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Biomedical subjects

S Mitra

Publications and source records attributed to S Mitra.

At least 289 records · Page 16Linked to original sources
PubMed1986

Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis.

Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.

Alanine↗
PubMed1986

Higher order structure of chromatin: evidence from photochemically detected linear dichroism.

We have used photochemically detected linear dichroism to measure the separate average angular orientations of nucleosomes and linker DNA in 30-nm chromatin fibers of varying linker size (20-80 base pairs). Our results indicate that the average tilt angles vary with linker size, but not in a monotonic manner, suggesting that the constancy of geometry of the 30-nm fiber is maintained by compensatory changes of nucleosomal tilt which accommodate packing of variable lengths of linker DNA. We discuss the compatibility of our results with the various classes of models that have been proposed for the 30-nm fiber, including the continuous solenoid model and models built from the basic unit of the zig-zag ribbon. Many models can be eliminated, and all have to be modified to fit our results for chromatins with very long linkers.

Animals↗
PubMed1986

Cell cycle-dependent modulation of O6-methylguanine-DNA methyltransferase in C3H/10T1/2 cells.

O6-methylguanine-DNA methyltransferase (MGMT) was measured in partially synchronized cultures of C3H/10T1/2 mouse embryo cells as a function of cell cycle. The degree of synchrony and progression of the cell cycle were monitored by flow cytometry. The MGMT level was significantly reduced prior to the onset of S-phase. This reduction was concomitant with the inhibition of in vivo repair of O6-methylguanine in DNA of S-phase cells as observed earlier. The recovery of the MGMT level paralleled the progression of synchronized cells into G2. S-phase cells purified by cell sorting contained approximately 15% of the MGMT present in G0 or early G1 cells. A comparison of the in vivo repair of O6-methylguanine and MGMT levels suggests that the lack of repair of O6-methylguanine in DNA of the mouse embryo cells is due only in part to a temporal loss of MGMT.

Animals↗
PubMed1986

Metabolic reactions responsible for glucose stimulation of alkaline phosphatase in Vibrio cholerae.

Alkaline phosphatase activity in Vibrio cholerae strain 569B grown in low-phosphate medium was stimulated if glucose or glycerol was used as the carbon source. No such stimulation was observed, however, if tricarboxylic acid cycle intermediates like succinate or citrate were used. Experiments using specific enzyme inhibitors strongly indicated that the metabolic reactions of the glycolytic pathway from glyceraldehyde 3-phosphate to 2-phosphoglycerate play a key role in the stimulation process.

Alkaline Phosphatase↗
PubMed1986

A measure of mortality based on life expectancies.

"Two alternative measures of mortality have been proposed in this paper. These are (a) the average age of the stationary population, and (b) the area covered by the life expectancy function. The former has been shown to be equal to the average number of years remaining to be lived by the stationary population and as such can be used as a measure of mortality.... The usefulness of the latter is also obvious, based as it is on the values of the life expectancies at all ages instead of that at birth alone." The measures are estimated for seven selected countries.

Age Factors↗
PubMed1985

Spatial contrast sensitivity in macular disorder.

Spatial contrast sensitivity functions were evaluated in 30 patients with macular disorders and 16 age-matched control subjects. Eighteen of the affected eyes had macular holes and the rest had isolated macular problems. A loss in spatial contrast sensitivity in the central visual field indicated a relative scotoma resulting from macular dysfunctions. The nature and size of a localized scotoma such as the type caused by a macular lesion was estimated by measuring spatial contrast sensitivity as a function of retinal area centered on the fovea. Such spatial-contrast-threshold perimetry was found to be helpful in differential classification of macular holes. Spatial contrast sensitivity and visual resolution improved with increasing retinal area even in the eyes with macular holes, but never reached normal sensitivity for spatial frequencies above 5 cycles per degree (cpd) within our experimental conditions. The loss in sensitivity to low spatial frequencies (less than or equal to 5 cpd) was found to be apparent and a function of the retinal area stimulated. The summation curves (contrast sensitivity vs. stimulus size) were abnormal for all spatial frequencies. The 'critical' sizes (i.e. the stimulus size above which no increase in sensitivity occurred) were always larger in the affected eyes than those in the normal. Thus 'critical' areas of complete summation in the parafovea may be a better measure of functional integrity of central retina in the presence of a foveal lesion than the visual resolution that varies with the retinal area and location activated. Evaluation of contrast sensitivity of the parafoveal regions with nonfunctional fovea also indicated existence of similar mechanism of spatial vision in the fovea and the parafovea.

Aged↗
PubMed1985

Study of ascorbate status in murine and human leukaemias.

Since mice can synthesize ascorbic acid but man cannot, the ascorbate status in murine and human leukaemia was compared. The decline in plasma ascorbate concentration in both cases indicates that vitamin C deficiency occurs in malignancy. Analysis of tissue ascorbate values in mice also indicated that an enhanced rate of utilization of this vitamin occurs during malignancy, as does an increased rate of excretion, and both events may be responsible for vitamin C deficiency. The hepatic ascorbate values suggest an endeavour by the animals to compensate for the loss through increased synthesis and storage of the vitamin, at least in the early stages of the disease.

Animals↗
PubMed1985

Proteins tightly associated with the termini of replicative form DNA of Kilham rat virus, an autonomous parvovirus.

Revie et al. [Revie, D., Tseng, B. Y., Grafstrom, R. H. & Goulian, M. (1979) Proc. Natl. Acad. Sci. USA 76, 5539-5543] have proposed that the double-stranded replicative form (RF) DNA of the autonomous rodent parvovirus H-1 has protein of 60 kDa covalently bound at its 5' termini. We present evidence that the RF DNA of a similar rodent parvovirus, Kilham rat virus (KRV), also has covalently bound protein. NaDodSO4/polyacrylamide gel electrophoresis of purified, 125I-labeled RF DNA shows that proteins of 68-72, 66, 64, and 55 kDa copurify with the DNA during velocity and equilibrium sedimentation in the presence of detergents and 4 M guanidine HCl. Phenol extraction in the presence of 2-mercaptoethanol removes the 68- to 72-kDa proteins, but the 66-, 64-, and 55-kDa proteins remain tightly, but noncovalently, bound. The latter polypeptides also appear to associate with protease-treated RF DNA when mixed with uninfected cell extract. Following removal of these proteins by electrophoresis in NaDodSO4/agarose gels, two proteins (called RF TP-90 and RF TP-40), of about 90 and 40 kDa, become evident. These remain bound to the DNA and are released only after nuclease digestion of the DNA. These two proteins, apparently not of viral origin, are associated with terminal restriction fragments of the RF DNA and appear to be covalently bound to the 5' termini of both strands.

DNA Replication↗
PubMed1985

1H NMR studies of electron exchange rate of Pseudomonas aeruginosa azurin.

T1 values of the His-35 C-2 proton resonance of reduced Pseudomonas aeruginosa azurin were determined at 25 degrees C and pH values 4.5, 7.3, and 9.0 in the presence of different fractional amounts of oxidized azurin. The C-2 proton of His-35 undergoes very rapid spin relaxation in oxidized azurin because of its close proximity to the paramagnetic copper. In the presence of oxidized protein, the T1 values of this proton in reduced azurin depend on the lifetime of the reduced protein. From the T1 data, the electron self-exchange rate constant for azurin was calculated to be 1.4 X 10(4) M-1 X s-1, 4.3 X 10(3) M-1 X s-1, and 6.0 X 10(3) M-1 X s-1 at pH values 4.5, 7.3, and 9, respectively. At pH 7.3, the C-2 proton of His-35 is in slow exchange between the imidazole and imidazolium forms and gives rise to two separate resonances at 9.39 and 8.00 ppm. By using these two resonances, the electron self-exchange rate constants were determined separately for the two species of azurin for which the His-35 residue is in the imidazole or the imidazolium forms; results showed that both species participate in self-exchange of electrons with equal efficiency.

Azurin↗
PubMed1984

Structure of the capsid of Kilham rat virus from small-angle neutron scattering.

The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 X 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H2O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, we propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H2O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H2O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H2O. We propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions.

Amino Acids↗
PubMed1984

Kinetics of incorporation of O6-methyldeoxyguanosine monophosphate during in vitro DNA synthesis.

O6-Methyldeoxyguanosine triphosphate (m6dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers. A study of the kinetic behavior of m6dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m6dGTP acts as an analogue of dATP with Kappm of about 6 microM for Escherichia coli DNA polymerase I (Klenow fragment) compared to the Kappm of about 0.8 microM for dATP. m6dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor). m6dGTP also inhibits in vitro DNA synthesis. Different DNA polymerases behave differently in utilization and turnover of m6dGTP. T4 DNA polymerase shows stronger discrimination against m6dGMP incorporation than either T5 DNA polymerase or E. coli DNA polymerase I. The possibility that m6dGTP is unlikely to contribute significantly to in vivo mutation is discussed.

DNA Polymerase I↗
PubMed1984

Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication.

The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6-methylguanine (m6Gua), was analyzed during in vitro replication of m6Gua-containing synthetic polydeoxynucleotides by T4 and T5 phage DNA polymerases and Escherichia coli DNA polymerase I. When poly(dT,m6dG) and poly(dC,m6dG) with covalently attached primers were replicated, O6-methylguanine paired with either thymine or cytosine but with a much higher preference for thymine. dCTP and dTTP acted as competitive inhibitors of each other during DNA synthesis. O6-Methylguanine also directed incorporation of dAMP by T5 DNA polymerase. This dAMP incorporation was not inhibited by dTTP. Contrary to theoretical predictions that the m6dG X dT pair should be comparable to the dA X dT pair, the presence of m6dG in the template inhibited DNA synthesis. Based on Kappm values, E. coli DNA polymerase I showed a much higher preference for dTMP incorporation over dCMP opposite m6dG in the template than T4 and T5 DNA polymerases. At the same time, there was a higher turnover of dCTP than of dTTP by the E. coli enzyme. However, in all cases, the turnover of deoxynucleotides during replication of m6Gua-containing templates was more than that observed with templates without the alkylated base.

Animals↗
PubMed1984

O6-Methylguanine-DNA methyltransferase in human cells.

O6-Methylguanine-DNA methyltransferase activity was measured in extracts of human tumor cells and was partially purified from human placenta. Repair of O6-methylguanine in DNA inactivated the methyltransferase, and treatment of cells with MNNG, which produces this alkylated base in DNA, depleted the cells of active methyltransferase. RNA and protein synthesis were required for restoration of methyltransferase activity, which transiently exceeded the original levels by 50% 48 h after treatment. One species of methyltransferase of Mr = 22 kd was present in human tumor cells and human placenta.

Astrocytoma↗
PubMed1984

Lack of induction of O6-methylguanine-DNA methyltransferase in mammalian cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

A synthetic DNA substrate containing O6-methyl[8-3H]-guanine was used to assay demethylation of the premutagenic base by O6-methylguanine-DNA methyltransferase in extracts of HeLa cells, Chinese hamster ovary cells and normal rat kidney cells which had been treated with multiple doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). No induction of methyltransferase activity was observed in any of the cell lines tested. Constitutive levels of methyltransferase in cell lines proficient (Mex+) in O6-methylguanine repair were decreased in a dose-dependent fashion by either single or multiple treatments with MNNG over a broad range of dose levels. Recovery of constitutive levels of activity required 24- to 48-h incubation periods. Repair deficient (Mex-) cell lines lacked both constitutive and inducible methyltransferase activity.

Animals↗