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Biomedical subjects

N K Sinha

Publications and source records attributed to N K Sinha.

At least 19 recordsLinked to original sources
PubMed1995

Field trial of an ecological approach for the control of Phlebotomus argentipes using mud & lime plaster.

A pilot study for the control of Ph. argentipes, a known vector of kala-azar in India, was carried out using an ecological approach. Of the 15 houses selected for the study 10, including the cattle sheds and latrines, were plastered with a mixture of mud and lime, up to a height of 1.22 m taking care to seal all cracks and crevices. The remaining five houses were left unplastered and were considered as control areas. The pre-treatment and post-treatment resting densities of the sandfly were monitored both in treated and untreated houses. A sudden drop in the sandfly density was noticed in the treated houses, whereas there was no significant reduction in the check houses, suggesting an effective control.

Animals↗
PubMed1993

Basic trypsin-subtilisin inhibitor from marine turtle egg white: hydrodynamic and inhibitory properties.

A basic trypsin-subtilisin inhibitor has been isolated from the egg white of marine turtle (Caretta caretta Linn.) and purified to homogeneity by gel filtration followed by ion-exchange chromatography. It has a single polypeptide chain of 117 amino acid residues, having a molecular weight of 13,600. It lacks methionine and tryptophan. Its isoelectric point is at pH 10.0 and the sedimentation coefficient (S20,w) value of 1.62 S is independent of protein concentration. It has a Stokes radius of 18.8 A, an intrinsic viscosity of 0.048 dl g-l and a diffusion coefficient of 10.17 x 10(-7) cm2 sec-1. Its fluorescence emission spectrum is similar to that of free tyrosine and the bimolecular quencing rate constant of its tyrosine residues with acrylamide is 3.15 x 10(9) M-1 sec-1. The inhibitor strongly inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio of unity. The nature of inhibition toward both enzymes is not temporary. It has independent binding sites for inhibition of trypsin and subtilisin. Chemical modification with tetranitromethane suggests the presence of three tyrosine residues on the surface of the inhibitor molecule.

Amino Acids↗
PubMed1993

Surface hydrophobicity of a low molecular weight basic trypsin subtilisin inhibitor from marine turtle eggwhite.

Surface hydrophobicity has recently been emphasized as an important parameter for functional correlation of proteins. However, evaluations of the parameter by different experimental techniques often do not correlate well with each other. In this paper we have compared surface hydrophobicity of a basic protein with those of beta-lactoglobulin, ovalbumin and lysozyme by fluorescence probe method using ANS as an external probe. Two different fluorimetric approaches to determining the surface hydrophobicity parameter, namely, the slope method and the binding parameter method, follow the same relative order. Denaturants, urea, and guanidine hydrochloride disrupted the hydrophobic clefts of the inhibitor on the surface, causing a drastic reduction of surface hydrophobicity.

Anilino Naphthalenesulfonates↗
PubMed1991

Effect of propranolol on ECG of Myna, Acridotheres tristis, and Goh, Varanus bengalensis.

Effect of propranolol (1 and 3 mg/kg body wt), a sympathetic blocking agent, on ECG patterns was studied in Varanus and Acridotheres. ECG was recorded before and after 5 min (immediate), 15 min and in some cases 25 min of drug infusion. All animals responded to propranolol with bradycardia. The effectiveness is dose dependent and it is also associated with the high heart rate both in Acridotheres and in Varanus. The P-R or P-S interval increased in all cases of Varanus after infusion. In Acridotheres height and duration of P-wave were increased slightly with the lower dose and decreased with the higher dose. The Q-S shortened with the lower dose and widened late with the higher dose in Varanus whereas in Acridotheres it is widened with lower and higher doses of propranolol. The Q-T interval has been increased in both groups of animals. An increased amplitude of T-wave height was observed in Varanus after 5 and 15 min of drug infusion. But it was noted with decrease in amplitude under high dose after 15 min of drug infusion. In Acridotheres it was on increase with lower dose and decrease with higher dose. The delta-wave disappeared after the administration of propranolol in Acridotheres.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Melting behavior of a covalently closed, single-stranded, circular DNA.

We synthesized the 26-residue deoxynucleotide sequence d(TTCCT5GGAATTCCT5GGAA) which folds intramolecularly to form a dumbbell-shaped, double-hairpin structure with a gap between the 3' and the 5' ends. We used T4 polynucleotide kinase to phosphorylate the 5' end followed by T4 DNA ligase to close the 3' and 5' ends. Melting of the dumbbell structure formed by this ligated sequence produces a covalently closed, single-stranded, circular final state. We employed calorimetric and spectroscopic techniques to characterize thermodynamically the melting behavior of the ligated molecule and compared it with the corresponding melting behavior of its unligated precursor. This comparison allowed us to characterize uniquely the influence of single-stranded ring closure on intramolecular duplex melting. The data reveal that ring closure produces a thermally more stable structure which exhibits significantly altered melting thermodynamics. We rationalize these thermodynamic differences in terms of differential solvation and differential counterion association between the ligated and unligated molecules. We also note the importance of such constrained dumbbell structures as models for hairpins, cruciforms, and locally melted domains within naturally occurring DNA polymers.

Base Sequence↗
PubMed1989

Physical and chemical characterization of macroglobulin from marine turtle eggwhite.

Macroglobulin was purified from the eggwhite of the marine turtle (Caretta caretta Linn.) by gel filtration through Sephadex G-200 and Sepharose 4B. It was characterized by physical techniques including sedimentation velocity, diffusion and viscosity. Its molecular weight (Mr) was determined as 724,000 with four subunits of equal molecular weight. A large amount of water was hydrodynamically associated with the macroglobulin. It inhibited the activities of trypsin and papain and did not cross-react with human alpha 2-macroglobulin. Its amino acid composition was similar to that of human alpha 2-macroglobulin. Results suggest that turtle eggwhite macroglobulin is a homologous but distinct protein from human alpha 2-macroglobulin.

Amino Acids↗
PubMed1988

Purification and characterization of a low molecular weight basic protein from marine turtle egg white.

The egg white of marine turtle (Caretta caretta Linn.) and one species of tortoise (Geomyda trijuga trijuga Schariggar) contain a low molecular weight basic protein. It has been purified to homogeneity from the egg white of marine turtle and characterized in terms of its major physicochemical and chemical properties. The molecular weight of this protein calculated from gel filtration, sodium dodecyl sulfate-gel electrophoresis in the presence of urea, sedimentation-diffusion data, and amino acid composition is 4300. Its isoelectric point is at pH 11.1 and intrinsic viscosity is 0.038 dl g-1 in 0.2 M NaCl. It has a Stokes radius of 12.6 A and a diffusion coefficient of 16.50 x 10(-7) cm2 s-1. Analysis of the far-ultraviolet circular dichroic spectrum has shown that the basic protein contains 27% beta-pleated sheet and little or no alpha-helix. It possesses a single polypeptide chain of 40 amino acid residues with three disulfide bonds. It lacks serine, methionine, phenylalanine and carbohydrate moiety. It binds to DNA and stimulates ATPase activity due to its strong basicity. The complex of DNA-basis protein is partially resistant to the action of DNase.

Amino Acids↗
PubMed1987

Specificity and efficiency of editing of mismatches involved in the formation of base-substitution mutations by the 3'----5' exonuclease activity of phage T4 DNA polymerase.

The specificity and efficiency of base-mispair editing by the 3'----5' exonuclease activity of phage T4 DNA polymerase has been measured using a sensitive infectivity assay. A series of oligodeoxynucleotide primer chains was synthesized chemically. These primers, when hybridized to phi X174 single-stranded DNAs containing an amber codon, result in a mispaired nucleotide at the 3'-hydroxyl end of the primer chain within the amber codon. DNA synthesis on these primer X templates without the removal of the mispaired terminal nucleotide results in the formation of heteroduplex molecules that yield viable revertants upon transfection into an amber nonsuppressor host. This method permits determination of the efficiency of editing of a mismatch to 1 in 10(6) mismatches that escape editing and allows all eight mispairs that can yield viable revertants at an amber codon to be studied. The results of experiments with primers hybridizing to phi X174 am16 and am3 codons show that the order of mispair editing by T4 DNA polymerase is Ttemplate X Gprimer less than (A X G, T X C) less than (T X T, G X A, G X G, A X C) less than A X A. The efficiency of editing depends upon the mispair, as well as the neighboring DNA sequence. Under these conditions of synthesis, the 3'----5' exonuclease activity, depending upon the mispair and DNA sequences beyond the nearest neighbors, is estimated to contribute a factor of from 2.3 X 10(3)- to greater than 10(6)-fold to the accuracy of T4 DNA polymerase.

Base Sequence↗
PubMed1984

Comparative studies on calotropins DI and DII from the latex of Calotropis gigantea.

Autodigestion of two cysteine proteinases, calotropins DI and DII isolated from the latex of Calotropis gigantea, has been studied at pH 7.5 and 37 degrees C in the presence of an activating agent. Calotropin DI is more susceptible to autodigestion than calotropin DII. During autodigestion no interconversion of one calotropin to another has occurred, as verified by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Immunologically, both calotropins are closely related, but they differ from papain and ficin. Both calotropins have blocked N-terminal amino acid residues. Their C-terminal amino acid sequences, determined by treatment with carboxypeptidase Y, are -(Pro, Ala)-Ala-Val-Tyr for calotropin DI and -(Ala, Val)-Ala-Pro-Tyr for calotropin DII. The tryptic peptide maps of their reduced and S-carboxymethylated derivatives suggest that both calotropins share a high proportion of common regions in their amino acid sequences. Calotropins DI and DII are two distinct proteinases, and they do not appear to be produced by autodigestion of a single precursor. Although they are inert to the common synthetic substrates of papain and ficin, their specificities toward oxidized insulin B chain are comparable to those of papain and ficin.

Amino Acids↗
PubMed1983

Products of bacteriophage T4 genes 32 and 45 improve the accuracy of DNA replication in vitro.

The six "accessory" proteins of the bacteriophage T4 specified by replication genes 32, 41, 44, 45, 61, and 62 were studied for their ability to enhance the accuracy with which phage T4 DNA polymerase (product of gene 43) replicates synthetic homopolymer duplexes in vitro. Two of these proteins, gene 32-protein (helix-destabilizing protein) and gene 45-protein, inhibited the selection of incorrect, but not correct, precursors, at the growing strand end. Gene 32-protein is shown to enhance replication fidelity by interacting with the DNA, whereas gene 45-protein exerts its fidelity-enhancing effect by interacting with the DNA polymerase. This is the first example to our knowledge of a DNA polymerase's accuracy being altered through interaction with another protein. Possible mechanisms by which gene 32- and gene 45-protein act to enhance replication fidelity are discussed.

DNA Replication↗
PubMed1982

Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white.

Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomyda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11,700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100 degrees C for 4 hr at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation constants are 7.2 x 10(-9) M for bovine trypsin and 5.5 x 10(-7) M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than that of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.

Amino Acids↗
PubMed1982

Are DNA precursors concentrated at replication sites?

We have asked whether the effective concentrations of deoxyribonucleotide 5'-triphosphates (dNTPs) at sites of DNA replication in vivo might be higher than the concentrations of dNTPs averaged over the entire cell volume. The approach involved determination of the dependence of DNA replication rate upon thymidine triphosphate concentration, both in vivo and in vitro system that closely approximates the intracellular replication apparatus. In T4 phage-infected Escherichia coli maximal rates of DNA synthesis were attained with dTTP pools of approximately 1.2 x 10(5) molecules per cell, corresponding to an average intracellular concentration of about 65 microM. When DNA synthesis was measured in the T4 purified protein system [Sinha, N. K., Morris, C. F. & Alberts, B. M. (1980) J. Biol. Chem. 255 4290--4303], maximal rates were observed at dTTP concentrations of 200--240 microM. This represents a minimal estimate, therefore, of dTTP concentration at replication sites and suggests that at least a 3- to 4-fold concentration gradient exists near these sites. We discuss why such concentration gradients might be needed and how they might be generated. We also discuss the implications of these results for understanding the relationship between intracellular dNTP pools and mutation rates. A by-product of our study was the finding that exogenous thymidine is used for T4 DNA synthesis in preference to endogenous pathways to thymidine nucleotides; at high thymidine concentrations in vivo the endogenous pathways can be completely bypassed.

DNA Replication↗
PubMed1981

Molecular mechanisms of substitution mutagenesis. An experimental test of the Watson-Crick and topal-fresco models of base mispairings.

The proteins coded by bacteriophate T4 replication genes 32, 41, 43, 44, 45, 61, and 62 together can replicate phi X174 DNA templates very efficiently. The fidelity of this in vitro replication reaction has been measured using an infectivity assay. The product molecules have the same specific infectivity as the template DNA. When an amber mutant DNA template is used, no increase in the frequency of revertants is seen even after more than 60 duplications in vitro. By using imbalances in the concentrations of deoxynucleotide substrates, the error rate during DNA replication in vitro can be greatly increased. Control experiments indicate that the increased mutagenesis is not due to the presence of dITP or dUTP as contaminants in the deoxynucleotide substrates used. The increase in the frequency of revertants is linearly related to the ratio of the correct and the incorrect deoxynucleotides. Determination of the DNA sequence of the revertants induced shows that a change in DNA sequence of the amber site predicted from the nucleotide bias occurs. DNA synthesis in vitro resembles in vivo replication in that the error rate depends not only upon the base change required for reversion but also upon the neighboring DNA sequences. The error rate is estimated to be 5 X 10(-6) at am3 site, 6.4 X 10(-7) at am86 site, and less than 2.9 X 10(-7) at am9 site. Comparison of the frequency of G-T and A-C mispairs reveals that most AT leads to GC transition mutations occur through G-T mispairs. Measurement of the frequency of the mispairs required to induce transversion mutations reveals that these occur primarily through purine-purine mispairs. Transition mutations are more frequent than transversion mutations at both the am3 and the am86 sites. These observations support the models for base pairing errors proposed by Watson and Crick ((1953) Nature 171, 964-967) and Topal and Fresco ((1976) Nature 263, 285-289).

Bacteriophage phi X 174↗
PubMed1980

Molecular basis for substitution mutations. Effect of primer terminal and template residues on nucleotide selection by phage T4 DNA polymerase in vitro.

The DNA-dependent conversion of incorrect deoxynucleoside triphosphate precursors to monophosphates (turnover) by bacteriophage T4 DNA polymerase was determined using either poly(dA) x (dT) or poly(dG) x (dC) homopolymer templates. Competition between correct and incorrect triphosphates for incorporation into DNA, and the use of chain-terminating dideoxynucleoside triphosphates enabled us to determine the amount of turnover occurring at the end of each strand of the homopolymer duplex (e.g. amount of turnover of dATP occurring at the 3'-OH of poly(dG) and the 3'-OH of poly(dC)). These determinations suggest that nearest neighbor interactions between incoming dNTPs and the growing strand terminal residue play a major role in the occurrence of substitution errors during DNA synthesis in vitro byDNA polymerase. When considered together with existing evidence from studies of turnover (Gillin, F. D., and Nossal, N. G. (1976) J. Biol. Chem. 251, 5225-5232) and direct incorporation (Hall, Z. W., and Lehman, I. R. (1968) J. Mol. Biol. 36, 321-333) these results demonstrate that pyrimidine-pyrimidine and purine-purine as well as purine-pyrimidine oppositions have a role in error production at least during DNA replication in vitro. The implications of these results for the role of the "accessory" replication proteins in maintaining accuracy during the DNA biosynthetic process are discussed.

Base Sequence↗
PubMed1980

Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system.

A wide variety of double-stranded DNA templates are replicated extensively in an in vitro DNA replication system containing the purified proteins specified by seven T4 bacteriophage DNA replication genes (32, 41, 43, 44, 62, 45, and 61). In favorable conditions, this multiprotein system catalyzes the synthesis of several copies of the input DNA template in a 30- to 60-min incubation. The replication forks produced in vitro move in a highly processive fashion, at approximately the in vivo rate of 500 nucleotides per s. The DNA synthesized on the lagging side of the in vitro replication fork is made discontinuously, as it is in vivo, giving rise to "Okazaki pieces" averaging some 10,000 nucleotides in length; in contrast, DNA is polymerized in a continuous manner on the leading side of the in vitro fork. Although the mechanism by which the seven-protein in vitro DNA replication system propagates replication forks closely resembles the in vivo mechanism, it lacks the capacity to remove RNA primers, to reseal Okazaki pieces, and to initiate replication forks at defined DNA origins; supplementation of the system with additional T4-specific replication proteins will be required to facilitate these latter three functions.

DNA, Viral↗