phosphate in Lee's medium was the reason for the reduced ability of the starved cells to form germ tubes at pH 4.5 (20% of cells compared to 88% at pH 6.8). However, when phosphate was replaced or its concentration reduced, germ tube formation occurred as frequently at pH 4.5 as at pH 6.8. This 'phosphate effect' was not observed in stationary phase cells, as they were already repressed by glucose.

Candida albicans↗
PubMed1991

Characterization of the inhibition of Escherichia coli pyruvate dehydrogenase complex by pyruvate.

The E. coli pyruvate dehydrogenase complex was inhibited by pyruvate in absence of its cofactor, NAD+. The inhibition was found to increase with pH and phosphate concentration of the buffer and decrease with its ionic strength. The inhibition profile was different with MOPS buffer. No radioactivity was found in the enzyme, when the latter was incubated with 2-14C-pyruvate. The results suggest that covalent adduct formation is not necessary for the observed inhibition.

Escherichia coli↗
PubMed1991

Induction of secretory acid proteinase in Candida albicans.

Candida albicans and some other pathogenic Candida species, when grown in a medium containing a protein as a sole source of nitrogen, secrete an acid proteinase. Culture supernatants were assayed for proteinase activity, and were also analysed by Western blotting with antibodies raised and affinity-purified against proteinase of C. albicans. Proteinases secreted by C. tropicalis and C. parapsilosis were antigenically related to that of C. albicans, but had different molecular masses. The proteinases secreted by C. lipolytica, C. rugosa and C. lusitaniae were not antigenically related. The kinetics of proteinase secretion by C. albicans were monitored by activity and by Western blotting. With BSA as the nitrogen source, proteinase secretion increased exponentially until about 16 h. Culture supernatants of BSA-grown cultures accumulated proteinase to about a 1000-fold higher level than those of ammonium-sulphate-grown cultures. In vivo labelling experiments showed that proteinase was not detectably accumulated in the cells, but was secreted immediately after synthesis. Immunoprecipitation of in vitro translated poly(A)-containing RNA identified a putative pre-protein of about 54 kDa. As well as BSA, other proteins (haemoglobin, ovalbumin, histone), peptone and tryptone, when used as nitrogen sources, could induce proteinase, but to different levels. When Casamino acids or an amino acid mixture (equivalent to the composition of BSA) was used as nitrogen source, no induction was observed. Ammonium sulphate, or any other ammonium salt, repressed secretion of proteinase.

Amino Acids↗
PubMed1991

Identification of a specific manganese peroxidase among ligninolytic enzymes secreted by Phanerochaete chrysosporium during wood decay.

The specific enzymes associated with lignin degradation in solid lignocellulosic substrates have not been identified. Therefore, we examined extracts of cultures of Phanerochaete chrysosporium that were degrading a mechanical pulp of aspen wood. Western blot (immunoblot) analyses of the partially purified protein revealed lignin peroxidase, manganese-dependent peroxidase (MnP), and glyoxal oxidase. The dominant peroxidase, an isoenzyme of MnP (pI 4.9), was isolated, and its N-terminal amino acid sequence and amino acid composition were determined. The results reveal both similarities to and differences from the deduced amino acid sequences from cDNA clones of dominant MnP isoenzymes from liquid cultures. Our results suggest, therefore, that the ligninolytic-enzyme-encoding genes that are expressed during solid substrate degradation differ from those expressed in liquid culture or are allelic variants of their liquid culture counterparts. In addition to lignin peroxidase, MnP, and glyoxal oxidase, xylanase and protease activities were present in the extracts of the degrading pulp.

Alcohol Oxidoreductases↗
PubMed1991

Molecular cloning of the secretory acid proteinase gene from Candida albicans and its use as a species-specific probe.

Candida albicans secretes an acid proteinase when grown with a protein as a sole nitrogen source. The gene encoding this proteinase was isolated from a genomic expression library of C. albicans constructed in lambda gt11 by screening with antiproteinase antibodies. The affinity-purified antibodies used to verify the clones are monspecific; these do not cross-react with any other protein in the culture supernatants or crude extracts of C. albicans but strongly react with fusion proteins encoded by recombinant clones, revealing that these are true proteinase clones. Genomic Southern blot analysis shows that the proteinase gene is present at a unique locus and that there is no other closely related gene in the C. albicans genome. The proteinase gene probe identified two transcripts on Northern blots (RNA blots), which are present at a much higher level in C. albicans cells induced for proteinase secretion than in uninduced cells. The aspartyl proteinase gene reported earlier (T.J. Lott, L.S. Page, P. Boiron, J. Benson, and E. Reiss, Nucleic Acids Res. 17:1779, 1989) is not that of secretory acid proteinase, since the N-terminal amino acid sequence of secretory acid proteinase does not correspond to the deduced amino acid sequence of the aspartyl proteinase gene. The secretory acid proteinase gene was used to probe Southern blots of genomic DNA of several medically important Candida species and Saccharomyces cerevisiae. Under hybridization and wash conditions of low stringency, Candida tropicalis and Candida parapsilosis, in addition to C. albicans strains, gave specific signals, implying that C. tropicalis and C. parapsilosis have homologous secretory acid proteinase genes. However, under under wash conditions of high stringency, signals were obtained only with C. albicans strains, suggesting that this gene can be used as a species-specific probe. A simple yeast colony hybridization technique is sufficient to distinguish C. albicans from other yeasts.

Animals↗
PubMed1990

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans.

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.

Calcimycin↗