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Biomedical subjects

M Sarkar

Publications and source records attributed to M Sarkar.

At least 91 records · Page 5Linked to original sources
PubMed1993

Purification and partial characterization of a N-glycolylneuraminic acid specific lectin from the clam Anadara granosa (L).

The N-glycolylneuraminic acid-specific lectin (AFL) from the foot muscles of the marine clam Anadara granosa has been purified to homogeneity by affinity chromatography on bovine submaxillary mucin-Sepharose 4B. The Ca(2+)-dependent lectin agglutinates rabbit erythrocytes. The purified lectin is a tetrameric protein of native M(r) 254 kDa having a pI value of 6.65. The M(r) of two subunits is 65 kDa each and that of the remaining two is 62 kDa each. The dominant amino acids of the lectin are aspartic acid, glutamic acid, serine and glycine. The lectin activity is inhibited only by N-glycolylneuraminic acid specially when it is present in the macromolecular structure of mucin viz., porcine submaxillary mucin, which is the most potent inhibitor. The binding site does not recognize N-acetylneuraminic acid. Due to this strict specificity, the lectin appears to be unique.

Amino Acids↗
PubMed1993

Nicotine and brain-stimulation reward: interactions with morphine, amphetamine and pimozide.

Using a rate-independent discrete trial method of determining thresholds for rewarding electrical intracranial stimulation in rats, we evaluated the pharmacological interaction of nicotine plus morphine, d-amphetamine, or the D2 receptor antagonist, pimozide. Both morphine and amphetamine shifted the dose-response curve for nicotine down and to the left, indicating increased efficacy and potency, respectively. Pimozide at doses that have no effect on performance and only minimal effect on brain-stimulation reward blocked the effect of nicotine. These data suggest that the same dopaminergic substrate that supports the positive reinforcing effects of other drugs of abuse also supports nicotine reward.

Amphetamine↗
PubMed1992

The transmembrane and flanking sequences of beta 1,2-N-acetylglucosaminyltransferase I specify medial-Golgi localization.

UDP-GlcNAc:alpha 3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (GnTI) is an N(in)/C(out) (type II) membrane protein, localized in the medial-Golgi, that initiates the conversion of high mannose N-glycans to complex N-glycans. Anti-rabbit GnTI antibodies were generated using a purified, enzymatically active, bacterial recombinant fusion protein as immunogen. Rabbit GnTI was effectively retained in the Golgi complex of transfected COS-1 cells and murine L cells, as assessed by indirect immunofluorescence using the species-specific anti-GnTI antibodies; no surface expression of rabbit GnTI could be detected in the transfected cells. Rabbit GnTI, stably expressed in murine L cells, was localized by immunoperoxidase electron microscopy to the medial-cisternae of the Golgi stack. The role of the transmembrane domain of GnTI in Golgi localization was examined by generation of a hybrid construct containing the amino-terminal 31 amino acids of GnTI, corresponding to the 25-residue transmembrane (signal/anchor) domain and flanking hydrophilic sequences, fused with ovalbumin; this ovalbumin/GnTI hybrid molecule was retained in the Golgi complex of transfected COS cells and stably transfected murine L cells. No surface expression of ovalbumin/GnTI was detected. In contrast, ovalbumin fused to the equivalent domains of the human transferrin receptor, a type II cell-surface protein, was efficiently expressed on the cell surface of transfected cells. The ovalbumin/GnTI hybrid molecules in the transfected L cells were N-glycosylated, indicating an N(in)/C(out) membrane orientation, and were localized by immunoperoxidase electron microscopy to one or two cisternae of the medial-Golgi (90% of stained Golgi profiles showed medial-cisternae staining). These results show that a signal contained within the transmembrane domain and flanking residues of GnTI specifies medial-Golgi localization.

Amino Acid Sequence↗
PubMed1992

A novel galactosyl-binding lectin from the plasma of the blood clam, Anadara granosa (L) and a study of its combining site.

The marine blood clam species Anadara granosa (L) belong to arcidae, a family with some extraordinary haematological features. The plasma of this species exhibited strong haemagglutinating activities, from which a galactosyl binding lectin, Anadarin P, was purified in a single step affinity chromatography using Sepharose 4B-asialofetuin as an affinity matrix. The purified lectin, eluted with lactose, was found to be homogeneous by alkaline polyacrylamide disc gels, gel-filtration and isoelectric focusing. Native M(r) of the lectin was 130,000 having a PI value of 6.82 and was composed of two subunits of M(r) 17,000 and M(r) 16,000 which were noncovalently bound. The lectin was remarkably thermostable; the agglutinating titre remained unchanged over a wide range of pH (from 5 to 10) but increased with neuraminidase treated rabbit erythrocytes. Anadarin P combining site has been proposed to be small pocket-like structure which recognised only C-3 and C-4 hydroxyl groups of D-galactose. Presence of bulky groups at C-2 and C-6 exert strong steric hindrance as L-arabinose, 2-deoxy-D-galactose and D-xylose are better inhibitors than D-galactose. The lectin fails to differentiate methyl substituted galactosides as both alpha- and beta- methyl galactosides are equally active; but in case of substituted phenyl glycosides, the lectin shows different affinity towards alpha and beta anomers. The avidity of the lectin to bind the aromatic aglycons of galactosides suggests the presence of a hydrophobic region in the combining site. Interactions with some disaccharides indicate the presence of an extended area near the monosaccharide binding site.

Animals↗
PubMed1992

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Man alpha 3R beta 2-N-acetylglucosaminyltransferase I using synthetic substrate analogues.

UDP-GlcNAc: Man alpha 3R beta 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The Vmax for the pure enzyme with [Man alpha 6(Man alpha 3)Man alpha 6](Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc beta-Asn as substrate was 4.6 mumol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta 1-2 linkage to the Man alpha 3Man beta-terminus of the substrate. Several derivatives of Man alpha 6(Man alpha 3)Man beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man alpha 6(Man alpha 3)Man beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha 3-linked mannose (Man) of the substrate increases the KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, e.g., substitutions of the Man alpha 6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.

Animals↗
PubMed1992

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I gene.

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.

Amino Acid Sequence↗
PubMed1992

Study of foetal antigen in haematological malignancy.

Forty patients with different varieties of leukaemia and lymphoma were studied before and after therapy. Red cells and lymphocytes from each patient were tested for foetal antigen by lectin-agglutination test. The antigen was detectable on red cells in all untreated cases, the highest titre being found in chronic myeloid leukaemia. The titre showed significant reduction after treatment in all cases. We conclude that foetal antigen on red cells is a useful diagnostic aid in haematological malignancy and is a good indicator of the outcome of therapy.

Antigens, Neoplasm↗
PubMed1991

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.

Agglutination↗
PubMed1991

N-glycolylneuraminic acid specific lectin from Pila globosa snail.

A N-glycolylneuraminic acid-specific lectin (PAL) has been purified from an albumin gland extract of the apple snail, Pila globosa. Purification is conducted on a bovine submaxillary mucin-Sepharose 4B affinity matrix followed by gel filtration on a Sepharose 6B column. The lectin agglutinates rabbit erythrocytes. The hemagglutination activity is dependent on Ca2+ concentration in a significant manner but with a remarkable behaviour. The lectin is a trimeric glycoprotein of native Mr 440 kDa with 25% carbohydrate and is composed of three nonidentical subunits of molecular weights 190, 145, and 105 kDa. It has a pI of 7.0. The lectin exhibits a unique and strict specificity toward N-glycolylneuraminic acid and this phenomenon discriminates it from other known sialic acid binding lectins. The uniqueness indicates the absolute need for a glycolyl substitution on the amino residue and of a glyceryl side chain on the exocyclic part and an axial -COOH group in neuraminic acid. The presence of an acetyl substitution on the exocyclic part impedes lectin-ligand interaction.

Animals↗
PubMed1991

Organization and localization to chromosome 5 of the human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I gene.

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is a medial-Golgi enzyme essential for the synthesis of hybrid and complex N-glycans. We have isolated two overlapping genomic DNA clones which span 18 kilobases (kb) containing a single 2.5 kb exon for GlcNAc-T I. The exon includes most of the 5'-untranslated region, the complete coding sequence (1335 bases) for GlcNAc-T I (445 amino acids) and the complete 3'-untranslated region. The remaining exon (or exons) is at least 2.0 kb upstream of the intron-exon junction. Transient transfection of either clone into Lec 1 Chinese hamster ovary cell mutants (which lack GlcNAc-T I) indicates the presence of a promoter responsible for expression of a truncated transcript. Southern blot analysis indicates that the gene exists in single copy in the human genome and is located on chromosome 5. The human and rabbit enzymes are 85% similar at the nucleotide sequence level and 92% similar at the amino acid sequence level.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of high-mannose to hybrid and complex N-glycans: UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.

UDP-GlcNAc:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyzes an essential first step in the conversion of high-mannose N-glycans to hybrid and complex N-glycans. Cloning of the gene encoding this enzyme was carried out by mixed oligonucleotide-primed polymerase chain reaction amplification of rabbit liver single-stranded cDNA using sense and antisense 20- to 24-base-pair (bp) primers. A rabbit liver library in phage lambda gt10 yielded a 2.5-kilobase (kb) cDNA with a 447-amino acid coding sequence. None of the nine asparagine residues were in an Asn-Xaa-(Ser or Thr) sequence, indicating that the protein is not N-glycosylated. There is no sequence homology to other previously cloned glycosyltransferases, but GnT I appears to have a domain structure typical of these enzymes--i.e., a short amino-terminal domain, a transmembrane domain, a "neck" region, and a large carboxyl-terminal catalytic domain. RNA was transcribed off the 2.5-kb cDNA, and in vitro translation with rabbit reticulocyte lysate yielded a 52-kDa protein with GnT I activity.

Amino Acid Sequence↗
PubMed1991

Immunoclinical correlation in diabetes mellitus. A preliminary report on the lectin-agglutination test.

Twenty cases with insulin dependent diabetes mellitus (IDDM) were studied and compared with a control group with non insulin dependent diabetes mellitus (NIDDM) and another group of nondiabetic healthy persons. Lymphocytes of each group were tested for agglutination with three sets of lectins: concavalin A (ConA), soyabean agglutinin (SBA) and peanut agglutinin (PNA). SBA test, being highly positive in IDDM and persistently negative in NIDDM, is the most significant of the three tests for differentiating between the two types of diabetes mellitus.

Adolescent↗