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S Mitra

Publications and source records attributed to S Mitra.

At least 235 records · Page 13Linked to original sources
PubMed1991

The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells transfected with human DNA.

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.

Animals↗
PubMed1991

Inhibition of human fetal brain acetylcholinesterase: marker effect of neurotoxicity.

Human fetal brains were obtained after medical termination of pregnancy at 8-10 wk from informed patients. A definite regionalization of AChE was found in the brain of the fetus, with cerebellum recording the highest and cerebral hemisphere the lowest activity. Optimum conditions for the cerebellar AChE activity were determined with respect to molarity of the buffer, pH, temperature, and concentrations of substrate (acetylthiocholine iodide), activators (NaCl, MgCl2), and thiol indicator (dithiobisnitrobenzoic acid). In vitro inhibition of cerebellar AChE with two commercial pesticides, Metacid-50 (O,O-dimethyl p-nitrophenyl phosphorothioate) and carbaryl (N-methyl naphthyl-1-carbamate), were compared with pure anticholinesterase agents, diisopropylfluorophosphate (DFP) and physostigmine (eserine). In general, organophosphates are more neurotoxic than carbamate compounds, as evidenced by higher degree of AChE inhibition by DFP and Metacid-50 as compared to eserine and carbaryl. Assays were also done with psychotropic drugs by employing the procedure of in vitro AChE inhibition kinetics, and it was found that psychotropic drugs are less potent than organophosphate and carbamate compounds. Results indicate that pure and commercial organophosphates and carbamates and psychotropic drugs are all able to significantly alter the AChE activity. Thus exposure of the mother to these environmental toxicants may adversely affect the fetal neural functions.

Acetylcholinesterase↗
PubMed1991

The role of O6-alkylguanine in cell killing and mutagenesis in Chinese hamster ovary cells.

Chinese hamster ovary cells with no detectable (less than 200 molecules/cell) O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) were transfected with human cell DNA and pSV2neo plasmid by electroporation. Two stable transformant clones, GC-1 and GC-2, containing 4 X 10(4) and 4-6 X 10(3) methyltransferase molecules/cell respectively were isolated by successive screening in the presence of G418 and 2-chloroethyl-N-nitrosourea (CNU). Only three or four copies of pSV2neo DNA and no repetitive human DNA sequence were detected in these isolates. Secondary transfection of parent cells with GC-1 DNA yielded several clones containing 2-10 X 10(3) methyltransferase molecules/cell. The rate of removal of O6-methylguanine in GC-1, GC-2 and parent cells in vivo reflected their methyltransferase levels, while the N-methylpurines were removed at similar rates in all three cell lines. The differential sensitivity of these cells to several alkylating agents, namely CNU, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methyl-methane sulfonate (MMS), known to yield different proportions of O6-alkylguanine among the alkyl adducts in DNA, varied widely. The largest and smallest differences in toxic response were observed with CNU and MMS respectively. These cell lines showed no difference in sensitivity to the DNA cross-linking agent psoralen. These data strongly suggest that alkylating agents produce two classes of lethal lesions, one of which is O6-alkylguanine. Induction of mutations at the hypoxanthine-phosphoribosyltransferase locus in these cells lines suggests that, regardless of its relative yield, O6-methylguanine is the major mutagenic lesion for all alkylating agents.

Alkylating Agents↗
PubMed1991

Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents.

O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.

Alkylating Agents↗
PubMed1991

Expression of O6-methylguanine-DNA methyltransferase in malignant human glioma cell lines.

When animals are treated with carcinogenic agents that alkylate O6-guanine residues, the incidence of tumors in specific tissues often relates inversely to the level of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) present in the tissue. Similarly, the hypersensitivity to anticancer chloroethylnitrosoureas of some human tumor cell lines is believed to result from their deficiency in MGMT. We have undertaken a comprehensive investigation of MGMT expression in a panel of nine characterized human glioma cell lines. Methyltransferase activity determined by incubating protein extracts of these glioma lines with [3H]methylated DNA ranged from undetectable in six lines (the Mer- phenotype) to greater than 0.8 pmol/mg in two lines (U-373 MG and D-392 MG). MGMT protein was undetectable in Western blots of the Mer- cell extracts probed with specific anti-MGMT monoclonal antibodies. Consistent with these results, steady-state levels of MGMT mRNA, determined by Northern blot analysis, were detectable only in the three Mer+ glioma lines (U-373 MG, D-392 MG, D-263 MG). Southern analysis of EcoRI-digested DNA probed with MGMT cDNA revealed no amplification, rearrangement or deletions of the MGMT gene in any of the glioma cell lines. This is the first report that examines MGMT expression at the biochemical, molecular and genetic levels in a particular tumor type. These studies suggest that transcriptional regulation is the basis of the Mer- phenotype in these malignant human glioma cell lines, since no gross structural or quantitative abnormalities of the MGMT gene were seen in the phenotypically Mer- lines.

Blotting, Northern↗
PubMed1991

Human cerebral malaria: characterization of malarial antibodies in cerebrospinal fluid.

Anti-malarial antibodies were quantified in cerebrospinal fluid (CSF) of 17 cases of cerebral malaria, 16 presumptive cases (no demonstrable parasitaemia in peripheral blood but responding to i.v. quinine therapy) of cerebral malaria, and 15 controls. A schizont-enriched Plasmodium knowlesi antigen was used in an ELISA. Anti-malarial antibodies of IgA and IgM isotypes were not detectable in most of the CSF samples analysed, although serum antibody titres were high. However, 88% of CSF from cerebral malaria and 56% of presumptive cerebral malaria cases had significant levels of IgG anti-malarial antibodies in comparison to control CSF. The antibody levels did not correlate with the severity of coma but correlated well with the duration of coma. The CSF malarial antibody titres were independent of degree of parasitaemia. The possible role of CSF anti-malarial antibodies in cerebral malaria in the light of recent demonstrations of intrathecal synthesis of immunoglobulins and deposition of immune complex in cerebral tissues is discussed.

Animals↗
PubMed1991

Inducibility of the DNA repair gene encoding O6-methylguanine-DNA methyltransferase in mammalian cells by DNA-damaging treatments.

The inducibility of the mammalian O6-methylguanine-DNA methyltransferase (MGMT) gene encoding the MGMT protein (EC 2.1.1.63) responsible for removal of the procarcinogenic and promutagenic lesion O6-alkylguanine from DNA was examined by an analysis of transcription of the MGMT gene following exposure of repair-competent (Mex+) and repair-deficient (Mex-) cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). While human and rodent Mex- cells (CHO-9, V79, HeLa MR) showed no detectable MGMT mRNA despite the presence of the gene in their genome, the amount of it in several Mex+ lines (NIH 3T3, HeLa S3, HepG2) paralleled their MGMT activity. However, none of these cell lines showed an increase in the MGMT mRNA level after treatment with various concentrations of MNNG. In contrast, MNNG-treated rat hepatoma cells, H4IIE and FTO-2B, both Mex+, had three- to fivefold more MGMT mRNA than the corresponding untreated controls as measured 12 to 72 h after alkylation. N-Methyl-N-nitrosourea, methyl methanesulfonate, N-hydroxyethyl-N-chloroethylnitrosourea, UV light, and X rays caused a similar accumulation of MGMT mRNA in rat hepatoma cells. Studies with inhibitors of RNA and protein synthesis indicate that the induced increase in the amount of MGMT mRNA was due to enhanced transcription of the gene. Furthermore, they revealed the turnover of the MGMT mRNA to be relatively low (half-life, greater than 7 h). Mutagen-induced increase of transcription of MGMT mRNA in H4IIE cells was accompanied by elevation of MGMT repair activity and resulted in reduction of mutation frequency after a challenge dose of MNNG. Although induction of MGMT mRNA transcription has been observed in two rodent hepatoma cell lines so far, this appears to be the first demonstration of inducibility of a mammalian gene encoding a clearly define DNA repair function. The transcription activation of the MGMT gene protects cells from the mutagenic effects of methylating agents.

Alkylation↗
PubMed1991

Derivations of complex roots of a stable model for a special distribution of net maternity rates: an alternative method.

"Several mathematical models representing the age distribution of net maternity rate p(x) have...tried in the past [to] generate estimates of the parameters of a stable population model.... In an earlier paper, Mitra and Levin (1990) experimented with a generalized model.... Among other things, that model generated a pair of nonlinear equations from which the intrinsic roots, real as well as complex, had to be solved. The purpose of this paper is to present an alternative method of solving the same pair of equations which reveals another set of interesting features characteristic of this different approach."

Age Factors↗
PubMed1991

Alteration of the ecto-protein phosphorylation profile of intact goat spermatozoa during epididymal maturation.

Intact cauda-epididymal mature and caput-epididymal immature goat spermatozoa were assessed for their capacity to phosphorylate the outer surface proteins upon incubation in a modified Ringer's solution containing [delta-32P]ATP. The immature spermatozoa possessed markedly greater (approximately 7-fold) efficacy to phosphorylate the ecto-proteins than the mature cells. Autoradiographic analysis of the 32P-labelled proteins resolved by SDS-PAGE, showed that multiple sperm ecto-proteins are phosphorylated by an endogenous ecto-cyclic AMP-independent protein kinase (CIK) and the phosphorylation profile of these proteins underwent marked alteration during the epididymal sperm maturation. The intact caput-sperm as well showed nearly 4-fold higher specific activity of ecto-CIK than the cauda-sperm when the kinase activity was estimated using phosvitin as the exogenous protein substrate. The data suggest that the ecto-CIK and its specific protein substrates located on the sperm outer-surface, may have important roles in regulating the epididymal maturation of the male gametes.

Animals↗
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↗
PubMed1991

Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA.

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.

Base Composition↗
PubMed1991

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations.

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.

Alpha Particles↗
PubMed1990

Physicochemical studies of human O6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O6-alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second-order rate constants of 2.20 x 10(8) and 0.067 x 10(8) lmol-1 min-1 at 37 degrees C for duplex and single-stranded DNA substrates, respectively. The corresponding value for the alkylated base in synthetic poly(dC, dG, m6dG) is 0.02 x 10(8) l mol-1 min-1. The native protein is monomeric with a molecular mass of 22-24 kDa. Methylation of the protein does not lead to a gross change in its conformation but causes a slight reduction in its isoelectric point of 6.2. Although DNA protects the protein from heat inactivation, both duplex and single-stranded DNAs inhibit its activity in a concentration-dependent manner. The transferase reaction rate is also strongly inhibited by salt with about 20% of the maximum rate observed in physiological ionic strength. This inhibition is nonspecific with respect to the ions of univalent salts.

Animals↗
PubMed1990

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies.

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.

Horseradish Peroxidase↗
PubMed1990

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study.

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.

Binding, Competitive↗
PubMed1990

N-methyl-N-nitrosourea-induced mutations in a shuttle plasmid replicated in human cells.

The supF gene of the recombinant shuttle plasmid pZ190 (modified pZ189) was used as a target to study the nature of mutations induced by N-methyl-N-nitrosourea (MNU) in human cells. Treatment of the intact plasmid with MNU followed by its replication in human lymphoblastoid cells led to extensive inactivation and no detectable mutations of the plasmid. However, exposure of the supF DNA fragment alone, followed by its ligation into the vector, caused a ten-fold increase in mutant frequency when replicated in O6-methylguanine-DNA methyltransferase-deficient cells (from 0.54 x 10(-3) to 5.8 x 10(-3)) and an 80-fold increase when replicated in cells containing normal levels of the enzyme (from 0.047 x 10(-3) to 3.8 x 10(-3)). About 45% of the mutant plasmid molecules recovered from human cells contained deletions and insertions. Sixty to 70% of the mutant molecules of wild-type size contained a single-base substitution. Most of these changes were of the G.C----A.T type, consistent with the hypothesis that O6-methylguanine is the primary mutagenic adduct induced by MNU. However, the distribution of mutation sites was highly nonrandom; more than half of all mutations were localized at the G.C position 123, and the rest were distributed in about a dozen sites. The high yield of mutations induced in the supF DNA in a host cell whose capacity for the removal of O6-methylguanine far exceeded the amount present in the supF suggests that the repair of damages in extrachromosomal DNA may be inefficient. This is supported by the observation that the yield of mutations in supF transfected into lymphoblastoid cells devoid of repair activity for O6-methylguanine was comparable to that observed with repair-proficient host cells. The present data, together with results of mutations induced in pZ189 by other agents, strongly suggest that one major determinant of mutational hot spots is the structure of the target DNA itself.

Bacterial Proteins↗
PubMed1990

Coordination geometry of heme in lactoperoxidase: pH-dependent 1H relaxivity and optical spectral studies.

Molar relaxivity of water proton in lactoperoxidase solution was studied as a function of pH in the range of 2-13 by spin-lattice relaxation time measurements on a Bruker AM 500 MHz nuclear magnetic resonance (NMR) spectrometer. It was shown by comparison with the molar relaxivities of met myoglobin (Mb) and horseradish peroxidase (HRP) solutions that the sixth coordination position of the heme pocket in lactoperoxidase (LPO) is vacant. Distance of the water proton in the heme pocket from ferric ion was deduced to be 2.7, 3.6 and 4.3 A for Mb, HRP, and LPO, respectively. Acid-alkaline transition for met myoglobin, horseradish peroxidase, and lactoperoxidase determined from the pH dependence of changes in the Soret absorptions were found to be characterized by pK of 8.8, 10.9, and 12.1, respectively. Proton NMR of LPO at pH = 12.2 was found to have single broad resonance considerably upfield shifted as compared to that of LPO at neutral pH. By comparison with the proton NMR of HRP and Mb at pH greater than their respective pK of acid-alkaline transition, the upfield shifted proton resonance of LPO at pH = 12.2 was assigned to be due to low-spin LPO.

Chemical Phenomena↗