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Biomedical subjects

M Kapoor

Publications and source records attributed to M Kapoor.

At least 73 records · Page 4Linked to original sources
PubMed1992

Increase in superoxide production by heat-shocked cells of Neurospora crassa, demonstrated by a fluorometric assay.

1. Increase in superoxide production by heat-shocked cells of Neurospora crassa was demonstrated by a fluorometric assay. 2. A sensitive fluorometric assay for the estimation of superoxide anion radical--based on the liberation of 4-methylumbelliferone from 4-methyl-beta-D-umbelliferyl glucopyranoside--is described. 3. Using this system the level of superoxide in the medium of heat-shocked Neurospora crassa cells was found to be consistently higher, in comparison with that of non-shocked cells, cultured at the normal growth temperature of 28 degrees C. 4. Addition of superoxide dismutase to the culture media suppressed the production of 4-methylumbelliferone.

Fluorometry↗
PubMed1992

Induction and intracellular localization of the 80-kilodalton heat-shock protein of Neurospora crassa.

The most abundant heat-shock protein of Neurospora crassa is a multimeric glycoprotein of 80-kilodaltons (i.e., HSP80), induced strongly by hyperthermia and at a lower level by sodium arsenite, ethanol, and carbon source depletion. Immunoelectron microscopy, using indirect immunogold labelling demonstrated that HSP80 was undetectable in mycelium cultured at the normal growth temperature of 28 degrees C, but it appeared rapidly following the commencement of heat-shock treatment at 48 degrees C. HSP80, visualized by the gold label, was observed almost exclusively in the cytoplasm, exhibiting a uniform distribution. Association of this protein with cellular membranes and (or) targeting to a particular subcellular compartment or organelle was not apparent.

Cytoplasm↗
PubMed1992

hsp80 of Neurospora crassa: cDNA cloning, gene mapping, and studies of mRNA accumulation under stress.

Using mRNA isolated from Neurospora crassa mycelium, grown for 14 h at normal growth temperature of 28 degrees C, and heat shocked for 1 h at 48 degrees C, a cDNA library was prepared in the expression vector lambda gt11. Following immunoscreening of this library with a polyclonal antiserum raised against a 80-kilodalton heat-shock protein (HSP80), cDNA clones containing 1.1- and 1.4-kilobase inserts were selected. Analysis of the partial nucleotide sequence and the deduced amino acid sequence of the cDNA clones revealed a remarkable extent of homology with other eukaryotic stress-90 family proteins; 85% identity of the amino acid sequence with that of yeast HSP90(82) was seen. The C-terminal end of the sequence contained the MEEVD motif, characteristic of eukaryotic stress proteins with a predominantly cytosolic localization. The gene for N. crassa HSP80 was mapped to the right arm of linkage group V, using restriction fragment length polymorphism mapping. Its expression during heat shock and recovery was monitored by probing Northern blots of RNA isolated from mycelium grown under various stress conditions.

Amino Acid Sequence↗
PubMed1992

Decreased aortic valve prosthesis-associated haemolysis after changing from haemodialysis to peritoneal dialysis.

We report here a chronic haemodialysis patient who had severe anaemia following replacement of his aortic valve. The investigations confirmed a traumatic valve effect. The surgeons were reluctant to replace the valve because of the high cardiothoracic risk and absence of significant leak or stenosis. Furthermore, the patient refused to consider additional surgery. We changed his modality of dialysis to continuous ambulatory peritoneal dialysis; subsequently, there was a dramatic decrease in his transfusion requirement, and the patient was symptomatically and functionally better. A literature search indicates that this therapeutic response has not been reported before.

Anemia, Hemolytic↗
PubMed1991

The quantitation of rabies-specific antibodies. I. Modified counter immunoelectrophoresis. A rapid and sensitive method.

Modified counter immunoelectrophoresis was standardized with respect to dilution of tissue culture antigen and indicator serum, the incubation time for neutralization and the effect of an electric current. The technique was found to be sensitive enough to detect a minimum level of antibodies (0.5 IU/ml) by using a 16 mA current per slide for 2 h, indicator serum of 15 IU/ml and the use of an antigen at a concentration of 1:35. Above all, the incubation period did not affect the neutralization of the virus. The test was also applied to the detection of rabies-specific antibody levels in 73 human sera. The test was found to be simple, quick and economical for titration of rabies antibodies.

Antibodies, Viral↗
PubMed1991

The quantitation of rabies-specific antibodies. III. A comparative evaluation of modified counter immunoelectrophoresis, haemagglutination inhibition and serum neutralization titres of human sera.

The rabies-specific antibodies of 73 serum samples from vaccinated humans were determined by the modified counter immunoelectrophoresis (MCIE), and the haemagglutination inhibition test (HAI) by using the conventional serum neutralization test (SN) as a yard-stick. Both MCIE and HAI were found to be sensitive and specific for the estimation of rabies antibodies. In general, the unitages obtained by the MCIE and SN showed statistically insignificant differences (P greater than 0.05) and the correlation coefficient between the two methods was 0.697 (P less than 0.05). Although the unitage of the sera detected by HAI tests was lower by a factor of 0.155 from the unitage of SN tests, there was statistically insignificant differences between the two techniques (P greater than 0.05) with a correlation coefficient of 0.556 (P less than 0.05).

Antibodies, Viral↗
PubMed1991

An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.

A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.

Ascomycota↗
PubMed1990

Heat shock response in Neurospora crassa: purification and some properties of HSP 80.

The heat shock response of Neurospora crassa was investigated. A 80-kilodalton heat shock protein (HSP 80) was purified to near homogeneity from heat-shocked mycelial extracts employing ammonium sulphate fractionation, gel filtration, and ion-exchange and affinity chromatography. It was observed to migrate as a single band on one-dimensional sodium dodecyl sulphate--polyacrylamide gels, with a molecular mass of approximately 83 kilodaltons (kDa). On two-dimensional gels it resolved into four polypeptide species with isoelectric points in the acidic range, which on staining with periodic acid--Schiff method were demonstrated to be glycosylated. In the native state, HSP 80 had a molecular size of approximately 610 kDa.

Fungal Proteins↗
PubMed1990

Oral versus axillary temperatures in human volunteers.

We measured the oral and axillary temperatures of 100 individuals (including 40 females) in the medical wards. Twenty six had fever ranging from 37.3 degrees C to 40.5 degrees C while the rest had normal temperature. Although the oral temperature was higher than axillary temperature in all the cases, there was no correlation between the two; in one case the difference was as high as 1 degrees C. We conclude that while recording temperature the site must be clearly stated, and no attempt must be made to extrapolate the axillary to the oral temperature.

Adolescent↗
PubMed1989

NAD-specific glutamate dehydrogenase of Neurospora crassa. cDNA cloning and gene expression during derepression.

The catabolic NAD-specific glutamate dehydrogenase of Neurospora crassa is one of the many enzymes regulated by carbon catabolite repression. To achieve an understanding of its regulation, cDNA and genomic clones were isolated. Total poly(A+) RNA from derepressed cells was used for the construction of a cDNA library in the expression vector, lambda gt11. By screening this library with a polyclonal antiserum against NAD-specific glutamate dehydrogenase, a positive clone with a 0.9-kilobase insert was isolated and the insert DNA sequenced. The insert was shown to code for approximately one-third of the known amino acid sequence, close to the carboxyl terminus. Using this truncated cDNA as a probe, the structural gene was shown to be transcriptionally activated approximately 60-fold during derepression, producing an approximately 4.7-kilobase mRNA transcript. N. crassa genomic clones, hybridizing to this cDNA probe, were isolated and the structural gene (on two BamHI fragments) was subcloned in pUC13.

Amino Acid Sequence↗
PubMed1988

The heat shock response of Neurospora crassa: stress-induced thermotolerance in relation to peroxidase and superoxide dismutase levels.

Heat shock and other treatments, including cadmium chloride, hydrogen peroxide and sodium arsenite, led to the induction of high levels of peroxidase activity as well as thermotolerance in Neurospora crassa. No correlation was apparent between superoxide dismutase levels and development of thermotolerance following exposure to these stress conditions. A prominent role for peroxidase in protection against damage by toxic products of oxygen is suggested.

Enzyme Induction↗
PubMed1988

A Neurospora crassa heat-shocked cell lysate translates homologous and heterologous messenger RNA efficiently, without preference for heat shock messages.

Cell-free protein synthesis systems were prepared from normally-grown (N-lysate) and heat-shocked (HS-lysate) Neurospora crassa mycelium. Although both lysates translated homologous mRNA, the HS-lysate was more active, yielding a higher incorporation of [35S]-methionine into hot TCA-insoluble material and a vastly superior protein synthesis profile. The optimal temperature for translation by both lysates was 21 degrees C; the HS-lysate did not translate heat-shock mRNA preferentially at any temperature tested. Fortuitously, heterologous messenger RNAs from diverse eukaryotic and viral sources - Drosophila, dog pancreas, rabbit globin mRNA, brome mosaic virus, tobacco mosaic virus - were translated by the HS-lysate with an efficiency comparable to that of the commercial rabbit reticulocyte system and superior to the wheat germ system. The cap analogues, m7G(5')ppp(5')G and m7G(5')Gm, inhibited translation significantly.

Animals↗
PubMed1988

pH-dependent glycine uptake in the presence and absence of sodium ions from rat small intestine.

Intestinal uptake of glycine in rats was stimulated 15-20% in the presence of 120 mM Na at pH 6.0 and below but around neutral pH, the amino acid uptake was augmented to 60% compared to that in the Na-free medium. Glycine uptake was 30% more at pH 5.5 compared to that at pH 7.3 in the absence of Na. Kinetic analysis revealed a decrease in Kt for glycine uptake (9.62 mM) at pH 5.5 compared to that at pH 7.3 (Kt = 16.67 mM) with no change in maximal velocity (1.51 mumole/10 min/g tissue) in Na-free buffer. Addition of -SH group reacting reagents to the incubation medium produced 36-58% inhibition of glycine uptake in the presence of Na. However, in absence of Na, inhibition of the order of 21-35% and 8-23% was observed at pH 5.5 and 7.0, respectively. These findings suggest that glycine uptake in rat intestine is influenced by pH and -SH groups are implicated in the process(es).

Animals↗
PubMed1988

Ethanol and carbon-source starvation enhance the accumulation of HSP80 in Neurospora crassa.

In Neurospora crassa, heat shock results in the induction of 9 to 11 heat shock proteins (HSP), of which HSP80 is the most abundant and the first to be synthesized. The induction of HSP80 was investigated during normal growth (2% sucrose) and under sucrose starvation. Transfer of mycelium to a medium supplemented with ethanol stimulated the synthesis of HSP80, even at the normal growth temperature of 28 degrees C. It was also synthesized under carbon starvation conditions, where the medium was supplemented with 0.02% sucrose, 0.3% acetate, 0.2% lactate, or ethanol. A 30-35 kilodalton polypeptide was induced by heat shock in carbon-sufficient media, but in 0.02% sucrose and 0.3% acetate containing media it was synthesized at normal temperatures. While the overall heat shock response remained unaltered in these cultures, the abundance of HSP90 and HSP70, relative to HSP80, was greater. HSP80 appears to be controlled by carbon-catabolite repression as well as heat shock. Another high molecular mass protein (tentatively designated alc'80') was observed to be induced by heat shock, provided carbon starvation conditions prevailed concurrently.

Carbohydrate Metabolism↗
PubMed1988

Expression of heat shock genes of Neurospora crassa: effect of hyperthermia and other stresses on mRNA levels.

Neurospora crassa mycelium was heat shocked for intervals varying from 15-180 min. Heat shock mRNA was monitored by hybridization of Northern blots with the Drosophila hsp-70 gene probe and an inducible member of the yeast hsp-70 gene family, YG100. A 2.7 kilobase (kb) transcript, with homology to these two probes, was detected in cultures shocked for 15 min; its levels increased up to 60-90 min and declined thereafter. Sodium arsenite, too, induced the synthesis of this transcript. An additional, constitutively synthesized 2.4-kb transcript was revealed by hybridization with the yeast probe. The synthesis of this message was terminated during heat shock. Hybridization of Northern blots with the Drosophila actin gene probe demonstrated two size classes, 1.85 and 1.63 kb; the former decreased dramatically following heat shock. Recovery, as assessed by the disappearance of the 2.7-kb hsp-70-mRNA and restoration of the 1.85-kb actin message to the prestress levels, was essentially complete within 60 min of transfer to 28 degrees C. In vitro translations of RNA from stressed cells showed the heat shock messages to be stable and readily translatable. RNA of cells subjected to heat shock plus CdCl2 showed a higher content of messages for heat shock proteins of 70, 80, and 90 kilodaltons.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Binding of a 30-kDa protein to the pyruvate kinase gene of Neurospora crassa.

Extracts of a wild-type strain of Neurospora crassa, electrophoresed on SDS-polyacrylamide gels and electroblotted onto nitrocellulose sheets, were hybridized to an end-labelled pyruvate kinase (PK) gene fragment containing the 5' noncoding sequence and a large part of the coding region. A 30-kDa protein was found to bind strongly to the PK gene DNA, while binding weakly to plasmid pUC12 DNA and to total N. crassa DNA. Probing of blots with individual restriction fragments derived from the PK gene showed that the protein binding occurred primarily to the 5' noncoding region. Nonspecific DNA from pUC12, PK gene DNA from the recombinant plasmid pNP460 (pUC12 containing a 1.8-kilobase EcoRI insert of the PK gene DNA), along with a 0.7-kilobase EcoRI-AccI restriction fragment containing the 5' flanking region, were used in filter-binding experiments to analyze the kinetics of binding. Formation of protein-DNA complexes was demonstrated by monitoring the electrophoretic mobility of this fragment on nondenaturing gels.

Base Sequence↗