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A Basu

Publications and source records attributed to A Basu.

At least 181 records · Page 10Linked to original sources
PubMed1991

Structure and expression of the beta-platelet-derived growth factor receptor gene in human tumor cell lines.

Southern digests of DNA from 15 human connective tissue tumor-derived cell lines (and cell strains) and from peripheral blood lymphocytes from normal volunteers were prepared to compare structure of the beta-platelet-derived growth factor receptor gene in normal and tumor-derived cells. The tumor-derived samples had no alterations in gene structure, nor was gene amplification evident. Two restriction fragment length polymorphisms were detected within the beta-receptor gene. Expression of the alpha- and beta-platelet-derived growth factor receptor was quantified using Northern blots. Expression was not tumor-type specific. For example, one rhabdomyosarcoma cell line expressed only the alpha-receptor, whereas two others expressed the beta. In contrast, a human fibroblast cell strain expressed both receptor types. Alterations in receptor expression may be a result of the tumorigenic process.

Alleles↗
PubMed1991

Ferrate oxidation of murine leukemia virus reverse transcriptase: identification of the template-primer binding domain.

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.

Amino Acid Sequence↗
PubMed1991

Structural organization of nuclear gene for subunit Vb of mouse mitochondrial cytochrome c oxidase.

We have reported recently the isolation of a cDNA for nuclear encoded subunit Vb of mouse cytochrome c oxidase by screening mouse bone marrow and kidney cDNA libraries. In the present study, this cDNA was used as a probe to screen a mouse genomic library and isolate the complete gene encoding subunit Vb. Southern blot hybridization of mouse genomic DNA with the cDNA probe suggested the occurrence of multiple genes including many retroinserts. Restriction analysis followed by Southern blot hybridization of genomic clones was used to identify the putative retroinserts from the intron containing genes. Of the 10 initial genomic clones isolated, one clone (MG3) showing the most complex hybridization pattern was found to contain the complete gene for subunit Vb. The DNA sequence analysis show that the subunit Vb gene contains four exons of 149, 73, 99, and 189 bases interrupted by three relatively small introns of 520, 165, and 648 nucleotides in a gene spanning about 2.5 kilobase pairs. As determined by a combination of primer extension and S1 protection analyses, the major transcription start site appears to be located 49 nucleotides upstream of the translation initiation codon. The ability of the 5' upstream DNA to initiate transcription was studied using the chloramphenicol acetyltransferase (CAT) expression plasmids in NIH 3T3 cells. Using this system we observed that a segment of the gene spanning nucleotides -574 to +45 can drive the transcription of CAT gene in an orientation dependent manner. The upstream region of subunit Vb gene lacks the TATA and CAAT elements, although it contains several GC rich elements and a pyrimidine rich stretch around the transcription start site.

Amino Acid Sequence↗
PubMed1991

Cellular sensitization to cis-diamminedichloroplatinum(II) by novel analogues of the protein kinase C activator lyngbyatoxin A.

Protein kinase C (PKC) has been implicated in enhancing cellular sensitivity to cis-diamminedichloroplatinum(II) (CP). We have synthesized a series of novel analogues of lyngbyatoxin A (7-linalylindolactam V), a natural tumor promoter and a potent activator of PKC, and investigated the effects of these synthetic compounds on PKC activity and the antiproliferative activity of CP. Lyngbyatoxin A was as effective as phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate, in enhancing the sensitivity of HeLa cells to CP. A 24-h pretreatment of HeLa cells with 1 to 100 nM lyngbyatoxin A caused an approximately 9-fold sensitization to CP. All analogues of lyngbyatoxin A that retained the lactam ring portion of the molecule but contained different hydrophobic substituents at C-7 including indolactam V (ILV), tert-butyl-ILV, or n-hexyl-ILV increased cellular sensitivity to CP in a concentration-dependent manner. Maximum cellular sensitization to CP (9-fold) was seen with 10 nM n-hexyl or tert-butyl compounds, and ILV devoid of any C-7 substitution required higher concentrations (1 microM) for equivalent sensitization. The ability of lyngbyatoxin A analogues to sensitize cells to CP correlated directly with their ability to activate PKC in vitro. Synthetic analogues that lacked the lactam ring structure neither activated PKC nor sensitized cells to CP. The C-9 epi analogue of n-hexyl-ILV was less effective than the corresponding natural stereoisomer in activating PKC as well as sensitizing cells to CP. Exposure of HeLa cells to 100 nM lyngbyatoxin A for 24 h caused a substantial decrease in cellular PKC activity to 20% of the untreated control value, but a similar treatment of cells with n-hexyl- or tert-butyl-ILV led to only a 25% reduction in PKC activity. Concentrations of ILV (e.g. 1 microM) that sensitized HeLa cells to CP caused no down-regulation of PKC. Thus, on the basis of results with these novel lyngbyatoxin A analogues, we conclude that activation but not down-regulation of PKC is necessary for sensitization of HeLa cells to CP.

Cell Division↗
PubMed1991

The isoforms of yeast cytochrome c oxidase subunit V alter the in vivo kinetic properties of the holoenzyme.

One of the nuclear-coded subunits of yeast cytochrome c oxidase is specified by a gene family composed of two genes, COX5a and COX5b. These genes are regulated differentially by oxygen and encode isoforms of subunit V, designated Va and Vb, which have only 66% primary sequence identity. Yeast cells require one or the other isoform for a functional cytochrome c oxidase (Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell Biol. 7, 3520-3526). To determine if these isoforms of subunit V alter the catalytic properties of holocytochrome c oxidase, we have analyzed various aspects of cytochrome c oxidase function in intact yeast cells that produce only one type of isoform. From measurements of room temperature turnover numbers and low temperature rates of ligand binding, single turnover cytochrome c oxidation, and internal electron transfer (heme a oxidation), we have found that isozymes which incorporate the Vb isoform have both higher turnover rates and higher rates of heme a oxidation than isozymes which incorporate Va. These findings support the conclusion that the isoforms of subunit V modulate cytochrome c oxidase activity in vivo and suggest that they do so by altering the rates of one or more intramolecular electron transfer reactions.

Carbon Monoxide↗
PubMed1991

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite thymidine in a DNA duplex. Nonplanar alignment of epsilon dA(anti) and dT(anti) at the lesion site.

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dT 9-mer duplex) containing 1,N6-ethenodeoxyadenosine (epsilon dA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. Our NMR studies have focused on the conformation of the epsilon dA.dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5.epsilon dA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and epsilon dA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4.dC15 and dG6.dC13 pairs. Furthermore, the d(G4-T5-G6).d(C13-epsilon A14-C15) trinucleotide segment centered about the dT5.epsilon dA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and epsilon dA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the epsilon dA.dT 9-mer duplex. Two families of energy-minimized structures were identified with the dT5 displaced toward either the flanking dG4.dC15 or the dG6.dC13 base pair. These structures can be differentiated on the basis of the observed NOEs from the imino proton of dT5 to the imino proton of dG4 but not dG6 and to the amino protons of dC15 but not dC13 that were not included in the constraints data set used in energy minimization. Our NMR data are consistent with a nonplanar alignment of epsilon dA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4.dC15 base pair within the d(G4-T5-G6).d(C13-epsilon A14-C15) segment of the epsilon dA.dT 9-mer duplex.

Adenosine↗
PubMed1991

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site.

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1991

Suppression of dexamethasone-induced metallothionein expression and cis-diamminedichloroplatinum(II) resistance by v-mos.

Metallothionein has been implicated in resistance to anticancer drugs. We examined whether transient induction of metallothionein by dexamethasone causes resistance to cis-diamminedichloroplatinum(II) (cis-DDP) in malignant and nonmalignant cells. Normal rat kidney cells (6m2) were infected with a modified v-mos oncogene construct in which expression of v-mos and consequently transformation was temperature-sensitive occurring at the permissive temperature of less than 33 degrees C and not at the nonpermissive temperature of 37 degrees C. Temperature-sensitive oncogenic transformation by v-mos attenuated induction of metallothionein by dexamethasone. No induction of metallothionein was observed in a revertant 6m2 cell line (54-5A4), which expressed v-mos and was transformed at 37 degrees C. Only nontransformed 6m2 cells displayed resistance to cis-DDP after dexamethasone pretreatment for 24 h. Dexamethasone pretreatment did not cause marked resistance to doxorubicin or melphalan in nontransformed 6m2 cells. When 6m2 cells (37 degrees C) were pretreated with dexamethasone (0.5 microM) for 24 h and then incubated in dexamethasone-free medium for 24 h, both metallothionein levels and resistance to cis-DDP decreased significantly. Thus, transient resistance to cis-DDP can be produced by a nonmetal inducer of metallothionein in nontransformed cells. Glucocorticoid-induced protection is suppressed in cells expressing v-mos and this might form the basis of future strategies to improve the therapeutic index of cis-DDP.

Animals↗
PubMed1991

Synthesis of structural analogues of lyngbyatoxin A and their evaluation as activators of protein kinase C.

Syntheses of several new analogues of lyngbyatoxin A from a single common intermediate are described. These compounds bear a carbon chain at the 7-position of the indolactam V (ILV) nucleus which contains either a hydrophilic or a lipophilic group. The effect of these minor structural alterations on the ability of the ILV analogues to activate the enzyme protein kinase C (PKC) was determined by measuring the extent of phosphorylation of calf thymus histone (III-S). Introduction of a hydroxyl group on the C-7 appendage was found to dramatically decrease compound 3's ability to activate PKC. This result is interpreted in terms of the decreased ability of 3 to associate with the membrane bilayer.

Adenosine Triphosphate↗
PubMed1991

Intravenous gammaglobulin therapy in the thrombocytopenia of haemorrhagic varicella.

We report the case of a teenage boy with thrombocytopenia complicating varicella infection, which responded promptly to treatment with intravenous immunoglobulin therapy. Such treatment is well recognized in the management of immune thrombocytopenia, particularly where steroids are contra-indicated, but we have not found any previous case reports of the use of immunoglobulin in this condition.

Adolescent↗
PubMed1991

Metallothionein expression and transient resistance to electrophilic antineoplastic drugs.

Intrinsic and acquired resistance to antineoplastic agents remains an important impediment to cancer therapy. Intracellular metallothioneins appear to be one factor in determining the responsiveness of malignant and normal cells to electrophilic anticancer agents. Metallothioneins are not only constitutively expressed but their expression can be transcriptionally activated by a host of endogenous and exogenous substances; this is a reversible phenomenon. This inducibility affords the possibility that cells can respond quickly to toxic substances and that under certain conditions transient drug resistance may occur. We review the evidence implicating metallothioneins in anticancer drug resistance and discuss this in the context of the possibility of transient resistance.

Animals↗
PubMed1990

Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site.

Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 1648-1653). To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys103 and Lys421 to leucine and alanine, respectively. Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect. Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers. The enzyme with mutation at codon 421 (Lys----Ala) exhibited properties that were indistinguishable from the wild type with respect to its mode of catalysis, i.e. preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA polymerase activity, RNase H activity, and the processive mode of DNA synthesis. These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase.

Base Sequence↗
PubMed1990

Nucleotide sequence of cDNA for nuclear encoded subunit Vb of mouse cytochrome-c oxidase.

We have used a polyclonal antibody probe to isolate cDNA clones for mouse cytochrome oxidase subunit Vb from a bone marrow tumor cell mRNA library in lambda gt11 expression vector. The mouse cDNA contains an open reading frame of 128 amino acids, which shows 81% positional identity with the predicted amino acid sequences of the human subunit. Northern blot analysis and sequencing of cDNA from a mouse kidney library show no tissue specific variations in subunit Vb.

Amino Acid Sequence↗
PubMed1990

Involvement of protein kinase C in phorbol ester-induced sensitization of HeLa cells to cis-diamminedichloroplatinum(II).

We have investigated the effect of tumor promoting phorbol esters on the antiproliferative actions of several antitumor agents. Pretreatment of HeLa cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a significant (9-fold) increase in cellular sensitivity to cis-diamminedichloroplatinum(II) (CP). TPA also sensitized HeLa cells to melphalan (2.5-fold) but had no effect on the antiproliferative activity of bleomycin, doxorubicin, vincristine, or mitomycin C. The sensitization of HeLa cells by TPA was concentration-dependent up to 1 nM and paralleled the activation of protein kinase C by TPA measured in vitro. The maximum stimulation of protein kinase C (6-fold) was observed with 10 nM TPA. 4 alpha-Phorbol 12,13-didecanoate neither activated protein kinase C nor sensitized HeLa cells to CP. 4-O-Methyl-TPA, which does not affect cell cycle distribution of HeLa cells, also sensitized these cells to CP by 6-fold and activated protein kinase C by 3-fold. Inhibitors of protein kinase C, such as palmitoylcarnitine and sphingosine, antagonized PDBu-induced sensitization of HeLa cells to CP. The maximum sensitization of HeLa cells to CP required prolonged pretreatment (greater than or equal to 24 h) with phorbol esters but could not be explained by down-regulation of protein kinase C. For example, 4-O-methyl-TPA caused no down-regulation of protein kinase C. Moreover, TPA caused substantial down-regulation of protein kinase C (1% of control) in A-253 cells but failed to sensitize A-253 cells to CP. TPA (100 nM), however, activated protein kinase C in A-253 cells by 5.5-fold. Therefore, activation of protein kinase C by TPA appears to be necessary but not sufficient for cellular sensitization to CP. The sensitization of HeLa cells by TPA was associated with a concentration- and time-dependent increase in cellular platinum content. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) blocked sensitization of HeLa cells to CP as well as the increase in platinum content caused by a 24-h pretreatment with PDBu.

Cell Division↗