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Biomedical subjects

M Sarkar

Publications and source records attributed to M Sarkar.

At least 55 records · Page 3Linked to original sources
PubMed1999

Expression of three Caenorhabditis elegans N-acetylglucosaminyltransferase I genes during development.

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1, 2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of Asn-linked complex and hybrid glycans. Studies on mice with a null mutation in the GnT I gene have indicated that N-glycans play critical roles in mammalian morphogenesis. This paper presents studies on N-glycans during the development of the nematode Caenorhabditis elegans. We have cloned cDNAs for three predicted C. elegans genes homologous to mammalian GnT I (designated gly-12, gly-13, and gly-14). All three cDNAs encode proteins (467, 449, and 437 amino acids, respectively) with the domain structure typical of previously cloned Golgi-type glycosyltransferases. Expression in both insect cells and transgenic worms showed that gly-12 and gly-14, but not gly-13, encode active GnT I. All three genes were expressed throughout worm development (embryo, larval stages L1-L4, and adult worms). The gly-12 and gly-13 promoters were expressed from embryogenesis to adulthood in many tissues. The gly-14 promoter was expressed only in gut cells from L1 to adult developmental stages. Transgenic worms that overexpress any one of the three genes show no obvious phenotypic defects. The data indicate that C. elegans is a suitable model for further study of the role of complex N-glycans in development.

Amino Acid Sequence↗
PubMed1999

Dexamethasone suppresses vascular smooth muscle cell proliferation.

BACKGROUND: Experimental studies in vivo have demonstrated that dexamethasone inhibits neointimal hyperplasia following arterial injury. The mechanisms of this inhibition have not been clearly defined. Our objective was to test the hypothesis that dexamethasone directly suppresses smooth muscle cell (SMC) proliferation by inhibiting cell cycle progression and the expression of key cell cycle-dependent genes. METHODS: Cultured rat aortic SMC were treated with incremental concentrations of dexamethasone and cell number was determined after 72 h. To determine if dexamethasone inhibited cell cycle progression, cells were synchronized, then restimulated to enter the cell cycle, and treated with or without dexamethasone. DNA synthesis was determined 24 h after restimulation by measuring [3H]thymidine incorporation. To define the point of action of dexamethasone in the cell cycle, synchronized SMC were treated with dexamethasone (10(-7) M) at various time points after entry into the cell cycle. Flow cytometry and Northern blots were performed to examine cell cycle progression and the expression of smooth muscle cell cycle-dependent genes c-fos, c-myc, and thymidine kinase (TK). RESULTS: Dexamethasone treatment induced a concentration-dependent inhibition of SMC proliferation and DNA synthesis. The cell cycle progression of synchronized SMC from G1 into S phase was inhibited by dexamethasone, even when added as late as 16 h after restimulation. The expression of TK was suppressed by dexamethasone, while c-fos and c-myc were not affected. CONCLUSIONS: Dexamethasone inhibits the proliferation of SMC in a concentration-dependent fashion. This inhibition is associated with a block in cell cycle progression late in G1 phase of the cell cycle. Consistent with this finding, dexamethasone does not alter the expression of the early cell cycle-dependent genes c-fos and c-myc, but significantly inhibits the expression of TK, a marker of late G1 phase.

Animals↗
PubMed1999

Neovascularization during venous thrombosis organization: a preliminary study.

PURPOSE: Thrombus organization after venous thromboembolism leading to recanalization occurs at a variable rate. The angiogenic chemokine interleukin-8 (IL-8) has been found in thrombus months after thrombus initiation. We hypothesize that thrombus organization involves neovascularization and leukocyte influx and that IL-8 administered at thrombus induction will promote thrombus organization. METHODS: A group of rats underwent inferior vena caval occlusive thrombosis. At thrombus induction and every 24 hours, the rats were administered IL-8 (1 microgram) or serum albumin. The rats were killed at either day 4, day 8, or day 12, and, at death, colloidal carbon was perfused via the heart. The inferior vena cava was isolated, measured, weighed, and formalin fixed. The sections were stained with anti-polymorphonuclear leukocyte antibody, the endothelial marker factor VIII-related antigen, and with hematoxylin and eosin. Thrombus neovascularization (colloidal carbon) with morphometric analysis was normalized to the total thrombus area. In addition, the rats underwent perfusion with fluorescein isothiocyanate dextran (molecular weight, 150,000) at death to correlate with colloidal carbon perfusion, and thrombus fluorescence was determined. RESULTS: Thrombus cellularity initially involved neutrophils, followed by monocytes. Significantly more neutrophils, monocytes, and cells that were defined as spindle shaped (fibroblasts and endothelial cells) were noted in the animals treated with IL-8. Neovascularization was significantly increased at day 4 in the animals treated with IL-8 versus the animals treated with serum albumin and was corroborated with a significant increase in thrombus fluorescein isothiocyanate dextran fluorescence at day 4 in the rats treated with IL-8. Colloidal carbon perfusion was noted within vascular channels without extravasation and colocalized with factor VIII-related antigen. CONCLUSION: This study shows that thrombus organization involves neovascularization and that IL-8 augments thrombus organization.

Animals↗
PubMed1999

Comparison of tRNA activity under homologous and heterologous conditions during the reproductive cycle of Heteropneustes fossilis.

Rate of protein synthesis in ovary is analyzed throughout the annual reproductive cycle of H. fossilis. It is highest during previtellogenic phase and lowest during post spawning phase. The variation pattern matches with aminoacylation capacity of tRNA. The aminoacylation capacity of tRNA is compared in the two phases under homologous and heterologous conditions. Both tRNA and aRS are obtained from the same phase under homologous conditions and from different phases under heterologous conditions. Aminoacylation capacity is also compared in ovary and liver under homologous and heterologous conditions. Both tRNA and aRS show higher activity in previtellogenic phase. However, tRNA contributes more for higher aminoacylation activity. Transfer RNA fractionates into similar isoacceptors during the two phases. This indicates that the primary structure of tRNA may not change during the reproductive cycle. Therefore, it is suggested that the difference in aminoacylation activity may be due to post-transcriptional modifications of tRNA leading to conformational changes. Gm modification and 2-O' methylation of tRNA are reported earlier to vary during the reproductive cycle [2]. The results support the earlier suggestion of conformational changes in tRNA in 'active' and 'inactive' forms in previtellogenic and post spawning phases respectively. Aminoacylation capacity of tRNA shows organ specificity. It is high in ovary than in liver. Poor aminoacylation capacity is shown under heterologous conditions. This may be due to incompatible aRS related to specific amino acid pool in the tissue, or post transcriptional changes in tRNA, which may not allow cross acylation with full efficiency.

Algal Proteins↗
PubMed1999

Antitesticular effect of copper chloride in albino rats.

Copper chloride treatment adversely affects testicular activity in albino rats. To investigate its antitesticular effects mature (120 days) Wistar strain albino rats were treated intraperitoneally (i.p.) with copper chloride at doses of 1000, 2000 and 3000 micrograms/kg body weight/day for 26 days. Significant reduction of testicular and accessory sex organs (seminal vesicle, ventral prostate) weight, along with inhibition of testicular delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity and reduction in plasma testosterone level, were observed at the doses of 2000 and 3000 micrograms/kg body weight/day. The degree of inhibition in all the parameters were increased with the increase of dosage. But no significant change was observed in the above parameters when the animals were treated with 1000 micrograms/kg body weight/day dose. This suggests that copper produces a suppressive influence on male reproductive activity, mainly on testicular weight and steroidogenesis and accessory sex organ weight in a dose-dependent manner.

Animals↗
PubMed1998

Comparison of tRNA conformation during different phases of reproduction.

The present study is a comparison of tRNA conformation from ovary of Heteropneustes fossilis in its active phase of reproduction (when it is highly engaged in protein synthesis i.e. previtellogenic phase) with inactive phase (when tRNA is mainly stored in mature ovary i.e. spawning phase). Transfer RNA of active phase is shown to be compact, flexible and susceptible towards nuclease. Compact tRNA structure is evidenced by higher hyperchromicity and presence of relatively less Gm modifications thereby allowing adequate hydrogen bonding between D loop and T loop. Higher sensitivity of tRNA towards Mg++ reflects its higher flexibility towards internal environment. This structure of tRNA may be required for active protein synthesis. On the other hand tRNA of inactive phase is shown to be relaxed but resistant towards nuclease which may be favoured for storage in mature ova of a teleost as maternal carry over.

Animals↗
PubMed1998

Removal of 106 amino acids from the N-terminus of UDP-GlcNAc: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I does not inactivate the enzyme.

UDP-GlcNAc: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnTI, EC 2.4.1.101) plays an essential role in the conversion of oligomannose to complex and hybrid N-glycans. Rabbit GnTI is 447 residues long and has a short four-residue N-terminal cytoplasmic tail, a 25-residue putative signal-anchor hydrophobic domain, a stem region of undetermined length and a large C-terminal catalytic domain, a structure typical of all glycosyltransferases cloned to date. Comparison of the amino acid sequences for human, rabbit, mouse, rat, chicken, frog and Caenorhabditis elegans GnTI was used to obtain a secondary structure prediction for the enzyme which suggested that the location of the junction between the stem and the catalytic domain was at about residue 106. To test this hypothesis, several hybrid constructs containing GnT I with N- and C-terminal truncations fused to a mellitin signal sequence were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV), Sf9 insect cells were infected with the recombinant baculovirus and supernatants were assayed for GnTI activity. Removal of 29, 84 and 106 N-terminal amino acids had no effect on GnTI activity; however, removal of a further 14 amino acids resulted in complete loss of activity. Western blot analysis showed strong protein bands for all truncated enzymes except for the construct lacking 120 N-terminal residues indicating proteolysis or defective expression or secretion of this protein. The data indicate that the stem is at least 77 residues long.

Amino Acid Sequence↗
PubMed1997

FTIR spectroscopic studies of oligonucleotides that model a triple-helical domain in self-splicing group I introns.

Fourier Transform infrared (FTIR) spectroscopy was used to characterize the Mg2+ dependent association of a 23-mer mixed ribo-deoxyribonucleotide (23-mer RNA) and a 7-mer oligoribonucleotide (7-mer RNA) that models the triple-helical domain of a self-splicing group I intron [Sarkar et al. (1996) Biochemistry 35, 4678-4688]. To elucidate the effect of deoxyribose substitution in the entire backbone, as well as at specific positions, in the assembly of the triple-helical domain, parallel studies were carried out on the association of pure deoxyribonucleotides having base sequences corresponding to the oligoribonucleotides and also between 23-mer RNA and two 7-mer RNA variants. In the variants, either the ribose attached to G451 or the ribose attached to U453 was changed to deoxyribose. FTIR-monitored thermal denaturation of the two 23-mer hairpins shows two distinct melting regions in 1 M NaCl, in case of the RNA hairpin but not for the 23-mer DNA. Triple-helix association between the two strands (7-mer and 23-mer) studied by FTIR show that only when both strands are RNA, association takes place with the formation of the P6 helix. Our results also show that the interactions between the two RNA strands involve some participation of the riboses, which could also involve the 2'-OH groups of the RNA backbone. The assembly of the triple-helical domain is not possible with a deoxyribose backbone and is completely perturbed even when only one ribose at either G451 or U453 position is substituted by deoxyribose.

DNA↗
PubMed1997

Expression of stable human O-glycan core 2 beta-1,6-N-acetylglucosaminyltransferase in Sf9 insect cells.

UDP-GlcNAc:Galbeta1-3GalNAc-R (GlcNAc to GalNAc) beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) catalyses the formation of O-glycan core 2. Purification and characterization of C2GnT from natural sources has been hampered by the instability of this enzyme. We have been able to prepare a stable partly purified recombinant human C2GnT by expression of a truncated form of the enzyme in the baculovirus/Spodoptera frugiperda 9 (Sf9) insect cell system. C2GnT activity was secreted into the Sf9 culture medium (15 pmol/min per microl; approx. 0.2 mg/l) and was stable at 4 degrees C either in solution or after lyophilization. Endoglycosidase H and N-glycanase F treatment of the radiolabelled C2GnT indicated the presence of N-glycans at both potential N-glycosylation sites. The elimination of one or both of the two potential N-glycosylation sites or treatment of the virus-infected insect cells with tunicamycin resulted in loss of enzyme activity due in part to protein degradation.

Animals↗
PubMed1997

Distribution of 105-kDa sperm unique antigen on goat epididymal mature spermatozoa.

The sperm surface antigens are organized into topographic domains, specifically formed into the membrane during the sperm maturation in the epididymis. The present study was directed to determine the distribution of 105 KD sperm membrane antigen (SMA2) into the cell by affinity purified antibody, generated by 105 KD sperm antigen. The cytoimmunofluorescence study of the integrated sperm cell reveal that the antigen is localized in the anterior head overlying the acrosome. On immunoblotting of isolated head and flagellum polypeptides it has been demonstrated that this antigen is selectively present in the head membrane and absent in the flagellum. Because of its selective presence in the anterior head membrane overlying the region of acrosome on mature spermatozoa, the 105 KD sperm unique glyco-antigen may have a role in events leading to sperm-egg recognition. It is a first report of a high molecular mass membrane antigen being localized in the anterior head membrane of goat mature epididymal spermatozoa.

Animals↗