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S Basu

Publications and source records attributed to S Basu.

At least 19 recordsLinked to original sources
PubMed1992

Isolation of a cDNA clone for beta 1-4 galactosyltransferase from embryonic chicken brain and comparison to its mammalian homologs.

Based on the observed biochemical and immunological similarities between bovine beta 1-4 galactosyltransferase (GalT) and a developmentally regulated galactosyltransferase (GalT-4; UDP-Gal:Lc3 beta 1-4 GalT) from embryonic chicken brain, a genetic similarity between the two enzymes has been postulated. To test our hypothesis, we have employed a reverse transcription-polymerase chain reaction (RT-PCR)based approach and isolated a approximately 600 bp cDNA clone from embryonic chicken brain mRNA. Our results indicate that, within the approximately 600 bp fragment, the avian cDNA nucleotide sequence is 74% homologous to the human beta 1-4 galactosyltransferase cDNA. Similarity and identity between the predicted amino acid sequences of the two galactosyltransferases are 75% and 61%, respectively. Similar to its mammalian counterparts, the embryonic chicken brain galactosyltransferase gene appears to encode multiple mRNA transcripts (2.3 and 1.4 kb) and shows multiple bands on a Southern blot (18.6, 12.9, 10.5 and 3.7 kb) indicating that the avian gene is either polyexonic and/or it belongs to a multiple gene family.

Amino Acid Sequence↗
PubMed1992

In vitro biosynthesis of GbOse4Cer (globoside) and GM2 ganglioside by the (1-->3) and (1-->4)-N-acetyl beta-D-galactosaminyltransferases from embryonic chicken brain. Solubilization, purification, and characterization of the transferases.

(1-->4)-N-Acetyl-beta-D-galactosaminyltransferase (GalNAcT-1) and (1-->3)-N-acetyl-beta-D-galactosaminyltransferase (GalNAcT-2), which are involved in the in vitro biosynthesis of GM2 and GbOse4Cer glycosphingolipids, respectively, have been solubilized and separated by differential detergent extraction from a membrane preparation of 19-day-old embryonic chicken brain. The separated GalNAcT-1 activity had a pH optima of 7.8-8.0, and the separated GalNAcT-2 activity a single pH optimum of 7.2. Furthermore, the partially purified GalNAcT-2 preparation catalyzed the transfer of N-acetylgalactosamine from UDP-D-[3H]GalNAc to only GbOse3Cer and nLcOse5Cer. Both GalNAcT-1 and GalNAcT-2 activities were purified to approximately 316- and 428-fold, respectively, by use of UDP-hexanolamine-Sepharose 4B affinity-column chromatography. However, the partially purified GalNAcT-1 preparation appeared to be active only with GM3, lactosylceramide, and lactotriaosylceramide. The proposed linkage of the N-acetylgalactosamine unit incorporated into GM3 is beta-D-GalpNAc-(1-->4)-GM3 from the isolation of [3H]threitol after hydrolysis of the desialylated, lead tetraacetate-treated, enzymic product, beta-D-GalpNAc-(1-->4)-beta-D-[6-3H]Galp-(1-->4)-beta-D-Glcp-(1-->1)-Cer . In addition, beta-D-GalpNAc-(1-->3)-GbOse3Cer was produced, as shown by the identification of 2,4,6-tri-O-methyl-galactose after permethylation and hydrolysis of the GalNAcT-2 enzymic product, GalpNAc-[6-3H]Galp--->Gal-->Glc-->Cer.

Animals↗
PubMed1992

Expression of tissue factor pathway inhibitor by cultured endothelial cells in response to inflammatory mediators.

We recently proposed that endothelium may represent the primary physiologic site of synthesis of the tissue factor pathway inhibitor (TFPI). In support of this conclusion, we have now found that the poly(A)+ RNAs obtained from rabbit and bovine lung tissues contain abundant amounts of TFPI messenger RNAs (mRNAs), whereas the poly(A)+ RNAs obtained from the liver of these animals contain less than 5% of that found in the lung tissues. Because inflammatory mediators are known to upregulate tissue factor (TF) expression by the endothelium, we have examined the effect of these agents on the TFPI expression by the cultured endothelial cells. When cultured human umbilical vein endothelial cells were stimulated (in 10% fetal bovine serum) with phorbol myristate acetate (PMA), endotoxin, interleukin-1, or tumor necrosis factor-alpha, the TF mRNA increased approximately 7- to 10-fold within 2 to 4 hours. Unstimulated cells constitutively expressed TFPI mRNA and its levels either did not change or increased slightly (up to 1.5-fold) upon stimulation with these inflammatory agents. TF mRNA abruptly declined to a negligible level and the TFPI mRNA returned essentially to the basal level at approximately 24 hours. The membrane-bound TF clotting activity of induced cells peaked between 4 and 8 hours, and finally declined. The cumulative TFPI activity secreted into the media was either unchanged or slightly higher in the induced cell cultures as compared with that present in the noninduced cultures. Endothelial cells were also cultured in 10% heat-inactivated human serum derived from plasma or whole blood. TFPI secreted into the media containing whole blood serum was consistently higher (approximately 1.5-fold at 8 hours) than that secreted into the media supplemented with serum obtained from plasma lacking the formed elements; these cells also expressed similarly increased levels of TFPI mRNA. Moreover, PMA-stimulated cells cultured in whole blood serum expressed modestly increased levels of TFPI mRNA (approximately 1.5-fold); supernatants from these cells also contained similarly increased TFPI activity. Cumulatively, our data indicate that, unlike thrombomodulin and fibrinolytic enzymes synthesized by the endothelial cells, TFPI synthesis is not downregulated and may be slightly upregulated during an inflammatory response. Inspection of the 5' flanking region of the TFPI gene showed a conserved GATA-binding motif located approximately 400 bp upstream of the proposed transcription initiation site(s). This motif by binding to the GATA-2 transcriptional factor may keep the endothelium in an 'on' state for constitutive expression of TFPI.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Structure-activity analyses of HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase is a dimeric enzyme which can exist in both homodimeric (p66/p66) and heterodimeric (p66/p51) forms. The monomeric subunits are catalytically inert. However, during DNA synthesis by the dimeric enzyme, only one subunit (p66) appears to carry out the catalysis, while the second subunit serves only a supportive role. In the case of the p66/p66 homodimers, we find that both the subunits are catalytically competent as judged by the observation that a) primer binding occurs to both subunits and b) catalytically inert dimers can be partially activated by replacement of one of the two inactive p66 subunits.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase.

We have labeled the primer binding domain of HIV1-RT with 5'-32P-labeled (dT)15 primer using ultraviolet light energy. The specificity of the primer cross-linking to HIV1-RT was demonstrated by competition experiments. Both synthetic and natural primers, e.g., p(dA)15, p(dC)15, and tRNA(Lys), inhibit p(dT)15 binding and cross-linking to the enzyme. The observed binding and cross-linking of the primer to the enzyme were further shown to be functionally significant by the observation that tRNA(Lys) inhibits the polymerase activity on poly(rA).(dT)15 template-primer as well as the cross-linking of p(dT)15 to the enzyme to a similar extent. At an enzyme to p(dT)15 ratio of 1:3, about 15% of the enzyme can be cross-linked to the primer. To identify the domain cross-linked to (dT)15, tryptic peptides were generated and purified by a combination of HPLC on a C-18 reverse-phase column and DEAE-Sephadex chromatography. A single peptide cross-linked to p(dT)15 was identified. This peptide corresponded to amino acid residues 288-307 in the primary sequence of HIV1-RT as judged by amino acid composition and sequence analyses. Further, Leu(289)-Thr(290) and Leu(295)-Thr(296) of HIV1-RT appear to be the probable sites of cross-linking to the primer p(dT)15.

Amino Acid Sequence↗
PubMed1992

Aortoventriculoplasty in the management of an infected Cabrol graft.

This report describes use of a modified aortoventriculoplasty (Konno procedure) for reoperation on a patient with prosthetic aortic valve conduit endocarditis. The modified Konno procedure was necessary to expose the mid-left ventricular outflow tract to reconstruct an aortic annulus.

Aortic Valve↗
PubMed1992

Up-regulation of IL-2 induced lymphokine activated killer cell activity by cisplatin and FK-565: involvement of calcium ion.

As reported earlier, the IL-2 induced lymphokine activated killer (LAK) activity is significantly up-regulated in the presence of cisplatin/FK-565. Based on these observations, we have investigated whether calcium is involved in the generation of LAK activity by IL-2 alone or along with CP/FK-565. We have shown that treatment of PBMC with IL-2 for four days caused an increase in intracellular free calcium and in ATP levels, which were further significantly enhanced when LAK cells were generated in the presence of CP/FK-565. Depletion of calcium resulted in decreased cytotoxic activity. Addition of tumor cells to LAK cells, generated in the presence of IL-2 alone or along with CP/FK-565 caused an instant rise in intracellular free calcium which was significantly decreased when an increase in intracellular free calcium was observed in calcium-free, EGTA-containing buffer. These data suggest that calcium is required for the activation and manifestation of lytic activity by LAK cells. Further, we observed that the increase in intracellular free calcium is not associated with the blastogenic response of peripheral blood mononuclear cells in response to treatment of IL-2 alone or together with CP/FK-565.

Adenosine Triphosphate↗
PubMed1992

Increased release of interleukin-1 and tumour necrosis factor by interleukin-2-induced lymphokine-activated killer cells in the presence of cisplatin and FK-565.

The supernatants of peripheral blood lymphocytes cultured in interleukin-2 (IL-2) for 4 days contained tumour necrosis factor (TNF) and interleukin-1 (IL-1). Addition of cisplatin (CP) or FK-565 to these cultures increased the level of both of these cytokines in culture supernatants. Furthermore, when the LAK cells induced by culture with IL-2 in the presence or absence of CP/FK-565 were co-cultured with tumour cells, the production of these cytokines was significantly enhanced. The level of enhancement depended on the ratio of LAK cells to tumour cells used and the length of coincubation of the two cell types. Culture supernatants of LAK cells and LAK cells stimulated by tumour cells were also cytotoxic to MCF-7 and U937 cells in 72 h cytotoxic assay, and antibodies specific for TNF and IL-1 inhibited the supernatant-mediated cytotoxicity. These results suggest that, in addition to the up-regulation of IL-2-induced LAK activity, treatment of PBL with CP/FK-565 leads to enhanced production of various cytokines with potential antitumour and immunoregulatory activity.

Adjuvants, Immunologic↗
PubMed1992

Interaction between lymphokine activated killer cells generated in the presence of cisplatin/FK-565 and tumor cells: transmission electron microscopy analysis.

Lymphokine activated killer (LAK) cells generated in the presence of cisplatin/FK-565 along with interleukin-2 (IL-2) showed enhanced binding to tumor cells as compared with LAK cells generated in the presence of IL-2 alone. The LAK cells showed a reorientation of cellular organelles in the presence of tumor cells. LAK-tumor conjugate pairs showed vesicle-like structures in the extracellular pockets. The number of vesicles in the extracellular pockets was found to be increased in the case of LAK cells generated in the presence of cisplatin/FK-565 along with IL-2. The closer interaction between the LAK cell-target cell and the large number of vesicles in the extracellular space of conjugate pairs may explain the increased lysis of target cells by LAK cells generated in the presence of cisplatin/FK-565 along with IL-2 as compared with IL-2 alone.

Antibiotics, Antineoplastic↗
PubMed1992

Massive abdominal lymphadenopathy in hairy cell leukaemia: a report of 12 cases.

Lymphadenopathy is an uncommon finding in hairy cell leukaemia (HCL). We report 12 HCL patients in whom relapse was associated with massive abdominal lymphadenopathy. All but one had long-standing HCL (range 3-25 years; median 10 years); in one it was discovered at presentation. Nine patients had been splenectomized and seven had previously been treated with 2'deoxycoformycin (DCF) and/or alpha-interferon (alpha IFN): three had achieved complete remission and four a partial response. The computerized tomography (CT) scan appearances were similar in all cases with a primary lymph node mass centred around the coeliac axis and involving upper para-aortic and retropancreatic regions. Histology and/or cytology confirmed nodal involvement by HCL in six patients. Large immature hairy cells were seen in both lymph nodes and bone marrow, suggesting a degree of transformation. Nine patients were treated with DCF: one had complete resolution, six responded with 50-90% reduction of the lymphadenopathy, one did not respond and one is still on treatment; alpha-IFN was used concomitantly or sequentially in two of the responders. One responding patient died of sepsis after four injections of DCF. Three patients received either alpha- or beta-IFN alone with no response. One elderly patient was not treated. Abdominal lymphadenopathy could be part of the natural history of HCL and/or may represent a transformation analogous to that seen in other low-grade lymphoproliferative disorders. Routine abdominal CT scanning should be part of the work up of all patients with HCL.

Adult↗
PubMed1992

Parathyroid hormone related protein in hypercalcaemia of Hodgkin's disease.

The role of parathyroid hormone related protein (PTHRP) as a humoral mediator of hypercalcaemia was investigated in a patient with lymphocyte depleted Hodgkin's disease during an episode of hypercalcaemia, using an immunohistochemical staining technique for PTHRP on the tumour tissue and an immunoradiometric (IRMA) assay for PTHRP1-86 on the patient's plasma. The plasma PTHRP was less than 0.23 pmol/l in the range found in normocalcaemic controls, and the immunohistochemical staining was not positive for protein. PTHRP did not have a role in the pathogenesis of hypercalcaemia in this patient.

Aged↗
PubMed1991

Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes).

Antisense oligonucleotides have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. However, the mechanism by which many oligonucleotide analogs enter cells to exert the desired effects is unknown. In this study, we have used phospholipid model membranes (liposomes) to examine further the mechanisms by which oligonucleotide analogs cross biological membranes. Permeation characteristics of 32P or fluorescent labelled methylphosphonate (MP-oligo), phosphorothioate (S-oligo), alternating methylphosphonate-phosphodiester (Alt-MP) and unmodified phosphodiester (D-oligo) oligodeoxynucleotides were studied using liposomal membranes. Efflux rates (t1/2 values) at 37 degrees C for oligonucleotides entrapped within liposomes ranged from 7-10 days for D-, S- and Alt-MP-oligos to about 4 days for MP-oligos. This suggests that cellular uptake of oligonucleotides by passive diffusion may be an unlikely mechanism, even for the more hydrophobic MP-oligos, as biological effects are observed over much shorter time periods. We also present data that suggest oligonucleotides are unlikely to traverse phospholipid bilayers by membrane destabilization. We show further that MP-oligos exhibit saturable binding (adsorption) to liposomal membranes with a dissociation constant (Kd) of around 20nM. Binding appears to be a simple interaction in which one molecule of oligonucleotide attaches to a single lipid site. In addition, we present water-octanol partition coefficient data which shows that uncharged 12-15 mer MP-oligos are 20-40 times more soluble in water than octanol; the low organic solubility is consistent with the slow permeation of MP-oligos across liposome membranes. These results are thought to have important implications for both the cellular transport and liposomal delivery of modified oligonucleotides.

Base Sequence↗
PubMed1991

Use of exoglycosidases from Mercenaria mercenaria (hard shelled clam) as a tool for structural studies of glycosphingolipids and glycoproteins.

The hepatopancreatic extract of M. mercenaria (hard shelled clam) was found to be a rich source for at least 16 different glycosidases. These glycosidases were successfully employed for the degradation of oligosaccharides, glycolipids, and glycoproteins at analytical as well as preparative levels. The identified glycosidases differ considerably in their stability profiles with respect to time and temperature of storage and presence of glycerol. However, most of the enzymes show higher activity at pH 4.5 than at pH 7.0, and could be bound on a DEAE CL-6B Sepharose anion-exchange column suggesting similar charge characteristics on the protein surface. A Gal beta 1, 3R linkage-specific beta-galactosidase activity has also been detected in the glycosidase-enriched fraction and has been utilized to obtain quantitative conversion of the ganglioside GM1 to GM2 on a preparative scale. The glycosidase-rich extract does not have detectable protease activity at the pH of optimal glycosidase activity (pH 4.5) and, hence, can be safely used for specific hydrolysis of carbohydrate moieties of glycoproteins and glycopeptides. This is the first report to characterize a repertoire of glycosidases from an inexpensive, dependable and convenient source that can be easily employed for compositional studies involving glycoconjugates.

Animals↗
PubMed1991

Biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer by a novel glucuronyltransferase (GlcAT-1) from embryonic chicken brain.

A novel glucuronyltransferase (GlcAT-1) has been detected in embryonic chicken brains. This enzyme catalyzes the biosynthesis in vitro of glucuronic acid containing glycolipids starting from neolactotetraosylceramide (nLcOse4Cer) and neolactohexaosylceramide (nLcOse6Cer). The activity is present primarily in the Golgi-rich membrane fraction and can be extracted (60%) from the membrane using a neutral detergent, Nonidet P-40, at pH 7.0. The detergent-solubilized GlcAT-1 is stable (70%) at -20 degrees C for at least 4 months. Both membrane-bound GlcAT-1 and solubilized GlcAT-1 show similar pH optima, 6.5-7.0, in HEPES buffer. The Km values were 15 and 200 microM with UDP-[14C] GlcA and nLcOse4Cer, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The purified 14C radioactive product that comigrated with chemically characterized GlcA beta 1-3nLcOse4Cer (GlcA-nLc4) also yielded a positive immunostain with monoclonal antibody (human IgM-RI). The anomeric linkage was established as beta-linked GlcA to the terminal galactose of the substrate, as evidenced by 90-99% cleavage of the terminal [14C] GlcA by purified Helix pomatia and limpet glucuronidases. Permethylation studies of the radioactive product obtained from [6-3H]Gal beta 1-4LcOse3Cer and non-radioactive UDP-GlcA showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed enzymatic product. These studies established the structure of the biosynthesized product from nLcOse4Cer as GlcA beta 1-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4Glc-ceramide.

Animals↗
PubMed1991

Up-regulation of induction of lymphokine (IL-2)-activated killer (LAK) cell activity by FK-565 and cisplatin.

The role of cisplatin and FK-565 in up-regulation of lymphokine-activated killer (LAK) cell induction by IL-2 was examined. Treatment of blood mononuclear cells (MNC) of healthy donors with cisplatin or FK-565 in the presence of IL-2 resulted in a significant increase in LAK activity against natural killer (NK)-resistant Daudi cells as assessed by the 4 h 51Cr release assay. Blood MNC treated with cisplatin alone was not cytotoxic to Daudi cells. However, MNC treated with FK-565 showed some cytotoxicity against Daudi cells. Addition of cisplatin to IL-2-stimulated MNC did not increase proliferation but did enhance cytotoxicity. FK-565 together with IL-2 increased both proliferation and cytotoxicity of blood MNC. These data suggest the potential of cisplatin and FK-565 in LAK adoptive immunotherapy for cancer treatment.

Antibiotics, Antineoplastic↗