PubMed Health⌕ Search

Biomedical subjects

S Mitra

Publications and source records attributed to S Mitra.

At least 19 recordsLinked to original sources
PubMed1995

Enhanced host cell reactivation capacity and expression of DNA repair genes in human breast cancer cells resistant to bi-functional alkylating agents.

Human breast carcinoma (MCF7-MLNr) cells resistant to the bifunctional drugs L-phenylalanine mustard (L-PAM, 5-fold resistance), mechlorethamine (9-fold), cisplatin (3-fold), and BCNU (3-fold) were used to investigate the role of DNA repair in the development of resistance to alkylating agents. We have previously shown that neither L-PAM transport and metabolism nor glutathione-associated enzymes were altered in MCF7-MLNr cells, compared to the sensitive cells MCF7-WT. This study shows that treatment of pRSV-CAT plasmid with L-PAM at concentrations up to 1 microM proportionally inhibit the expression of chloramphenicol acetyl transferase (CAT) activity, while higher concentrations abolished CAT activity. pRSV-CAT reactivation was significantly increased when plasmid was transfected into MCF7-MLNr cells, compared to MCF7-WT cells. This indicates that resistant cells have more efficient capacity to recognize and repair L-PAM induced DNA damage. The mRNA expression of DNA nucleotide excision repair genes ERCC1, XPD (ERCC2), XPB (ERCC3), and polymerase beta was found to be similar in both the MCF7-WT and MCF7-MLNr cells. Western blot analysis also reveals no difference in the expression of ERCC1, AP endonuclease, poly (ADP-ribose) polymerase, and alkyl-N-purine-DNA glycosylase proteins. The lack of correlation between enhanced host cell reactivation capacity in resistant cells, and the expression of these specific DNA repair genes suggests that proteins encoded by these genes are not rate limiting steps for resistance to bi-functional alkylating drugs in human breast cancer cells.

Adenocarcinoma↗
PubMed1995

Application of the associated particle technique for the whole-body measurement of protein, fat and water by 14 MeV neutron activation analysis--a feasibility study.

A small and compact sealed tube neutron generator with an integral alpha particle detector has been used for applying the associated particle technique for prompt-gamma 14 MeV neutron activation analysis of total body carbon (TBC), total body nitrogen (TBN) and total body oxygen (TBO). Ground sheep meat samples in the weight range 20-40 kg and of varying composition have been scanned using two 12.5 cm diameter x 10 cm Nal(Tl) crystals for gamma-ray detection. The content of protein, fat and water was calculated from their fractional content of C, N and O using a four-compartment model of body composition, which included minerals. The precision for measuring TBC, TBN and TBO has been obtained from the mean count rates of ten repeat irradiations of the same sample. The accuracy has been confirmed by comparison against chemical analysis. The reproducibilities for measuring TBN have been found to be comparable to those obtained when the same samples were analysed using prompt-gamma thermal-neutron activation analysis in an existing body composition facility. Based on the results obtained, we conclude that an instrument comprising the neutron generator and four 15 cm x 15 cm x 45 cm NaI(Tl) gamma ray detectors can be assembled to determine, in vivo, protein, fat and water in an approximately 41 kg sample with precisions of 4.4%, 5.0% and 2.1% (CV) respectively within a 15 min scan. The radiation dose equivalent delivered due to neutrons would be approximately 0.03 mSv.

Animals↗
PubMed1995

Structural organization of the mouse DNA repair gene, N-methylpurine-DNA glycosylase.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.

3T3 Cells↗
PubMed1995

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases.

The O6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic and carcinogenic O6-alkylguanine in DNA by accepting stoichiometrically the alkyl group from the base. Although the mouse MGMT is larger than the human protein because of an additional tetrapeptide sequence, these proteins are 70% homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified to homogeneity from Escherichia coli. The N-terminal amino acid sequences of these proteins are identical to those predicted from the nucleotide sequences, and their molecular masses determined by SDS-PAGE agreed with the predicted values. However, the observed isoelectric points of 9.3, 9.2 and 9.3, for the human, mouse and mutant mouse proteins respectively were significantly different from the values, 8.09, 7.47 and 7.49 calculated from the amino acid composition. The extinction coefficients E280 nm1% of human, mouse and mutant mouse protein were calculated from amino acid composition to be 18.2, 11.1 and 11.3 respectively. These values agree fairly well with calculated values. Human and wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m6dG) with comparable second-order rate constants of 2.2 x 10(8) and 3.7 x 10(8) l/M/min at 37 degrees C respectively and were inactivated by O6-benzylguanine at similar rates. The initial reaction rate (Kin) and rate of inactivation (kinact) constants for reaction with the base were calculated to be 1.8 x 10(-4) M and 1.4 x 10(-3)/s for the human protein, 2.3 x 10(-4) M and 1.1 x 10(-3)/s for the wild-type mouse protein, and 2.1 x 10(-4) M and 1.4 x 10(-3)/s for the mutant mouse protein respectively. The MGMTs were inactivated to the extent of 55-65% after heating at 50 degrees C in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However, in the presence of DNA (200 micrograms/ml), only 25-35% of the protein was inactivated. Both DNA and RNA inhibited all three enzymes in a concentration-dependent fashion, although DNA was a better inhibitor than RNA. High salt (0.2 M NaCl) inhibited human MGMT by 80%, while the wild-type and the mutant mouse MGMTs were inhibited by 55%. The human protein had higher affinity for binding to duplex DNAs than the mouse proteins. Immunoprecipitation (69%) and affinity constant (19.4 nM) of human MGMT with a human-specific monoclonal antibody 4.A1 significantly discriminated the human protein from either of the mouse proteins.

Amino Acid Sequence↗
PubMed1995

Cyclic AMP mediates change in superoxide dismutase activity to monitor host-parasite interaction in Leishmania donovani.

The present study is designed to understand the role of cyclic AMP (cAMP) in host-parasite interaction involving Leishmania donovani, the causative agent for Kala-azar. When Leishmania promastigotes or macrophages were pretreated with dibutyryl cAMP or theophylline and epinephrine, which are well defined initiators for cAMP release, a key enzyme of the oxygen defense system, superoxide dismutase (SOD), was inhibited. At the same time, parasite interaction was considerably reduced to the level of 54.5% and 46.2%, respectively, for pretreated promastigotes. Internalization of the organisms in phagolysosomes was similarly affected. Dibutyryl cAMP-treated promastigotes in the presence of SOD, on the other hand, restored in vitro infection to the normal level. At least 50% less cAMP entered into Leishmania promastigotes when SOD was added to the incubation system containing dibutyryl cAMP. Data reveal that cAMP perturbs the Leishmania-macrophage interaction through inhibition of SOD, pointing to the importance of a promastigote enzyme for the survival of this pathogen within phagolysosomes.

Animals↗
PubMed1994

Purification and biochemical characterization of recombinant N-methylpurine-DNA glycosylase of the mouse.

The mouse N-methylpurine-DNA glycosylase (MPG), responsible for the removal of most N-alkyladducts in DNA, was purified to homogeneity as a recombinant nonfusion protein from Escherichia coli. Only 10-15% of the protein was present in the soluble form in E. coli cells. The N-terminal amino acid sequence of the purified protein which lacks 48 residues from the amino terminus of the wild type protein was identical to that predicted from the nucleotide sequence. The glycosylase hydrolyzes 3-methyladenine (m3A), 7-methylguanine(m7G), and 3-methylguanine (m3G) from DNA, and the Km and kcat values were 130 nM and 0.8 min-1 for m3A, and 860 nM and 0.2 min-1 for m7G, respectively, when methylated calf thymus DNA was used as the substrate. A comparison of kcat/Km values for different bases indicates that the enzyme was more efficient in excising both m3A and m3G than m7G from methylated DNA. The enzyme showed moderate binding affinities (KA) for both methylated (5.8 x 10(7) M-1) and nonmethylated DNAs (4.2 x 10(7) M-1). The mouse protein has an extinction coefficient E280nm1% of 10.5 and a pI of 9.3. The enzyme activity was optimal in the presence of 100 mM NaCl, with a broad pH optimum of 8.5-9.5. The enzymatic release of both m3A and m7G was stimulated 50-75% by 0.5 mM MgCl2 and 0.02 mM spermine but inhibited by higher concentrations of these agents. Product inhibition by 40-50% of the reaction occurred in the presence of 10 mM m3A or m7G. However, 1.0 mM m3A stimulated release of m7G. The enzyme was inhibited by 60% in the presence of 0.9 mg/mL DNA which, at the same time, protected it from thermal inactivation.

Adenine↗
PubMed1994

Substrate specificity of an RNase III-like activity from Bacillus subtilis.

Bacillus subtilis bacteriophage SP82 codes for several early RNAs that were shown previously to be cleaved by an RNase III-like enzyme called "Bs-RNase III." Cloning of DNA fragments that encode these RNA sequences downstream of a T7 RNA polymerase promoter allowed the synthesis of substrates that were used to test the cleavage specificity of Bs-RNase III, which was purified from a protease-deficient strain of B. subtilis. Single nucleotide changes at or near the cleavage site and deletions upstream and downstream of the cleavage site were constructed. The effects of these changes on the rate of Bs-RNase III cleavage were measured. The activity of Bs-RNase III and Escherichia coli RNase III on heterologous substrates was also tested. Although the local environment of the site of Bs-RNase III cleavage appears very similar to that of E. coli RNase III, there are important differences in their substrate specificity.

Bacillus subtilis↗
PubMed1994

Cloning of a marsupial DNA photolyase gene and the lack of related nucleotide sequences in placental mammals.

Photoreactivating enzyme, DNA photolyase, reduces lethal, mutagenic and carcinogenic effects of ultraviolet light (UV) by catalyzing near UV or visible light-dependent repair of cyclobutane pyrimidine dimers (CPDs) in DNA. The enzyme activity has been detected in a wide variety of organisms ranging from bacteria to nonplacental mammals. However, the evidence for photoreactivation in placental mammals, including humans, is controversial. As a first step to identify the presence and activity of the gene in mammalian species, we isolated a cDNA clone of this gene from a marsupial, the South American opossum Monodelphis domestica. Photolyase activity was expressed in Escherichia coli from the cDNA which is predicted to encode a polypeptide of 470 amino acid residues. The deduced amino acid sequence of this protein is strikingly similar to those of photolyases from two metazoans; the opossum photolyase shares 59% and 63% sequence identity with the Drosophila melanogaster and goldfish Carassius auratus enzymes, respectively. However, no closely related nucleotide sequence was detected in higher mammals and a homologous transcript was undetectable in a number of human tissues. These results strongly suggest that humans, as well as other placental mammals, lack the photolyase gene.

Amino Acid Sequence↗
PubMed1994

Factors in the sociocultural environment of child labourers: a study in a small scale leather goods industry in Calcutta.

OBJECTIVES: During a study of the occupational health of the child workers in several small leather workshops, investigations were also made of the sociocultural factors that made it necessary for the children, aged between 7 and 14 years, to work. The psychosocial effects of the same factors were also investigated. METHODS: Personal interviews were carried out with a prepared questionaire, and the findings were compared between the working children and a control group of the same number of non-working children from the neighbourhood, matched for age, sex, and religion. RESULTS: It was found that the foremost cause of taking up employment by the children was poverty. The overall effects found were deprivation of education and lowering of aspirations, that indicated a blunting of mental faculties. CONCLUSION: Prolonged, monotonous work in childhood that precludes education and healthy recreation, is harmful to the children.

Adolescent↗
PubMed1994

Effect of flurbiprofen sodium on pupillary dilatation during scleral buckling surgery.

Maintenance of pupillary dilatation is necessary for success of scleral buckling procedures. The efficacy of 0.03% flurbiprofen in preventing intraoperative miosis was evaluated by a prospective randomized, double-masked controlled trial of 60 patients. Thirty patients received 0.03% flurbiprofen 6 times at 15 minute intervals 90 minutes preoperatively in addition to the routine dilation regimen. The treated group had a mean pupillary decrease of 1.88 mm and the control group had a decrease of 1.57 mm (p > 0.05). Flurbiprofen did not affect the pupillary size at any step of the surgery. Factors such as age of the patient, lens status, number of cryo applications, duration of surgery, and the size and extent of buckle were assessed. The use of flurbiprofen did not affect the mean pupillary change for any of these groups. Preoperative use of flurbiprofen does not significantly decrease intraoperative miosis during scleral buckling procedures.

Adult↗
PubMed1994

Serum IgE levels and its clinical correlation with varying patterns of bronchial asthma and nasal allergy.

In a study of 847 cases of bronchial asthma and nasal allergy frequent association with raised serum IgE was noticed. Degree of atopy (as judged by skin allergy tests), age, sex and duration of disease tended to influence IgE levels in asthmatics. Both extrinsic and intrinsic varieties of asthma showed similar raised serum IgE levels. In subjects with nasal allergy alone, the values were considerably lower but still much higher than found in control patients.

Adult↗
PubMed1993

Cleavage at site A, Glu-642 to Ser-643, of the gizzard myosin heavy chain decreases affinity for actin.

The actin-activated ATPase activities of subfragment 1 (S1) produced from gizzard myosin by papain or Staphylococcus aureus protease are different. The activity of the latter is lower, in spite of the presence of intact 20,000-dalton light chains. To study this difference, the S. aureus protease S1 was subjected to further proteolysis by papain. This second stage of proteolysis markedly increased actin-activated ATPase, due to a decrease in K(actin) with no change in Vm and increased the affinity of S1 for actin in the presence of ATP. Treatment with papain caused degradation of the 20-kDa light chain, a decrease in the 26-kDa C-terminal domain of S1 and the 68-kDa fragment containing the N-terminal and central domains, and in the appearance and progressive increase of a 94-kDa fragment. The increase in actin-activated ATPase activity was due to the production of the 94-kDa fragment but not due to light chain degradation. Analyses of N-terminal sequences following papain digestion showed that the 94-kDa fragment was formed from a combination of the 68- and 26-kDa fragments. The bond formed probably involved the N-terminal residue of the 26-kDa fragment (Ser-643) and a side chain carboxyl (Glu-642) or amine (Glu-636). From the sequence data site A was identified as Glu-642-Ser-643. These results confirm the importance of site A in actin-binding of gizzard myosin. It is suggested that the sequence Ser-643 and Val-659, as well as the 3 lysine residues, are important for actin binding.

Actins↗
PubMed1992

Expression of O6-methylguanine-DNA methyltransferase in six human medulloblastoma cell lines.

Six well characterized human medulloblastoma cell lines (D283 Med, Daoy, D341 Med, D384 Med, D425 Med, and D458 Med) were examined for the expression of O6-methylguanine-DNA methyltransferase (MGMT) by activity and Western and Northern blot analysis. High levels of MGMT activity were present in D283 Med, Daoy, D341 Med, and D384 Med (1.36, 0.80, 1.68, and 1.62 pmol/mg of protein, respectively), but negligible MGMT activity was detected in D425 Med and D458 Med (0.06 and 0.05 pmol/mg of protein, respectively), which were derived separately at different times from the same patient. The presence of MGMT protein and its transcript was demonstrated in D283 Med, Daoy, D341 Med, and D384 Med, but both the protein and the mRNA were undetectable in D425 Med and D458 Med. Nevertheless, all six cell lines contained an apparently unaltered MGMT gene, as determined by Southern blot analysis. The absence of MGMT activity in D425 Med and D458 Med is likely due to the absence of the protein, resulting from a lack of transcription of the MGMT gene. The varying levels of expression of MGMT in medulloblastoma cells found in this study should provide a molecular basis for drug design and selection in chemotherapy of this tumor.

Blotting, Northern↗
PubMed1992

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.

A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.

Amino Acid Sequence↗
PubMed1992

Correlation between DNA methylation and expression of O6-methylguanine-DNA methyltransferase gene in cultured human tumor cells.

Approximately 20% of human tumor cell strains are deficient in a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), and are called Mer- strains. In an attempt to determine the molecular basis for the extinction of MGMT expression in Mer- human cells, the distribution of DNA methylation sites in and around the exon sequences of the repair gene was compared in 6 Mer+ (repair-proficient) and 12 Mer- cell lines. Southern blot analysis of the genomic DNA digested with isoschizomeric restriction endonucleases MspI and HpaII to detect 5-methylcytosine in CCGG sequences indicated that the DNA of all the Mer+ cells but of none of the Mer- cells is heavily methylated in the exon-containing regions. The methylation pattern contradicts the general belief that inactive genes are hypermethylated compared to hypomethylation of transcriptionally active genes. It appears that the regulation of the MGMT gene in human cells is much more complex than simply dictated by its methylation level.

Blotting, Northern↗
PubMed1992

Overexpression of human DNA repair protein N-methylpurine-DNA glycosylase results in the increased removal of N-methylpurines in DNA without a concomitant increase in resistance to alkylating agents in Chinese hamster ovary cells.

N-Alkylpurines induced in DNA by simple monofunctional alkylating agents are known to be cytotoxic and possibly indirectly mutagenic. These adducts are removed by the ubiquitous N-methylpurine-DNA glycosylase (MPG) in a multistep repair pathway. Chinese hamster ovary (CHO) cell clones expressing 2- to 16-fold enhanced levels of MPG activity were isolated from cells stably transfected with human MPG cDNA expression plasmids. The in vivo removal of 3-methyladenine and 7-methylguanine from some of these lines was analyzed and was observed to reflect their MPG levels. These cell lines did not develop increased resistance, as compared to the control, in regards to cytotoxic, mutagenic and sister chromatid exchange inducing effects of the alkylating agents that induce 3-alkyladenine and 7-alkylguanine as the major alkyl adducts in DNA. These results suggest that the MPG activity is not limiting in the multi-step repair pathway of N-alkylpurines in CHO cells.

Alkylating Agents↗