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S Dube

Publications and source records attributed to S Dube.

At least 19 recordsLinked to original sources
PubMed1992

Adverse effects of oral amiodarone therapy.

Oral amiodarone was administered to 38 patients (25 males, 13 females) with mean age of 43.6 years. Ventricular and supraventricular arrhythmias were present in 30 and 8 patients respectively. Amiodarone was given as 400-1200 mg/day for 1-2 weeks as loading dose and then it was maintained as 100-600 mg/day. The mean duration of therapy was 12.4 months. Adverse effects were noted in 21 (55.3%) cases. The commonest adverse effects observed were asymptomatic corneal microdeposits followed by gastrointestinal, cardiac, neurological and cutaneous disturbances. The drug was withdrawn in 2 (5.3%) patients because of nausea and vomiting. One patient died of pulmonary infiltrations. It is concluded that adverse effects are common with amiodarone but are tolerated well, making this drug an excellent choice for treatment of cardiac arrhythmias.

Administration, Oral↗
PubMed1991

Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10.

Glutamic acid decarboxylase (GAD; glutamate decarboxylase, L-glutamate 1-carboxy-lyase, EC 4.1.1.15), which catalyzes formation of gamma-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, we describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different from that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows less than 65% identity to previously published, highly conserved GAD-1 brain sequences, which show greater than 96% deduced amino acid sequence homology among the three species. The function of this additional islet GAD isoform and its importance as an autoantigen in insulin-dependent diabetes remain to be determined.

Amino Acid Sequence↗
PubMed1991

Effects of sodium butyrate on proliferation-dependent insulin gene expression and insulin release in glucose-sensitive RIN-5AH cells.

A rat islet tumor subclone, RIN-5AH-T2-B, was cultured with 2 mmol/liter of the proliferation-arresting compound sodium butyrate (NaB). Insulin gene expression and glucose-stimulated insulin release were analyzed and compared with logarithmically proliferating and confluent control cells cultured without NaB. Logarithmically proliferating control cells revealed high insulin gene expression. In the presence of amino acids, these cells showed a dose-dependent insulin response to glucose with a half-maximal and maximal 6.5-fold stimulation by 0.8 and 5.6 mmol/liter D-glucose, respectively. However, as the control cells approached growth arrest, insulin gene expression subsided to below detectability, an occurrence that is associated with decreased insulin release and accumulation of cells in the G1 phase of the cell cycle. In contrast, NaB-arrested cells showed continuous insulin gene expression throughout the experiment. Despite this, insulin release in response to glucose was lost. NaB revealed a biphasic effect on the cell-cycle: after an initial leaky G1 arrest during the first 24 h, the 5AH-B cells were arrested in G2 during the following 3 days. These data suggest that insulin gene expression and glucose-stimulated insulin release are affected by the cell cycle. These glucose-sensitive RIN-5AH-T2-B cells may be useful in studies of insulin secretion and gene regulation.

Animals↗
PubMed1991

Autoimmunity of diabetes.

Insulin-dependent diabetes mellitus is associated with a growing number of immune abnormalities. At the time of clinical onset, most patients developing the disease as children or young adults have autoantibodies reactive with islet beta cells. Current autoantibody markers for IDDM are not sufficient to predict the disease in the general population. Studies in first-degree relatives indicate the presence of a subclinical disease characterized by beta cell dysfunction, which may or may not progress to overt IDDM. Although IDDM is genetically linked to certain HLA-DQ class II molecules, it needs to be clarified whether these molecules determine the propensity to react to certain antigens, the failure to maintain tolerance, or the ability to produce disease-associated autoantibodies. Circumstantial evidence suggests that yet another gene outside the HLA complex on chromosome 6 is more important. The interaction with the environment needs to be clarified, and the etiologic role of viruses has not been substantiated. An underlying systemic autoimmune propensity may influence environmental insults and perpetuate islet beta cell destruction. Until these mechanisms are understood, clinicians should periodically check their patients with IDDM for other organ-specific as well as non--organ-specific autoimmune diseases. Our understanding of these phenomena is poor, which may explain why clinical trials with immunosuppressive agents have been of limited success. Further studies on the molecular biology of the immune response against islet beta cell-specific antigens are necessary for the development of both predictive tests and novel measures to prevent IDDM.

Autoantibodies↗
PubMed1989

Amplification of a polymorphic dihydrofolate reductase gene expressing an enzyme with decreased binding to methotrexate in a human colon carcinoma cell line, HCT-8R4, resistant to this drug.

A methotrexate (MTX)-resistant human colon carcinoma cell line was obtained by growing HCT-8 cells in stepwise increasing concentrations of the drug. The resistant subline (HCT-8R4) was able to grow in the presence of 1 x 10(-4) M MTX and was found to have a 25-fold increase in the level of the target enzyme dihydrofolate reductase (DHFR), with a corresponding increase in DHFR gene copies as well as DHFR transcripts. Southern blot analysis of DNA from HCT-8R4 cells revealed the amplification of an altered gene. The amplified DHFR gene lacks an EcoRI restriction enzyme site in the coding region, normally present in other human cell lines. Sequence analysis of cDNA synthesized from transcripts in the MTX-resistant cell line revealed a base transition T----C at nucleotide position 91 resulting in a substitution of serine for phenylalanine. The dissociation constant for MTX binding to the HCT-8R4 enzyme was 1.25 nM, an 8-fold increase from the Kd 150 pM of purified wild type human DHFR. This decrease in binding of MTX to the HCT-8R4 DHFR is consistent with the predicted involvement of phenylalanine in the DHFR active site in hydrophobic interactions with MTX. This mutation plus the 25-fold increase in DHFR activity explains the high level of resistance of this subline to MTX.

Base Sequence↗
PubMed1988

The effect of 5'-amino-5'-deoxythymidine on the pattern of polyadenylation of herpes simplex virus type 1 ribonucleic acid.

Treatment of herpes simplex virus type 1 (HSV-1) infected Vero cells with 5'-amino-5'-deoxythymidine (AdThd) causes a 65% reduction in the amount of total viral RNA present at late times of infection. This decrease is apparent as early as 6 hours post infection, affecting the levels of viral cytoplasmic polyadenylated (poly(A+] species to a greater extent than non-polyadenylated (poly(A-] RNA. Cytoplasmic viral poly(A+) RNA is present in AdThd-treated cultures at only 10% control poly(A+) levels, yet there is no evidence of a direct inhibition of RNA polyadenylation. Underrepresentation of RNA species in the cytoplasm is not sequence-specific. The results suggest that the antiviral activity of AdThd may involve not only incorporation into viral DNA, with a resultant decrease in viral transcription, but also perturbation of the relative amounts of viral poly(A+) RNA and poly(A-) RNA.

Animals↗
PubMed1987

[Genetic transformation of somatic cells. XII. Mutations in human satellite DNA introduced into Chinese hamster cells].

Data are presented about detailed restriction mapping, hybridization and sequence analysis of plasmid pRT301 rescued into E. coli 25-30 cell divisions after transfection of Chinese hamster cells. This plasmid contains an insert of human satellite III fragment (HS3), which was replicated many times in mammalian cells, and we found multiple point mutations in this non-selectable fragment, mainly single inserts of GC base pair. Specificity of mutagenesis of HS3 induced by transfection and replication in Chinese hamster cells differs from mutagenesis specificity found by other authors for selectable genes.

Animals↗
PubMed1978

Phosphoglycerate mutase. Kinetics and effects of salts on the mutase and bisphosphoglycerate phosphatase activities of the enzyme from chicken breast muscle.

The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.

Animals↗
PubMed1977

Formation of renal medullary lysosomes during potassium depletion nephropathy.

The biochemical correlates of droplet formation in renal inner medullary cells of potassium-deficient rats were studied. An increase in the activities of five hydrolytic enzymes typical of lysosomes was associated with an increase in the number and size of droplets observed during progressive potassium depletion. Acid phosphatase activity increased 7-fold whereas beta-glucuronidase, beta-galactosidase, cathepsin, and acid DNase increased 2- to 4-fold in medullary homogenates at 25 days of depletion. Following potassium repletion the activities returned to normal at a rate dependent upon the duration of potassium depletion. The decreases in enzyme activities were associated with a concomitant rapid disappearance of the droplets from medullary cells. Protein synthesis for new droplet enzyme formation was studied by measuring the rate of [14C]leucine incorporation into protein in medullary slices. The rate increased at 1 day of depletion and reached a maximum which was 139 per cent higher than control after 7 days of depletion. In droplets isolated from medullary tissue during progressive potassium depletion the rate of protein labeling with [14C]leucine and acid phosphatase specific activity increased in parallel. When droplet proteins were separated by gel electrophoresis, acid phosphatase activity was detected in a protein band which had been labeled with [14C]leucine, thereby suggesting new enzyme protein formation. The increase in enzyme and protein synthesis and a previously demonstrated increase in phospholipid synthesis and membrane formation indicate that potassium depletion induces specific alterations in renal inner medullary cell metabolism which result in increased lysosome formation.

Acid Phosphatase↗
PubMed1976

Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase.

Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.

Animals↗
PubMed1976

Size, subunit composition, and secondary structure of the Friend virus genome.

Electron microscope and gel electrophoresis studies show that the high-molecular-weight (50 to 70S) RNA extract from Friend virus (FV) is a dimer with the same basic structure previously observed for the RNAs from RD-114 virus, baboon virus, and woolly monkey virus. This observation greatly strengthens the inference that the dimer structure is a general characteristic of the RNAs of all mammalian type C viruses. The FV dimer is slightly less stable than the RNA dimer of woolly monkey virus, which is, in turn, much less stable than those of RD-114 and baboon virus. There are three FV monomer components, small (S), medium (M), and large (L), with molecular lengths of 6.7 +/- 0.6, 7.7 +/- 0.6, and 9.5 +/- 0.6 kilobases, respectively. There are approximately equal amounts of the S and M components and much less of the L component. Most of the dimers are homodimers (SS, MM, and LL). The frequency of heterodimers (SM, SL, ML) is much less than expected for a random assortment model.

Friend murine leukemia virus↗