Search PubMed⌕ Search

Biomedical subjects

S Kochhar

Publications and source records attributed to S Kochhar.

At least 37 records · Page 2Linked to original sources
PubMed1995

Purification and characterization of homoserine dehydrogenase from spinach leaves.

Homoserine dehydrogenase (HSDH) has been purified to homogeneity from spinach leaves using ammonium sulphate fractionation followed by ion exchange chromatography, gel filtration and FPLC techniques. The purified enzyme has a relative molecular mass of 220,000 and subunit molecular mass of 55,000 and probably occurs as a tetramer. The enzyme was found to be sensitive to threonine and also exhibited aspartate kinase (AK) activity, which was also sensitive to threonine suggesting that it is a bifunctional protein. The enzyme protein also gave a positive cross reaction with antibodies raised against purified AK isoenzymes. Both HSDH and AK activities were stimulated by calcium and calmodulin.

Aspartate Kinase↗
PubMed1995

Disability patterns among SSI recipients.

In December 1993, about 3.8 million persons under age 65 received Supplemental Security Income (SSI) payments because of a disability. More than half of these recipients had some form of mental disorder. In recent years, the number of disabled SSI recipients has climbed sharply. At the same time, there has been a change in the disability patterns among these recipients. The proportion of recipients with mental disorders, particularly those with psychiatric illness, is increasing steadily. Many of these recipients enter the SSI program in their youth and may stay in the program for many years. Similar increases and disability patterns in the Social Security Administration's Disability Insurance (DI) program imply program related causes, including recent changes to the disability requirements and outreach efforts. These changing disability patterns have implications for the size and shape of future SSI caseloads.

Adolescent↗
PubMed1994

Bilateral renal angiomyolipomatosis in tuberous sclerosis presenting with chronic renal failure: case report and review of the literature.

A 45-year-old patient with tuberous sclerosis and bilateral renal angiomyolipomas who presented with chronic renal failure is reported. Although bilateral renal angiomyolipomas are commonly associated with tuberous sclerosis, the occurrence of chronic renal failure in the absence of associated cystic disease of the kidneys as seen in this patient is very rare.

Angiomyolipoma↗
PubMed1994

Fine needle aspiration biopsy of pancreas.

Percutaneous fine needle aspiration biopsy (FNAB) were performed in 22 cases of pancreatic lesions. FNAB was done with the help of ultrasonographic guidance in 14 cases and intraoperatively in 2 cases. In rest of the cases it was done without any radiological guidance. Diagnosis was confirmed either by histopathology or exploratory laparotomy by follow-up data in rest of the cases. There was no false positive or negative diagnosis. Nuclear crowding, nuclear enlargement and prominent nucleoli were the important cytological criteria for pancreatic adenocarcinoma. Cytology smears of the islet cell tumour show round to oval monomorphic cells with central to eccentric nuclei and fine reddish granulations in cytoplasm. There was no complication following aspiration. Thus FNAB of pancreas is a relatively safe, reliable and quick technique which can be performed under ultrasound guidance.

Adolescent↗
PubMed1993

Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site.

Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527-2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse-phase HPLC. Mass spectroscopy, 1H- and 13C-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of the two lysine residues: [formula: see text]

Amino Acids↗
PubMed1993

Lysine- and threonine-sensitive aspartokinase isoenzymes from spinach leaves share common antigenic determinants.

The lysine- and threonine-sensitive isoenzymes of aspartate kinase were purified to homogeneity from spinach leaves and polyclonal antibodies were raised in rabbits. The antibodies were characterized by various immunological tests like Ouchterlonys-double-diffusion, titrations of the inhibition of enzyme activity and ELISA. The antibodies against the lysine-sensitive isoenzyme could recognise as little as 50 ng of the pure antigen protein and that against the threonine-sensitive form could recognise 200 ng of the protein in the ELISA tests. The immunological tests have also shown that the lysine and threonine sensitive isoenzymes of aspartate kinase share some common antigenic determinants and differ in others.

Animals↗
PubMed1992

Glutamate 264 modulates the pH dependence of the NAD(+)-dependent D-lactate dehydrogenase.

Recently, we amplified the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene by the polymerase chain reaction, cloned and overexpressed it in Escherichia coli (Kochhar, S., Chuard, N., and Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 185, 705-712). Polymerase chain reaction-amplified DNA fragments may contain base changes resulting in mutant gene products. A comparison of specific activities of D-lactate dehydrogenase in the crude extracts of 50 recombinant clones indicated that one of the clones had drastically reduced enzyme activity. Nucleotide sequence analysis of the insert DNA showed an exchange of A to G at position 795 resulting in substitution of Glu264 to Gly in the D-lactate dehydrogenase. The purified mutant D-lactate dehydrogenase showed a shift of 2 units in its optimum pH toward the acidic range. The dependence of kcat/Km on the pH of the mutant enzyme showed that the pKa of the free enzyme was around 4, at least 2 pH units lower than that of the wild-type enzyme. Both the wild-type and the mutant enzyme at their respective optimum pH values showed similar kcat and Km values. The data suggest that the highly conserved Glu264 is not critical for enzyme catalysis, but it must be situated within hydrogen bonding distance to amino acid residue(s) involved in substrate binding as well as in catalysis.

Amino Acid Sequence↗
PubMed1992

Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli.

NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.

Alcohol Oxidoreductases↗
PubMed1992

Cloning and overexpression of the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene in Escherichia coli:purification and characterization of the recombinant enzyme.

The Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene was amplified by the polymerase chain reaction and cloned into an Escherichia coli expression plasmid pKK223.3. Attempts to clone the full-length chromosomal DNA encoding D-lactate dehydrogenase from a partial Sau3AI lambda phage library or an enriched clone bank in E. coli were unsuccessful. The recombinant plasmid pKBULDH containing the amplified gene overexpressed D-lactate dehydrogenase (greater than 30% of total soluble protein) following induction of the tac promotor with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by two chromatographic steps with 76% recovery of enzyme activity. All the properties of the recombinant protein, e.g., optimum pH and temperature, Km and k(cat) for pyruvate as well as for other 2-oxo acids and the subunit structure were identical to the wild-type enzyme.

Amino Acid Sequence↗
PubMed1992

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.

Amino Acid Sequence↗
PubMed1992

Evolutionary relationship of NAD(+)-dependent D-lactate dehydrogenase: comparison of primary structure of 2-hydroxy acid dehydrogenases.

A comparison of the primary structures of NAD(+)-dependent D-lactate dehydrogenase with L-lactate dehydrogenase and L-malate dehydrogenase failed to show any sequence similarity. However, D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, glycerate dehydrogenase from cucumber, D-3-phosphoglycerate dehydrogenase and erythronate 4-phosphate dehydrogenase from Escherichia coli showed 38%, 24%, 24% and 22% amino acid identity, respectively. The profile analysis of the aligned sequences confirmed their relatedness. The hydropathy profiles of the aligned dehydrogenases were almost identical between residues 100-300 indicating largely preserved folding patterns of their polypeptide chains. The data suggest that L- and D-specific 2-hydroxy acid dehydrogenase genes evolved from two different ancestors and thus represent two different sets of enzyme families.

Alcohol Oxidoreductases↗
PubMed1992

Mechanism of racemization of amino acids by aspartate aminotransferase.

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur. J. Biochem. 175, 433-438]. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3-5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase-(27/32-410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the rate-limiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0 degrees C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three-dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of C alpha of the coenzyme-substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate-limiting step in aspartate-aminotransferase-catalyzed racemization.

Amino Acids↗
PubMed1992

Multiple congenital arteriovenous malformations with involvement of the vagina and profuse hemorrhage from vaginal ulcer.

Congenital arteriovenous malformation (AVM) of the female pelvis is a rare but potentially life-threatening condition. An unusual presentation of AVM involving the vagina, abdominal wall, spleen and thigh, with profuse hemorrhage from a vaginal ulcer is described. Doppler, real time ultrasound and computerized tomography were useful in confirmation and delineation of the extensive AVM in this case. Angiography and embolization were thought risky and possibly unnecessary. Bleeding stopped with bed rest and vaginal packing with gauze soaked in 3% hypertonic saline. The ulcer healed after 3 months with conservative management. The risk of future problems was explained to the patient and follow-up was advised. Undertreatment of AVM may be preferable to overtreatment, and the aim should be 'control' rather than 'cure'.

Adult↗
PubMed1991

Development of diagnostic data in the 10-percent sample of disabled SSI recipients.

The Social Security Administration has created a 10-percent sample data base of blind and disabled recipients from the Supplemental Security Income (SSI) program. Codes showing the primary medical diagnosis were obtained for the sample by matching several files, and by imputing codes to sample cases where no diagnostic codes were found. The data base is updated each year by repeating this matching operation and by bringing forward the diagnostic codes from the previous year's file. This article describes the sources of diagnostic information in the administrative record system; the methodology used in the development of the 10-percent disability data base, and the technique chosen to compensate for missing values.

Databases, Factual↗
PubMed1991

Pyogenic meningitis and cerebral abscesses after endoscopic injection sclerotherapy.

Infectious complications of endoscopic injection sclerotherapy (EIS) are rare. We report here a case in which pyogenic meningitis and multiple cerebral abscesses developed after a session of EIS. The patient was successfully treated with a combination of parenteral antibiotics and repeated bedside pus aspirations. Prophylactic antibiotics may be considered in such situations for subsequent sessions of EIS.

Adult↗
PubMed1990

Modified ultrasound-guided percutaneous transgastric drainage of pancreatic pseudocysts.

We describe a modified ultrasound-guided transgastric drainage technique for pancreatic pseudocysts. Using a water-filled stomach to improve visualization, we have successfully drained pseudocysts in ten patients. This report also describes the use of a stiffening cannula to assist transgastric catheter placement. We emphasize the value of constant real-time tracking of the dilator and guidewire to ensure correct positioning of the drainage catheter.

Adolescent↗