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Biomedical subjects

P Sinha

Publications and source records attributed to P Sinha.

At least 145 records · Page 8Linked to original sources
PubMed1992

Immobilized pH 2.5-11 gradients for two-dimensional electrophoresis.

Extremely wide immobilized pH gradients, pH 2.5-11, for isoelectric separation of complex protein mixtures are described. These pH gradients are theoretically and practically the maximum that can be achieved at present with the available acrylamido buffers and titrants. Conditions are described for reducing conductivity and electroendosmosis in extreme pH ranges. Furthermore, new conditions are described for the separation of proteins in the second dimension. Using this protocol, nearly all the possible cellular products can be separated in one single two-dimensional map.

Electric Conductivity↗
PubMed1992

Rapid and simple method for the identification of apolipoprotein E isomorphic phenotypes from whole serum.

For the identification of apolipoprotein E isomorphic phenotypes, fresh or thawed serum was analyzed without prior delipidation or other pretreatment. Using 5% polyacrylamide gels with a 40 mm interelectrode distance, the isoforms were separated by isoelectric focusing in immobilized pH gradients ranging from pH 5 to 6.5, and transferred onto polyvinylidene difluoride membranes by contact blotting for 1 h. The apolipoprotein E isoforms were identified following immunostaining. The electrophoresis required less than 2 h and the entire procedure could be completed within 6 h.

Antibodies, Monoclonal↗
PubMed1992

An investigation into pellet dispersion ballistics.

Existing works on pellet dispersion ballistics are confined to some data-based models derived from statistical analysis of observed patterns on targets but the underlying process causing the dispersion lacks due attention. The present article delves into the relatively unexplored areas of dispersion phenomena, and attempts to develop a theoretical model for general application. The radial velocity distribution of pellets has been worked out by probing into the physical process of dispersion based on transfer of momentum from undispersed shot mass to dispersed pellets. The ratio 2u/v0 (u = root mean square (r.m.s.) radial velocity and v0 = muzzle velocity of the pellets) is found to be fairly constant for a fixed gun-ammunition combination and has been suitably designated as 'Dispersion Index' (DI) characterising its dispersion capability. The present model adequately accounts for pellet distribution on targets and it appears that 'Effective Shot Dispersion' (ESD) as introduced by Mattoo and Nabar [ESD = [(4/N0)sigma Ri2]1/2, where N(0) is the total number of pellets and Ri is the radial distance of the i-th pellet from centre of pattern], gives a faithful numerical measure of overall dispersion at a given distance. A relationship between ESD and firing distance, incorporating the effects of air resistance and gravity has been worked out, which reveals that DI controls the dispersion at a given distance. For small distances (less than 20 m) the relation reduces to a linear one, as already observed empirically and looks like ESD = E0+DI x firing distance, E0 being a parameter dependent on gun and ammunition. The present model, unlike earlier ones, is versatile enough to explain the natures of the dependence of dispersion on firing distance as well as on gun-ammunition parameters, which are essential for a faithful reconstruction of a crime scene. The model has been tested with such experimental data as are available and reasonable agreement is observed.

Firearms↗
PubMed1991

Isoelectric focusing in immobilized pH gradients: applications in clinical chemistry and forensic analysis.

The applications of isoelectric focusing in immobilized pH gradients in clinical chemistry and forensic analysis are reviewed. Strong emphasis is given to the separation of serum proteins, in particular alpha 1-acidic glycoprotein, acid phosphatase, alkaline phosphatase, alpha 1-antitrypsin, apolipoproteins, complement component, factor B, factor XIIIB, group-specific component, lecithin:cholesterol acyltransferase, phosphoglucomutase, prealbumin, protein C and transferrin. The analysis of human parotid salivary proteins is discussed and an assessment is given of the state of the art in thalassaemia screening.

Blood Proteins↗
PubMed1990

On the application of a constitutive equation for whole human blood.

In consideration of the pulsatile blood flow in a conduit, the constitutive equation for the whole human blood of F. J. Walburn and D. J. Schneck (Biorheology, Vol. 13, 1976, pp. 201-210) is utilized. Governing equations are solved numerically yielding the velocity and the shear stress distributions. These results are discussed and compared with the Newtonian fluid, Casson's fluid, and Bingham fluid applications.

Blood Flow Velocity↗
PubMed1989

Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients.

A new technique is described for in situ visualization of the activity of intestinal disaccharidases after isoelectric focusing in immobilized pH gradients using their physiological substrates. The reaction principle is based on the oxidation of D-glucose, liberated by the disaccharidases, into D-gluconolactone and the production of NADH by glucose dehydrogenase. At the sites of enzymatic activity, tetrazolium salts present in the reaction mixture are reduced to relatively water-insoluble formazans by NADH. The rate of formazan production is increased by the presence of phenazine methosulfate. An additional modification of the technique involves the use of polyvinyl alcohol in the substrate solution. Due to the increase in the viscosity of the substrate solution, leakage of the enzyme from the IPG gels is minimized. Incubation times can thus be prolonged without loss of resolution and band-blurring.

Disaccharidases↗
PubMed1989

Gastric ulcer is accompanied by a decrease of epidermal growth factor in gastric juice and saliva.

Numerous studies have indicated a role of epidermal growth factor in the maintenance of the gastrointestinal mucosa. In the present study epidermal growth factor concentrations in saliva and gastric juice of patients with gastric or duodenal ulcer or gastritis are compared with those of healthy controls. For this purpose a novel ELISA system has been developed and shown to be sensitive and specific. It is demonstrated that gastric juice and saliva of patients with gastric ulcer contain less epidermal growth factor than the samples of healthy controls (p less than 0.01). Epidermal growth factor concentrations and outputs (product of epidermal growth factor concentration and the volume secreted in 15 min) in the gastric juice of patients with duodenal ulcer do not differ from those of healthy controls.

Duodenal Ulcer↗
PubMed1988

Alkaline phosphatase. Laboratory and clinical implications.

Alkaline phosphatase (ALP), which was discovered in 1907, is extensively reviewed. The first section deals with biochemical aspects of ALP, e.g. the anchorage of ALP to cell membranes via a phosphatidylinositol linkage, the charge and molecular mass heterogeneities and their causes, and methods for the separation and analysis of ALP isoforms using the newest electrophoresis techniques, such as affinity electrophoresis with wheatgerm lectin and isoelectric focusing in immobilized pH gradients. The second section deals exclusively with the clinical implications of ALP analysis in diseased states, e.g. cholestasis, hyper- and hypophosphatasemias and neutrophil ALP. Extensively discussed is the involvement of ALP in numerous bone diseases. The role of ALP and its isoforms in tumours and their applicability as potential tumour markers is critically evaluated.

Alkaline Phosphatase↗
PubMed1987

Mitotic chromosome organization of neuroblastomas induced by the recessive oncogene Lethal(2)giant larvae4 of Drosophila melanogaster.

Mitotic chromosomes in neuroblastomas of Drosophila melanogaster homozygous for the recessive oncogene Lethal(2)giant larvae4 were studied at different larval ages. There was a progressive increase in mitotically active cells with increasing period of tumorous growth. The karyotype of the neuroblastomas generally remained stable. The metaphase chromosomes of the neuroblastomas, however, showed widely varying levels of condensation ranging from highly condensed to highly extended. Staining with the AT-specific fluorescent dye Hoechst 33258 showed extensive resolution of AT-rich heterochromatic blocks in the third and Y chromosomes suggesting their lack of coalescence in the extended metaphases of neuroblastomas. Our results suggest aberrant condensation as a major chromosomal lesion in Drosophila neuroblastomas.

Animals↗
PubMed1986

A mutant that affects the function of autonomously replicating sequences in yeast.

We previously reported the isolation of a series of mcm mutants that are defective in the maintenance of minichromosomes in yeast. These minichromosomes are circular plasmids, each containing an autonomously replicating sequence (ARS) and a centromere. One of the mcm mutants, mcm2, has the following phenotype: at room temperature it affects the stability of only some minichromosomes depending on the ARS present, while at high temperature it affects all minichromosomes tested irrespective of the ARS present. Here we show that the mcm defect as well as its temperature-dependent specificity for ARSs can be demonstrated with circular as well as linear plasmids that do not contain centromeric sequences. Larger chromosomes containing multiple ARSs are also unstable in this mutant. Further analyses indicate that the mcm2 mutation causes the loss, rather than the aberrant segregation, of the circular minichromosomes. In addition, this mutation appears to stimulate mitotic recombination frequencies. These properties of the mcm2 mutant are consistent with the idea that the mcm2 mutation results in a defect in the initiation of DNA replication at ARSs, the putative chromosomal replication origins in yeast.

Centromere↗
PubMed1986

Host factors in nuclear plasmid maintenance in Saccharomyces cerevisiae.

In yeast, the initiation of DNA replication on chromosomes is believed to occur at specific sequences known as autonomously replicating sequences (ARSs). We previously isolated a number of mutants that are defective in the maintenance of minichromosomes. Analysis of these mutants suggests that although ARSs differ greatly from one another in their primary sequences, they appear to share a common enzyme complex for the initiation of DNA replication. However, this initiation enzyme complex probably binds with differential affinity to different ARSs. This idea is corroborated by our identification of an ARS-binding protein that binds to different ARSs with different efficiencies.

Cell Nucleus↗
PubMed1985

The alkali burned cornea: electron microscopical, enzyme histochemical, and biochemical observations.

The early phase of wound healing after small central alkali burns of the guinea pig cornea was studied using electron microscopical, enzyme histochemical, and biochemical techniques. In the first phase, which was morphologically characterized by the destruction of the epithelium and keratocytes and by the infiltration of the cornea with polymorphonuclear leukocytes, an increase in the activity of lysosomal phosphatases and glycosidases (beta-D-glucuronidase, acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase) was noticed. In the second phase, the cornea was invaded by capillaries and fibroblasts. In this phase, the activity of proteases (aminopeptidase M, dipeptidyl peptidase IV) increased intra- and extracellularly, suggesting that these enzymes may be involved in the turnover of the collagenous matrix and the ground substance. Using synthetic 4-methoxy-2-naphthylamine substrates and fluorescence-band detection techniques after isoelectric focusing, an increase in the activity of endopeptidases was demonstrated. The decreased activity of gamma-glutamyl transpeptidase may be linked with the activation of latent collagenase.

Alkalies↗
PubMed1985

Detection of alkaline phosphatase activity after conventional isoelectric focusing by an indoxyl-tetrazolium salt technique.

Alkaline phosphatase was solubilized from human and rat tissues using papain in the presence of TRITON X-100 and subjected to isoelectric focusing (IEF) in polyacrylamide or agarose gels. Up till now, usually 1- and 2-naphthylphosphates have been used as substrates in order to specifically stain molecular forms of this enzyme by the azo-dye technique. In this paper, the use of another histochemical substrate, 5-bromo-4-chloro-3-indoxyl phosphate, in combination with tetrazolium salts [McGadey, J. (1970) Histochemie 23, 180-184] is presented. After hydrolysis, the released indoxyl moieties reduce tetrazolium salts to insoluble formazans at the zones of alkaline phosphatase activity. Zymogrammes showing molecular forms of alkaline phosphatase from 20 rat organs and the application of this staining technique for the detection of alkaline phosphatase activity in non-dialyzed human plasma after IEF are presented.

Acid Phosphatase↗
PubMed1984

Recent advances in protease research using synthetic substrates.

2-Naphthylamine, 4-methoxy-2-naphthylamine, 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin and X peptides have become a useful tool to study proteases by a battery of complementary methods; they now include biochemistry, section biochemistry, location cytochemistry, ultracytochemistry and isoelectric focusing. By the use of these procedures we hope that more insight will be possible in the functional role of proteases in health and disease in the near future.

Animals↗
PubMed1984

Fluorescence detection of proteases with AFC, AMC and MNA peptides using isoelectric focusing.

The fluorescence detection of proteases is not only possible with MNA but also with AFC and AMC peptides which were used up till now only for biochemical protease investigations. The MNA method is more sensitive due to the coupling capability of MNA with NSA than the AMC and AFC procedure. In comparison with the simultaneous azo-dye method the fluorescence technique may be inferior as far as the sharpness of the bands is concerned. The fluorescence staining is superior, however, if proteinases have to be investigated that are inhibited by diazonium salts, or where interference of diazonium salts and activators or inhibitors occurs; finally, it is a simple procedure to elucidate the value of MNA, AFC and AMC peptides for one and the same protease. In the field of applied isoelectric focusing the azo-dye and fluorescence procedure are now used together by us to detect possible differences in protease isoenzyme patterns in normal and diseased human biopsies, e.g. from liver, kidney and small intestine.

Animals↗
PubMed1984

Investigation of proteases with chromogenic substrates after isoelectric focusing (IEF).

A technique is described for the detection of protease isoenzymes which is more sensitive than disc electrophoresis. Supernatants of crude rat and human organ homogenates are subjected to analytical isoelectric focusing (IEF) and the gel strips are finally incubated in histochemical media containing 4-methoxy-2-naphthylamine amino acids or peptides and diazonium salts for simultaneous or post-coupling. The incubation media are identical with those used for section histochemistry of proteases. This combination of IEF and proteases histochemistry yields excellent and reproducible data which cannot be obtained by protease histochemistry alone. Post-coupling delivers less and more diffuse bands than simultaneous coupling. For simultaneous coupling, Fast Blue B and Fast Black K are the most suitable diazonium salts. More bands are found in agarose gels compared with polyacrylamide. Sex-differences exist for endopeptidases in the submandibular gland, but are absent in other rat organs. Despite their uniform membrane localization in tissue sections, aminopeptidase (AP) A and M and dipeptidylpeptidase (DPP) IV and gamma-glutamyltranspeptidase (GGT) show striking heterogeneous band patterns depending on the investigated organ. The similar band patterns of APA and APM can be specified by the use of activators or inhibitors. In rat kidney, up to 26 bands are obtained with DPP II and IV substrates, 3 for APA and APM and up to 12 for GGT. DPP IV of human liver is different from that in rat liver.

Animals↗
PubMed1984

Isoelectric focusing (IEF) and band detection with fluorogenic protease substrates.

In a previous paper, combined dye histochemistry and analytical isoelectric focusing (IEF) of supernatants from organ homogenates have been shown to yield good results for the detection of protease isoenzymes. Difficulties arise when the protease to be studied is partially or completely inhibited by diazonium salts and when synthetic peptide substrates different from 4-methoxy-2-naphthylamine (MNA) amino acids and peptides are to be used. In this paper a technique is described in which cellulose acetate foils impregnated with MNA, 7-amino-4-methyl-coumarin (AMC) and 7-amino-4-trifluoromethylcoumarin (AFC) substrates are overlaid on electrophoresis strips after IEF. After incubation, the foils are viewed with an UV-lamp and photographed. The MNA, AMC and AFC peptides are equally suitable for fluorescence band detection. Using this technique, occasionally protease isoenzymes are found which are more sensitive towards diazonium salts, e.g. aminopeptidase A and M. Sometimes it is also possible to detect thiolproteases which is not the case when employing dye histochemistry.

Aminopeptidases↗