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Biomedical subjects

M Sarkar

Publications and source records attributed to M Sarkar.

At least 73 records · Page 4Linked to original sources
PubMed1997

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction.

The biosynthesis of complex asparagine (N)-linked oligosaccharides in vertebrates proceeds with the linkage of N-acetylglucosamine (GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:beta-D-mannoside beta 1-4 N-acetylglucosaminyltransferase III (GlcNAc-TIII, EC2.4.1.144) catalyzes the addition of GlcNAc to the mannose that is itself beta 1-4 linked to underlying N-acetylglucosamine. GlcNAc-TIII thereby produces what is known as a 'bisecting' GlcNAc linkage which is found on various hybrid and complex N-glycans. GlcNAc-TIII can also play a regulatory role in N-glycan biosynthesis as addition of the bisecting GlcNAc eliminates the potential for alpha-mannosidase-II, GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, and core alpha 1-6-fucosyltransferase to act subsequently. To investigate the physiologic relevance of GlcNAc-TIII function and bisected N-glycans, the mouse gene encoding GlcNAc-TIII (Mgat3) was cloned, characterized, and inactivated using Cre/loxP site-directed recombination. The Mgat3 gene is highly conserved in comparison to the rat and human homologs and is normally expressed at high levels in mammalian brain and kidney tissues. Using fluorescence in situ hybridization (FISH), the Mgat3 gene was regionally mapped to chromosome 15E11, near the Scn8a sodium channel gene at 15F1. Following homologous recombination in embryonic stem cells and Cre mediated gene deletion, Mgat3-deficient mice were produced that lacked GlcNAc-TIII activity and were deficient in E4-PHA visualized GlcNAc-bisected N-linked oligosaccharides. Nevertheless, GlcNAc-TIII deficient mice were found to be viable and reproduced normally. Moreover, such mice exhibited normal cellularity and morphology among organs including brain and kidney. No alterations were apparent in circulating leukocytes, erythrocytes or in serum metabolite levels that reflect kidney function. We thus find that GlcNAc-TIII and the bisecting GlcNAc in N-glycans appear dispensable for normal development, homeostasis and reproduction in the mouse.

Acetylglucosamine↗
PubMed1997

Thrombocytopenic coagulopathy (Kasabach-Merritt phenomenon) is associated with Kaposiform hemangioendothelioma and not with common infantile hemangioma.

Children with a large vascular tumor and associated Kasabach-Merritt coagulopathy respond inconsistently to therapy and have a high mortality rate. For this reason, we undertook a retrospective study of 21 such patients, and focused on clinical, radiographic, and histopathologic features. The male to female ratio was 1:1.6. Tumor was noted at birth in 50 percent of patients; the remainder appeared throughout infancy. The location was cervicofacial (n = 2), shoulder/upper limb (n = 4), trunk including retroperitoneum (n = 11), and lower limb (n = 4). These tumors grew rapidly to large size and were characterized by cutaneous purpura, edema, and an advancing ecchymotic margin. In contrast to common hemangioma, magnetic resonance imaging showed diffuse enhancement with ill-defined margins, cutaneous thickening, stranding of subcutaneous fat, hemosiderin deposits, and small feeding and draining vessels. All tumors were Kaposiform hemangioendothelioma (KHE); none were infantile hemangioma. Light microscopy showed irregular lobules or sheets of poorly formed, small vascular channels infiltrating and entrapping normal tissues. Characteristic features included spindle-shaped endothelial cells, diminished pericytes and mast cells, microthrombi, and hemosiderin deposits. Wide endothelial intercellular gaps and incomplete basement membranes were seen by electron microscopy. Dilated, hyperplastic, lymphaticoid channels were prominent in one tumor. KHE in 14 infants was treated with interferon alpha-2a: 6 had accelerated regression; 2 had stabilization of growth; and 6 evidenced no response. The mortality rate was 24 percent (5 of 21); this included three infants with retroperitoneal KHE. Kasabach-Merritt phenomenon does not occur with common hemangioma. Rather it is associated with the more aggressive KHE and rarely with other vascular neoplasms. Variable response to current pharmacologic therapy underscores our inadequate knowledge of the pathogenesis of thrombocytopenia in KHE.

Adipose Tissue↗
PubMed1997

Major goat sperm 105 kDa maturation antigen: purification, characterization, and effect of its antiserum on acrosin activity.

A ConA binding membrane glycoantigen of 105 kDa molecular mass was purified from mature goat sperm by ion exchange and gel filtration chromatography. Of the detergents examined, the anionic deoxycholate was found to be highly effective in maximum solubilization of this sperm membrane antigen (SMA2). The analysis of the saccharide components by gas liquid chromatography revealed that the 105 kDa antigen (SMA2) contained the highest amount of mannose, followed by galactose and glucose in a ratio of 4:3:1. One amino sugar, N-acetylglucosamine, was also found to be present in the polysaccharide branching of the SMA2 antigen. The internal sulfydryl linkage is essential for the maintenance of the protein backbone of 105 kDa antigen. The antigen selectively resides on the anterior head of goat sperm. The binding of anti-SMA2 antibody to the integrated mature goat spermatozoa inhibited the release of acrosin after the induction of spermatozoa with Ca-ionophore.

Acetylglucosamine↗
PubMed1996

A synthetic model for triple-helical domains in self-splicing group I introns studied by ultraviolet and circular dichroism spectroscopy.

Structural studies were performed on synthetic oligonucleotides with sequences corresponding to the P4/P6 and J3/4, J6/7 regions of the self-splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena thermophila intron. A 23-mer RNA was synthesized as a mixed ribo-deoxyribo oligonucleotide, modeling an expected base-paired region of P4 along with the J3/4 and P6 (5'-end bases of P6) regions. strand modeling the 3'-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA). The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy. The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region. Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg2+ (>5mM) but not in presence of a monovalent cation like Na+ (up to 500 mM). Studies on selected variants of both 7-mer and 23-mer RNAs were carried out. The results show that for the association of the two partner strands not only the formation of P6 helix but also triplet interactions between two strands are required. The association of the two strands in general follow a pattern predicted by comparative sequence analysis. Parallel studies on pure oligoribonucleotides having base sequence corresponding to those of the oligoribonucleotides showed no evidence of association under similar conditions, which could indicate that the 2'-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples. Molecular modeling studies on the proposed "plaited triple helix" formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.

Animals↗
PubMed1996

Prophylactic surgery for multiple endocrine neoplasia type IIa after genetic diagnosis: is parathyroid transplantation indicated?

Identification of germline mutations in the RET proto-oncogene predisposing to multiple endocrine neoplasia type IIa (MEN-IIa) has allowed a DNA-based approach to diagnosis and treatment by prophylactic thyroidectomy in children testing genetically positive. Although total thyroidectomy is the accepted operation for C cell disease, the necessity of routine total parathyroidectomy and autotransplantation as previously described in these asymptomatic children is questionable, particularly given the low occurrence of hyperparathyroidism in MEN-IIa (10-20%). Thirty-six children (ages 1 month to 12 years) from four MEN-IIa kindreds at risk for disease underwent genetic testing. Mutational analysis was done using a highly sensitive PCR-based denaturing gradient gel electrophoresis technique. Parathyroid or serum calcium concentrations were determined preoperatively. Of the 36 children at risk, 18 were found to have a MEN-IIa mutation; 11 have undergone prophylactic thyroidectomy at ages ranging from 2 to 12 years (mean 7.5 years). In each case, there was no biochemical evidence of hypercalcemia preoperatively, and all parathyroid glands were identified and were found to be grossly normal at exploration. Glands were carefully dissected and left in situ. Postoperatively, 10 of the 11 children maintained normocalcemia, allowing discharge within 24 to 36 hours. Resected thyroid glands contained C cell hyperplasia in nine, medullary carcinoma in one, and normal histology in one. We conclude that an alternative to routine parathyroidectomy may be desirable for prophylactic treatment of MEN-IIa. In situ parathyroid preservation can be safely achieved without compromising the completeness of the thyroid resection. This conservative approach obviates the potential morbidity associated with total parathyroidectomy and autotransplantation.

Calcium↗
PubMed1996

Effects of passive immunization against alpha-2u-globulin and supplementation with alpha-2u-globulin on serum gonadotrophins and testicular activity.

The purpose of the present study was to evaluate the effects of anti-alpha-2U-globulin on pituitary-gonadal functions in male rats. Adult male rats were given injections of anti-alpha-2u-globulin (1 mg/day) for 14 days, when they were killed 7 days after the last injection, serum levels of gonadotrophins and testosterone measured by radioimmunoassays, were less, testicular delta 3 beta- and 17 beta-hydroxy-steroid dehydrogenase (3 beta- and 17 beta-HSD) activities were suppressed, spermatogenesis was inhibited and serum level of alpha-2u-globulin was decreased in anti-alpha-2u-globulin treated rats. Administration of alpha-2u-globulin (1.5 mg/day) for 7 days to anti-alpha-2u-globulin treated rats reversed the 3beta-HSD and 17beta-HSD activities and serum levels of gonadotrophins, testosterone and alpha-2u-globulin, while spermatogenesis was restored to normal. The results indicate that changes in testicular steroidogenesis and spermatogenesis in rats after passive immunization against alpha-2u-globulin may be due to decrease in availability of endogenous alpha-2u-globulin.

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

Retroviral-mediated transduction of endothelial cells with the lac Z gene impairs cellular proliferation in vitro and graft endothelialization in vivo.

PURPOSE: An endothelialized lumen within a synthetic graft that expresses recombinant proteins with anticoagulant or antiproliferative activity has the potential to improve graft function. However, preliminary data suggest that genetic modification of endothelial cells (ECs) impairs their proliferation. The purpose of this investigation was to test the hypothesis that retroviral transduction of cultured ECs with the lac Z gene encoding for beta-galactosidase would decrease EC proliferation in vitro and graft endothelialization in vivo. METHODS: In vitro studies compared canine EC proliferation over a 14-day period among early-passage ECs (two and three) and late-passage ECs (six and nine) transduced with the BAG vector (containing the lac Z gene and the neomycin resistance gene), ECs transduced with the neomycin resistance gene only, the nontransduced ECs. In vivo canine studies assessed endothelialization of expanded polytetrafluoroethylene thoracoabdominal grafts seeded with autologous lac Z-transduced ECs (n = 7) or nontransduced ECs (n = 3) compared with that of nonseeded grafts (n = 3). Histochemical staining and DNA polymerase chain reaction was used 6 weeks after implantation to detect the presence of the lac Z gene in the grafts' cellular linings and perigraft tissues. Endothelialization was assessed by light microscopy and electron microscopy. RESULTS: Proliferation of late-passage lac Z-transduced ECs in vitro was significantly decreased compared with neomycin resistance-transduced ECs or nontransduced ECs. Among early-passage ECs smaller but significant decreases in proliferation were noted among lac Z-transduced cells compared with nontransduced cells. Six of seven expanded polytetrafluoroethylene grafts seeded with transduced ECs showed lac Z gene expression. Lac Z gene expression was not found on grafts seeded with nontransduced ECs or nonseeded grafts. The endothelialized luminal surface area was significantly less in grafts seeded with lac Z-transduced ECs compared with grafts seeded with nontransduced ECs. CONCLUSIONS: Retroviral-mediated transduction of canine ECs with the lac Z gene encoding for beta-galactosidase impairs EC proliferation in vitro and the ability of transduced ECs to form a confluent EC monolayer on the luminal surface of synthetic grafts in vivo.

Animals↗
PubMed1995

The human UDP-N-acetylglucosamine: alpha-6-D-mannoside-beta-1,2- N-acetylglucosaminyltransferase II gene (MGAT2). Cloning of genomic DNA, localization to chromosome 14q21, expression in insect cells and purification of the recombinant protein.

UDP-GlcNAc:alpha-6-D-mannoside [GlcNAc to Man alpha 1-6] beta-1,2-N-acetylglucosaminyltransferase II (GlcNAc-T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligomannose to complex N-glycans. A 1.2-kb probe from a rat liver cDNA encoding GlcNAc-T II was used to screen a human genomic DNA library in lambda EMBL3. Southern analysis of restriction endonuclease digests of positive phage clones identified two hybridizing fragments (3.0 and 3.5 kb) which were subcloned into pBlueScript. The inserts of the resulting plasmids (pHG30 and pHG36) are over-lapping clones containing 5.5 kb of genomic DNA. The pHG30 insert (3.0 kb) contains a 1341-bp open reading frame encoding a 447-amino-acid protein, 250 bp of G + C-rich 5'-upstream sequence and 1.4 kb of 3'-downstream sequence. The pHG36 insert (3.5 kb) contains 2.75 kb of 5'-upstream sequence and 750 bp of the 5'-end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e. a short 9-residue putative cytoplasmic N-terminal domain, a 20-residue hydrophobic non-cleavable putative signal-anchor domain and a 418-residue C-terminal catalytic domain. Northern analysis of human tissues showed a major message at 3 kb and minor signals at 2 and 4.5 kb. There is no sequence similarity to any previously cloned glycosyltransferases including human UDP-GlcNAc:alpha-3-D-mannoside [GlcNAc to Man alpha 1-3] beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-T I) which has 445 amino acids with a 418-residue C-terminal catalytic domain. The human GlcNAc-T I and II genes (MGAT1 and MGAT2) map to chromosome bands 5q35 and 14q21, respectively, by fluorescence in situ hybridization. The entire coding regions of human GlcNAc-T I and II are each on a single exon. There is 92% identity between the amino acid sequences of the catalytic domains of human and rat GlcNAc-T II. Southern analysis of restriction enzyme digests of human genomic DNA indicates that there is only a single copy of the MGAT2 gene. The full-length coding region of GlcNAc-T II has been expressed in the baculovirus/Sf9 insect cell system, the recombinant enzyme has been purified to near homogeneity with a specific activity of about 20 mumol.min-1.mg-1 and the product synthesized by the recombinant enzyme has been identified by high-resolution 1H-NMR spectroscopy and mass spectrometry.

Amino Acid Sequence↗
PubMed1995

Analysis of transfer RNA during the early embryogenesis of the freshwater teleost, Heteropneustes fossilis.

Total RNA as well as transfer RNA were quantified from mature ova apart from four different embryonic stages namely mid-cleavage, early gastrula, mid-gastrula and organogenesis of the freshwater teleost Heteropneustes fossilis. Total RNA as well as transfer RNA quantity follow a similar variation pattern, being maximum during mid-gastrulation. When analysed by total amino acid acceptance capacity, transfer RNA shows its maximum activity during mid-gastrulation. This coincides with the higher ratio of tRNA to total RNA at this stage. The relative aminoacylation capacity for Ser, Gly, Asn and Thr are found to be higher (9-34%) compared to that for other amino acids. Total tRNA, resolved into three peaks upon HPLC fractionation, shows a high cumulative peak area during mid-gastrulation and organogenesis. These results indicate a switch over of maternal to embryonic translation machinery during gastrulation.

Animals↗
PubMed1994

Purification and partial characterization of a heparin-binding lectin from the marine clam Anadara granosa.

The heparin-binding lectin, Anadarin MS, from the plasma of the marine clam Anadara granosa is purified through affinity chromatography on heparin-Sepharose 4B followed by gel filtration on a Sepharose 6B column. The purified lectin is a pentameric protein of native M(r) 300 kDa and is composed of identical subunits of 60 kDa. The pI value of this Ca(2+)-dependent lectin is 6.2. Anadarin MS agglutinates normal rabbit erythrocytes but not that of human. Aspartic acid, glutamic acid, histidine and glycine are the predominant amino acids. Unlike other reported heparin-binding lectins, Anadarin MS exhibits a unique and strict specificity for iduronic acid containing glycosaminoglycans. This lectin agglutinates infective promastigotes of Leishmania donovani exclusively and can therefore be used as a novel biochemical surface marker for this parasite.

Agglutination↗
PubMed1994

Synthesis of tetrasaccharide analogues of the N-glycan substrate of beta-(1-->2)-N-acetylglucosaminyltransferase II using trisaccharide precursors and recombinant beta-(1-->2)-N-acetylglucosaminyltransferase I.

Recombinant rabbit UDP-GlcNAc: alpha-Man-(1-->3R) beta-(1-->2)-N-acetylglucosaminyl-transferase I (EC 2.4.1.101, GlcNAc-T I) produced in the Sf9 insect cell/baculovirus expression system has been used to convert compounds of the form 3-R-alpha-Man(1-->6)(alpha-Man(1-->3)) beta-Man-O-octyl to 3-R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha-Man(1-->3)) beta-Man-O-octyl where R is OH (14), O-methyl (17), O-pentyl (18), O-(4,4-azo)pentyl (19), O-(5-iodoacetamido)pentyl (20) and O-(5-amino)pentyl (21); 2-deoxy-alpha-Man(1-->6)(beta-GlcNAc(1-->2) alpha-Man(1-->3)) beta-Man-O-octyl (16), 4-O-methyl-alpha-Man(1-->6) (beta-GlcNAc(1-->2) alpha-Man(1-->3)) beta-Man-O-octyl (22), 6-O-methyl-alpha-Man(1-->6)(beta-GlcNAc(1-->2) alpha Man(1-->3)) beta-Man-O-octyl (23) and alpha-Man(1-->6)[beta-GlcNAc(1-->2)(4-O-methyl) alpha-Man(1-->3)] beta-Man-O-octyl (15) were also synthesized by this procedure. The yields ranged from 80 to 99%. Products were characterized by high resolution 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 14, 15, 17, 22, and 23 are excellent substrates for UDP-GlcNAc: alpha-Man(1-->6R) beta-(1-->2)-N-acetylglucosaminyltransferase II and the other compounds are inhibitors of this enzyme.

Animals↗
PubMed1994

Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development.

Complex asparagine (N)-linked oligosaccharides appear late in phylogeny and are highly regulated in vertebrates. Variations in these structures are found on the majority of cell-surface and secreted proteins. Complex N-linked oligosaccharide biosynthesis is initiated in the Golgi apparatus by the action of Mgat-1-encoded UDP-N-acetylglucosamine:alpha-3-D- mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI). To determine if these structures govern ontogenic processes in mammals, mouse embryos were generated that lacked a functional Mgat-1 gene. Inactivation of both Mgat-1 alleles produced deficiencies in GlcNAc-TI activity and complex N-linked oligosaccharides. Embryonic lethality occurred by day 10.5, thus establishing that complex N-linked oligosaccharides are required during post-implantation development. Remarkably, embryonic development proceeded into day 9 with the differentiation of multiple cell types. Complex N-linked oligosaccharides are important for morphogenic processes as neural tube formation, vascularization and the determination of left-right body plan asymmetry were impaired in the absence of a functional Mgat-1 gene.

Animals↗
PubMed1994

Expression of recombinant rabbit UDP-GlcNAc: alpha 3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I catalytic domain in Sf9 insect cells.

UDP-GlcNAc: alpha 3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyses a key reaction in the conversion of oligomannose to complex and hybrid N-glycans. The cytoplasmic tail and transmembrane segment of rabbit GnT I cDNA were replaced with an in-frame cleavable signal sequence and the hybrid construct was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) under the control of the polyhedrin promoter. Sf9 insect cells were infected with the recombinant baculovirus and the enzymatically active and soluble catalytic domain of GnT I was purified from the medium (1-5 mg l-1) in two steps to a specific activity of about 2 mumol min-1 mg-1 protein. Recombinant GnT I has been used for the chemical-enzymatic synthesis of analogues of Man alpha 1-6]GlcNAc beta 1-2Man alpha 1-3]Man beta-O-octyl.

Animals↗
PubMed1994

Transfer RNA analysis during the reproductive cycle of a freshwater teleost, H. fossilis.

Transfer RNA was analyzed qualitatively as well as quantitatively from ovaries of the fresh water teleost Heteropneustes fossilis for twelve months. The tRNA samples were found to be pure and devoid of any high molecular weight RNA or DNA contaminations. The quantity of tRNA as well as its biological activity, assayed by in vitro aminoacylation using homologous aminoacyl tRNA synthetases, were found to be higher during resting and preparatory (pre-vitellogenic) phases, i.e. from November to March, as compared to vitellogenic and spawning phases of the fish, i.e. from April to October. The highest tRNA pool and its activity was found in the month of February, which coincides with the early preparatory phase. The results indicate that the accumulation of active tRNA starts in the resting phase. Such an accumulation of tRNA may be a part of the enrichment of mature eggs with complete translational machinery before ovulation in order to cope with the high rate of protein synthesis after fertilization.

Amino Acyl-tRNA Synthetases↗