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Biomedical subjects

A K Abraham

Publications and source records attributed to A K Abraham.

At least 19 recordsLinked to original sources
PubMed1997

A novel pathway for the conversion of homocysteine to methionine in eukaryotes.

Activation of amino acid homocysteine was compared with that of methionine in rabbit crude liver extracts and purified multi-enzyme complex of aminoacyl-tRNA synthetases. Activation was studied by measuring the incorporation of radioactive amino acid into unlabelled trichloroacetic-acid insoluble materials in the absence of protein synthesis. Homocysteine synthetase activity was found in the crude extract and in the purified multi-enzyme complex of aminoacyl-tRNA synthetases. On a molar basis, the activation of methionine by the crude extract was five times higher than the activation of homocysteine. There was a partial loss of Hcy-tRNA synthetase activity in the purified multi-enzyme complex. Preliminary reconstitution experiments indicated a requirement for an additional factor for Hcy-tRNA synthetase activity. TLC of the amino acid released from tRNA charged with [14C]homocysteine, revealed radioactivity in homocysteine, methionine and homocysteine thiolactone, indicating a conversion of tRNA-attached homocysteine to methionine. Total tRNA was separated on a benzoylated cellulose column into a fraction enriched in initiator tRNA and a methionine-accepting, but initiator tRNA-deficient, fraction. Homocysteine-accepting activity was present only in the initiator tRNA-enriched fraction. Based on the above data we propose that homocysteine activation in reticulocyte lysates, reported previously, also occurs in liver. Activated homocysteine is attached to initiator tRNA and then converted to methionine by a methylating enzyme. In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone.

Animals↗
PubMed1989

Kinetics of ribosomal protein S6 phosphorylation by HepG-2 cells in response to insulin.

The effect of insulin on the phosphorylation of ribosomal protein S6 was studied in a human liver cell line (HepG-2), using [32P] inorganic phosphate. Increased rate of protein S6 phosphorylation was detected 8 min following the addition of insulin to serum starved cells. Maximum enhancement of phosphorylation was observed at 80 nM insulin. Minimum level of insulin required to produce measurable increase of S6 phosphorylation was 20 nM. Radioactivity of protein S6 increased most in the native subunit and polysome fractions. Significant increase in radioactivity of this protein was not observed in the monosome fraction during the first 30 min of insulin stimulation. Increase in the specific radioactivity of native 40S subunit was higher than that of polysomes. These results suggest that phosphorylation takes place in the subunit compartment and moves preferentially into the polysomes.

Carcinoma, Hepatocellular↗
PubMed1988

Translation of globin RNA spliced in vitro.

Pre-mRNA (precursor mRNA) enriched for globin-gene transcription products was prepared from murine erythroleukaemia cells induced to differentiate with dimethyl sulphoxide. The pre-mRNA was prepared from nuclear RNA by oligo(dT)-cellulose chromatography followed by sucrose-gradient centrifugation. The pre-mRNA thus obtained was utilized as the substrate for a splicing assay based on sequential splicing and translation reactions. Small nuclear ribonucleoprotein particles, which were shown to inhibit translation, were removed by an intermediate high-speed centrifugation step. The assay developed is based on a three-step procedure: (1) incubation of pre-mRNA with HeLa-cell nuclear extract in the presence of ATP; (2) incorporation of 3H-labelled amino acid into proteins by wheat-germ extract by using RNA from step 1; and (3) immunoprecipitation of globin chains with rabbit anti-(mouse globin) antibody. No incorporation of label into immune complexes was observed in the absence of HeLa-cell extract or ATP. Preincubation with these components was unnecessary for the incorporation of label into immune complexes when mature cytoplasmic RNA was used as a template for protein synthesis. The assay was further validated by fluorography after polyacrylamide-gel electrophoresis, when the only band detectable corresponded to globin chains. These results demonstrate, for the first time, translational activity of transcripts spliced in vitro. The current assay can be utilized with a few micrograms of pre-mRNA and may have potential for further characterization of the splicing system.

Cell Line↗
PubMed1988

A precaution when preparing very large plasmids by alkaline lysis procedure.

The extractabilities of plasmids of different sizes by the sodium lauryl sulfate (SDS)-alkali procedure were compared using either sodium acetate or potassium acetate buffer as the neutralizing agent. There was a selective loss of large plasmids (above 100 kb) when the potassium salt was used. When N-lauryl sarcosine instead of SDS was used as the detergent, no loss of large plasmids occurred in the presence of potassium salt. A comparison of the kinetics of precipitate formation with sodium acetate and potassium acetate indicated that the rate and the amount of lauryl sulfate precipitated were lower with the sodium salt. It is suggested that faster precipitation of lauryl sulfate with potassium acetate leads to trapping of large denatured plasmids that cannot renature as fast as the small ones.

Acetates↗
PubMed1987

Pulmonary atresia with ventricular septal defect in adult patients.

Hemodynamic data and angiograms of 15 adult patients with pulmonary atresia and ventricular septal defect were reviewed to assess the pulmonary circulation and other associated features. The most common variety of pulmonary atresia was that of pulmonary valve, main pulmonary artery, and the confluence of pulmonary arteries (6 cases, 40%). The collateral vessels to the lungs were well developed in all cases; selective injections into the collateral vessels were of great value in their delineation. The left ventricle was well developed in 11 cases (73.3%). Congestive heart failure was seen in 8 (53.3%), tricuspid regurgitation in 10 (66.7%), and aortic regurgitation in 7 (46.7%) cases. The long survival in these patients was related to the favorable anatomy of central pulmonary arteries (12 cases, 80%) and adequate pulmonary collateral circulation.

Adolescent↗
PubMed1987

Supernumerary right coronary artery.

A rare case of a patient with supernumerary right coronary artery in whom the two vessels arose from the right coronary sinus from two separate ostia adjacent to each other is presented. The smaller vessel gave off the sinoatrial nodal branch and the posterior descending artery whereas the larger one gave off the conus branch, the right ventricular branches, and continued as acute marginal branch. This is the first case report in the English literature.

Adult↗
PubMed1987

Quadrivalvular rheumatic heart disease.

Three cases of chronic rheumatic heart disease with involvement of all four valves are presented. The involvement of tricuspid and pulmonary valves was suspected clinically and was confirmed by two-dimensional echo, Doppler, hemodynamic and angiographic findings. These findings were also verified surgically and histopathologically in 2 cases. One of the cases died after cardiac catheterization; the other 2 cases were treated surgically with success.

Adult↗
PubMed1986

Nucleotide binding to elongation factor 2 inactivated by diphtheria toxin.

Binding of guanosine nucleotides to purified native and ADP-ribosylated wheat germ EF-2 was measured. Both forms of EF-2 bound [3H]GDP to the same extent. [3H]GDP binding to native but not to ADP-ribosylated EF-2 was reduced in the presence of GTP and ribosomes. Binding of [gamma-32P]GTP to EF-2 was significantly reduced upon ADP-ribosylation. ADP-ribosylation almost abolished both the stimulatory effect of ribosomes on GTP binding to EF-2 and the ability of EF-2 to form a high-affinity complex with GuoPP(CH2)P and ribosomes. Low-affinity complex formation between EF-2 X GDP and ribosomes was not influenced by ADP-ribosylation. The results indicate that the inhibition of the elongation process caused by the toxin is probably due to the inability of modified EF-2 to exchange GDP with GTP.

Adenosine Diphosphate Ribose↗
PubMed1986

Regulation of pancreatic islet cell replication: an inquiry into the controversy regarding the effects of steroid hormones.

The controversial issue of the effects of prednisolone and 17 beta-estradiol on replication of fetal rat pancreatic islets in culture was studied using 32P and [3H]thymidine as probes for studying DNA synthesis. DNA synthesis was not affected by the steroid hormones, as was evident from the rate of incorporation of 32P into total DNA. Decreased incorporation of [3H]thymidine into DNA found in islets treated with either of these steroids seemed to reflect an inhibitory effect of these hormones on thymidine kinase, leading to decreased phosphorylation of labeled thymidine. In addition, the hormones stimulated the activity of thymidylate synthetase, thus enhancing the endogenous synthesis of thymidine and thereby diluting the specific activity of the [3H]thymidine added to the cultured islets. Further support for a lack of inhibition of growth of islet cells treated with steroid hormones was provided by the observation that prednisolone increased uridine kinase activity and RNA biosynthesis, both of which may participate in the growth of cells preceding mitosis and (the latter) in protein hormone biosynthesis.

Animals↗
PubMed1984

Culture negative infective endocarditis.

Twenty cases of culture negative infective endocarditis admitted to the Cardiology Department of Green Lane Hospital from 1959 to 1980 out of a total of 265 cases (7.5%), were analysed retrospectively. Cases were included only when adequate proof of endocarditis was available at surgery or postmortem. Indiscriminate use of antibiotics before taking blood cultures was the most common association with failure to obtain positive cultures, seen in 16 of the 20 patients described. Failure to obtain positive cultures in four cases was attributed to inadequate bacteriologic techniques before 1967. Where no antibiotics were given prior to collecting blood cultures and bacteriologic techniques were adequate, proven culture negative endocarditis was virtually unknown. When antibiotics have been given, repeated blood cultures are recommended following withdrawal of antibiotic for at least four days.

Anti-Bacterial Agents↗
PubMed1983

Effect of protein synthesis inhibitors on the fidelity of translation in eukaryotic systems.

Factors influencing the accuracy of poly(U)-directed poly(Phe) synthesis in a wheat germ and in a reticulocyte system were studied. Addition of preformed phenylalanyl-tRNA, as well as increasing the ratio of poly(U) to ribosomes, significantly enhanced the poly(Phe) synthesis and concurrently reduced the misincorporation of leucine. The protein synthesis inhibitors cycloheximide, abrin and ricin had little or no effect on the misreading when the system was supplemented with 100 microM phenylalanyl-tRNA, but they reduced the relatively high error rate observed when the poly(U) system was not supplemented with the cognate substrate. Raising the incubation temperature enhanced the accuracy to the same extent whether or not ricin was present i.e., at widely different rates of elongation. The results show that the translational accuracy is not linked to the elongation rate as such. Translational inhibitors affect the fidelity by influencing the kinetics of the system. In systems containing limiting concentrations of cognate substrate, translational inhibitors will cause an increase in the limiting aminoacyl-tRNA species and thereby increase fidelity.

Abrin↗
PubMed1982

Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts.

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin-treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied, indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes. Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment. The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis, at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.

Animals↗
PubMed1982

Inhibition of mitochondrial protein synthesis and energy coupling by fragment A of diphtheria toxin.

The effect of intact diphtheria toxin and its fragment A on energy-dependent functions in mouse liver mitochondria/mitoplasts has been studied. Fragment A was found to inhibit protein synthesis in mitoplasts to the same extent (approximately 80%) as the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone, but had no similar effect in lysed mitoplasts. Intact diphtheria toxin had no effect in either case. Fragment A was found to function as a potent uncoupler in isolated mitochondria and mitoplasts, inhibiting oxidative phosphorylation by approximately 80% at a concentration (2 micrograms fragment A/mg of protein) that did not inhibit protein synthesis. In contrast, intact toxin slightly increased the tightness of energy coupling in isolated mitoplasts. 125I-labelled intact diphtheria toxin was bound to mitoplasts to about the same extent as labelled fragment A. At concentrations which efficiently inhibited mitochondrial protein synthesis, fragment A had no effect on the intramitochondrial concentration of nicotinamide adenine dinucleotides, nor was it capable of ADP-ribosylating mitochondrial proteins, indicating that the well known enzymatic activity of fragment A is not involved in the observed effect in mitochondria. The results indicate that fragment A of diphtheria toxin inhibits protein synthesis in mitochondria and mitoplasts by inhibiting mitochondrial energy transduction. The detailed mechanism of the uncoupling effect and its possible significance in intact cells remain to be elucidated.

Animals↗