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S Sharp

Publications and source records attributed to S Sharp.

At least 55 records · Page 3Linked to original sources
PubMed1990

Purification and immunochemical characterization of a rat liver sulphotransferase conjugating paracetamol.

Paracetamol sulphotransferase (ST) was purified 250-fold from male rat liver, and the pure enzyme used to elicit antibodies in rabbit. The enzyme was active towards paracetamol at pH 9.0, as well as towards several commonly used drugs, and formed sulphates at both O- and N-atoms. Comparison of the substrate specificity of paracetamol ST with that of aryl sulphotransferases isolated by other workers suggested that we have purified a previously unknown isoenzyme of rat liver ST, although the difficulties of characterization of STs based on their substrate specificities is noted. The antibody preparation recognized only one polypeptide (Mr = 35,000) on immunoblot analysis of rabbit liver cytosol, corresponding to purified paracetamol ST. Analysis of the tissue distribution of this protein demonstrated that its expression was restricted to the liver, as was the enzyme activity. The observed sex difference in paracetamol ST (males greater than females) was determined by immunoblot analysis to be the result of reduced enzyme protein levels in females. In human liver cytosol, the antibody recognized two polypeptides, probably corresponding to M- and P-phenol STs, suggesting significant sequence similarity between rat and human phenol sulphotransferases.

Acetaminophen↗
PubMed1986

The 5'-flanking sequences of Drosophila melanogaster tRNA5Asn genes differentially arrest RNA polymerase III.

Three tRNA5Asn genes have been subcloned from a tRNA gene cluster isolated from the cytogenetic locus 42A of Drosophila melanogaster. The three tRNAAsn genes, contained on plasmids pAsn6, pAsn7, and pAsn8, have identical mature tRNA coding regions but have different 5'- and 3'-flanking sequences. In vitro transcription in Drosophila Schneider S3 cell-free extracts showed the tRNAAsn genes had different transcription efficiencies. pAsn8 had a transcription efficiency of approximately 8 transcripts/gene/h, whereas pAsn6 was a less active template at 5 transcripts/gene/h. pAsn7 was the poorest template at 1.5 transcripts/gene/h. Exchanging 5'-flanking regions of the tRNAAsn genes showed that the differences in transcription efficiencies were attributable to the corresponding 5'-flanking region. Transcription of each of the tRNAAsn genes revealed a different optimum for KC1 concentration for each template which also was directly attributable to the corresponding 5'-flanking region. The "salt effect" is not related to the ability of the three tRNAAsn genes to sequester transcription factors as determined using the stable complex competition assay. Rather, this effect appears to be due to the ability of the respective 5'-flanking regions to interact with RNA polymerase III. The poorest transcription template, pAsn7, was a better competitor in the stable complex formation assay than either pAsn8 or pAsn6. We conclude that the pAsn7 stable complex binds and functionally arrests RNA polymerase III in the initiation reaction.

Animals↗
PubMed1986

Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas.

Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.

Animals↗
PubMed1986

Use of high-performance liquid chromatography in the measurement of in vitro acetylation in man.

Liquid chromatographic methods were developed for the study of the in vitro acetylation of the sulphonamide drug sulphamethazine and a series of aniline derivatives. The sensitivity of the methods have allowed data on the activity of the N-acetyltransferase enzyme(s) in man to be obtained. The use of bonded-phase columns with a series of organic mobile phases has been compared with reversed-phase separation of the acetanilide derivatives, produced in the acetylation reaction.

Acetanilides↗
PubMed1986

Kingella denitrificans prosthetic endocarditis.

Kingella species are rarely reported as a cause of endocarditis. We report two cases of K. denitrificans endocarditis occurring in patients with prosthetic valves. K. denitrificans has not been previously reported to cause prosthetic valve endocarditis. Both patients may have developed bacteremia from altered upper respiratory or oral mucosa with subsequent prosthetic valve infection. Treatment with penicillin G and streptomycin was successful in the first patient, whereas ampicillin alone resulted in clinical cure in the second case. Neither patient required further surgery. In vitro antibiotic susceptibility testing indicated that both organisms were highly sensitive to cefotaxime and ampicillin.

Adult↗
PubMed1986

Staphylococcus epidermidis ventriculitis treated with vancomycin and rifampin.

Two cases of ventriculitis with Staphylococcus epidermidis that failed on therapy with an antistaphylococcal penicillin are presented. Both infections responded to a combination of intravenous and intraventricular vancomycin and rifampin. Vancomycin and rifampin represent an important antibiotic regimen for the management of resistant infections of the central nervous system, especially with those due to S. epidermidis or methicillin-resistant Staphylococcus aureus.

Anti-Bacterial Agents↗
PubMed1985

Point mutations in the 5' ICR and anticodon region of a Drosophila tRNAArg gene decrease in vitro transcription.

We have examined the effects of various nucleotide substitutions in a Drosophila tRNAArg gene on in vitro transcription and stable transcription complex formation in Drosophila KcO and HeLa cell extracts. Substitutions in positions encoding the invariant G18 and G19 residues resulted in decreased transcription, however, the moderate decreases indicate that these nucleotides are not obligatory promoter recognition sites. An A21 to C21 mutation had no effect on transcription levels using homologous extract however, this mutant displayed decreased transcriptional abilities in HeLa cell extract. Nucleotide substitutions within the sequence encoding the anticodon led to a decrease in the transcription activity but not in the ability to form a stable transcription complex.

Animals↗
PubMed1984

Transcriptionally active and inactive gene repeats within the D. melanogaster 5S RNA gene cluster.

Transcription of isolated repeat units of D. melanogaster 5S DNA in a Drosophila KcO cell extract revealed three types of template activities. 5SI DNA encodes the known 5S rRNA of D. melanogaster and has a relatively high transcription efficiency. 5SII DNA is identical to 5SI DNA except for a two-nucleotide deletion at 5S rRNA positions 28 and 29; the efficiency of transcription is approximately 40% that of 5SI DNA and because of the deletion, the primary transcript is two nucleotides shorter. 5SIII DNA does not support in vitro transcription (less than 2% 5SI DNA), but has the same sequence as 5SI DNA except for a single G to A transition at position 86. This is the first reported point-mutation in a 5S RNA gene resulting in loss of transcription function. Of approximately 23 5S rRNA gene copies in a cloned 5S DNA sub-cluster (p12D1) 19 appear to be of the transcriptionally inactive 5SIII DNA type.

Animals↗
PubMed1984

The extent of a eukaryotic tRNA gene. 5'- and 3'-flanking sequence dependence for transcription and stable complex formation.

We have examined the 5'-and 3'-flanking sequence requirements for the "wild type transcription properties" of a Drosophila tRNA Arg gene through the use of transcription assays in cell-free extracts. Thirty-three base pairs of the 5' flank immediately adjacent to the sequence encoding the mature tRNA are necessary for efficient transcription in Drosophila Kc cell extract. Sequences affecting factor binding to form stable transcription complexes extend more than 60 base pairs into the 5' flank, and approximately 35 base pairs into the 3' flank. HeLa cell extract exhibits dependence, albeit reduced, on the same 5'-flanking sequence; it also has 3'-flanking sequence requirements for maximal stable complex formation. This requirement of in vitro transcription for flanking sequence is not dependent on the use of a homologous system, but is dependent on the cellular source of the extract.

Animals↗
PubMed1983

Partial purification of Drosophila Kc cell RNA polymerase III transcription components. Evidence for shared 5 S RNA and tRNA gene factors.

Class III transcription components were fractionated from Drosophila Kc cells. By the use of successive DEAE-Sephadex and CM-Sepharose column chromatographies, two fractions were identified to contain distinct positive transcription components. The reconstitution of these two fractions and RNA polymerase III was essential for faithful in vitro transcription of cloned Drosophila tRNA and 5 S RNA genes. Further chromatography of one of these fractions using phosphocellulose and DEAE-cellulose did not separate additional components required for reconstitution of transcription. These fractions may contain generalized class III transcription factors, as suggested by the cofractionation of required tRNA and 5 S RNA transcription components. We present further evidence that tRNA and 5 S RNA genes share at least one common transcription component other than RNA polymerase III by the use of stable transcription complex formation and competition experiments.

Animals↗
PubMed1983

Each element of the Drosophila tRNAArg gene split promoter directs transcription in Xenopus oocytes.

The intragenic control regions of a eukaryotic tRNA gene have been examined by transcribing mutant forms of a Drosophila tRNAArg gene either by injection into the nucleus of Xenopus oocytes or in extracts prepared from isolated oocyte nuclei. These experiments demonstrate that the selection of the transcription initiation site is a complex mechanism that involves the T-control region, the D-control region, and sequences 5' adjacent to the D-control region. In this study either "half" of the Drosophila tRNAArg gene promoted transcription in Xenopus oocytes. This finding supports a recent model for eukaryotic tRNA gene transcription (Dingermann et al., 1983, J. Biol. Chem. 258, 10395-10402) that proposes transcription initiation is dependent on the ability of specific DNA sequences to sequester two RNA polymerase III transcription factors.

Animals↗
PubMed1983

Stable transcription complex formation of eukaryotic tRNA genes is dependent on a limited separation of the two intragenic control regions.

We have examined the transcriptional role of the DNA region which lies between the two intragenic control sequences (D-control and T-control) of tRNA genes. Deletion templates (3' and 5') of a Drosophila tRNAArg gene, which contain either the D- or T-control region, were joined together through XhoI linkers such that the mutant tRNA genes formed now contain an internal cloning site. DNA fragments of different lengths were inserted into the newly formed cloning site to create a series of mutant tRNAArg genes which have an increased separation between the two intragenic control regions of 12 to 1530 nucleotides. Increased separation of the two intragenic control regions did not alter the transcription initiation or termination sites from those of the wild type tRNAArg gene. Transcription, while most efficient in the wild type gene, is still efficient when the two regions are further separated by a distance of 12-77 nucleotides. However, any further increase in length of the sequence separating the control regions resulted in a decreased transcription efficiency and in a decreased ability to compete in the binding of transcription factors in Drosophila Kc cell extracts. The reduction in transcription efficiency is directly related to the decreased ability of the insertion mutant tDNAs to form stable transcription complexes. The tRNAArg gene forms detectable stable complexes up to a separation of the two intragenic control regions by approximately 200 to 400 base pairs. These results suggest a model for tRNA gene transcription that involves factor recognition of sequences within each control region and that the control regions interact only via these factors.

Base Sequence↗
PubMed1983

Transcription of eukaryotic tRNA genes in vitro. I. Analysis of control regions using a competition assay.

The regions of a Drosophila tRNAArg gene responsible for the "wild type" in vitro transcription level were determined by a transcription-competition assay. Cell-free transcription extracts programmed with 5' and 3' deletion mutants of the tRNAArg gene were used to quantitate the efficiency of transcription and to measure the ability of these DNAs to compete for transcription factors compared to the wild type tRNAArg gene. The results show that those portions of the gene which code for the D-stem/D-loop and T-stem/T-loop of the tRNA product are the regions responsible for competitive ability. These regions were previously shown to contain the intragenic control sequences for eukaryotic tRNA gene transcription and are respectively referred to as the D- and T-control regions. The presence of both the D- and T-control regions is essential for maximum competitive strength. The 5'-flanking and 5' stem regions adjacent to the D-control region have a function in the competitive ability of the D-control region while the 3'-flanking and the 3' stem regions adjacent to the T-control region have a function in the competitive ability of the T-control region. These results are consistent with a model for promotion of tRNA gene transcription that involves recognition by transcription factors of the two control regions. Optimal binding of the transcription factors is dependent upon sequences adjacent to and flanking the intragenic control regions.

Animals↗
PubMed1983

Transcription of eukaryotic tRNA genes in vitro. II. Formation of stable complexes.

Drosophila tRNA genes form stable transcription complexes in vitro, as we have demonstrated by kinetic analyses of transcription experiments in Drosophila Kc cell extracts. tRNA genes added to transcriptionally active cell-free extracts rapidly and stably sequester a transcription factor, inhibiting transcription of a tRNA gene added later. We describe a simplified competition assay dependent on the ability of tRNA genes to form stable complexes. Through the use of this assay with deletion mutations of a Drosophila tRNAArg gene, we demonstrate that stable transcription complex formation is dependent on the DNA region extending from the 5' end of the sequence encoding the T-stem of the tRNA to more than 10 base pairs downstream from the transcription termination sequence. Stable transcription complex formation involves an initial, rapid factor binding followed by rearrangement of the gene-factor complex to a transcriptionally active state. Factor binding to form the stable transcription complex is kinetically dependent on the sequence 5' to the gene region encoding the D-stem, and thermodynamically dependent on the gene region encoding the D-stem and -loop.

Animals↗