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R W Bishop

Publications and source records attributed to R W Bishop.

10 recordsLinked to original sources
PubMed1998

Characterization of the mevalonate kinase 5'-untranslated region provides evidence for coordinate regulation of cholesterol biosynthesis.

Using a probe derived from the 5'-untranslated region of the human mevalonate kinase (MK) cDNA, we screened a lambda gt 11 genomic library and obtained a single clone containing the 5' untranslated region of the gene. Nucleotide sequencing identified several putative regulatory elements, including two Sp1 (GC box) elements and a CCAAT box. A canonical TATA box was not detected. Directly adjacent to one Sp1 element was a sterol regulatory element (SRE), 5'-CACCCCAG-3', which was a 7/8 base pair match to the consensus sequences identified in the genes encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase and reductase, and the LDL receptor. There was no Sp1 element upstream of the SRE. Northern blot analysis in human CRL1508T cells revealed that quantities of MK poly A+ RNA increased for cells grown in the presence of lipid-deficient calf serum, and further increased upon addition of 1 microM lovastatin. Primer extension analysis with human poly A+ RNA suggested at least 4 transcription initiation sites downstream from the CCAAT box. To assess sterol responsiveness of transcription initiation, a 1.4 kb genomic fragment upstream of the translational start site was fused to the pSV2cat vector for transient expression in COS-7 cells, with chloramphenicol acetyltransferase (CAT) as the reporter gene. This construct demonstrated modest levels of CAT expression which was induced > 2-fold when cells were grown in lipoprotein-deficient calf serum. Our data provide further evidence for coordinate regulation of cholesterol biosynthesis in response to sterol.

Animals↗
PubMed1993

Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme. Inhibition of sterol synthesis in S. cerevisiae and S. pombe cells or in cultured human fibroblasts by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin resulted in elevated levels of squalene synthetase mRNA in all three cell types.

Adult↗
PubMed1992

Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic disease mevalonic aciduria.

Mevalonic aciduria is the first proposed inherited disorder of the cholesterol/isoprene biosynthetic pathway in humans, and it is presumed to be caused by a mutation in the gene coding for mevalonate kinase. To elucidate the molecular basis of this inherited disorder, a 2.0-kilobase human mevalonate kinase cDNA clone was isolated and sequenced. The 1188-base pair open reading frame coded for a 396-amino acid polypeptide with a deduced M(r) of 42,450. The predicted protein sequence displayed similarity to those of galactokinase and the yeast RAR1 protein, indicating that they may belong to a common gene family. Southern hybridization studies demonstrated that the mevalonate kinase gene is located on human chromosome 12 and is a single copy gene. No major rearrangements were detected in the mevalonic aciduria subject. The relative size (2 kilobases) and amounts of human mevalonate kinase mRNA were not changed in mevalonic aciduria fibroblasts. Approximately half of the mevalonic aciduria cDNA clones encoding mevalonate kinase contained a single base substitution (A to C) in the coding region at nucleotide 902 that changed an asparagine residue to a threonine residue. The presence of this missense mutation was confirmed by polymerase chain reaction amplification and allele-specific hybridization of the genomic DNAs from the proband and the proband's father and brother. Similar analysis failed to detect this mutation in the proband's mother, seven normal subjects, or four additional mevalonic aciduria subjects, indicating that the mutation does not represent a common gene polymorphism. Functional analysis of the defect by transient expression confirmed that the mutation produced an enzyme with diminished activity. Our data suggest that the index case is a compound heterozygote for a mutation in the mevalonate kinase gene.

Amino Acid Sequence↗
PubMed1992

Structure of the hamster low density lipoprotein receptor gene.

The metabolism of low density lipoprotein (LDL) in the hamster is substantially similar to that of the human. To extend the usefulness of the hamster as an experimental model, the hamster LDL receptor gene was isolated and characterized. The gene is composed of 18 exons and 17 introns which span 26 kilobases. The introns occur at precisely the same positions as those previously determined for the human LDL receptor gene. The 18 exons of the hamster gene predict an LDL receptor protein of 854 amino acids that is similar in organization and sequence to those predicted from the cDNAs of rat, rabbit, cow, and human. Within the 5'-flanking region of the hamster LDL receptor gene are three highly conserved imperfect direct repeat sequences of 16 nucleotides each that in the human gene have been demonstrated to regulate transcription. In addition, a similar arrangement of direct repeat sequences was also isolated from the 5'-flanking region of the rat LDL receptor gene using the polymerase chain reaction. These results indicate a strong sequence and structural conservation of the LDL receptor among several species and further support the hamster as an experimental model for the study of human LDL-cholesterol metabolism.

Amino Acid Sequence↗
PubMed1989

Expression cloning and regulation of steroid 5 alpha-reductase, an enzyme essential for male sexual differentiation.

The conversion of testosterone into the more potent androgen, dihydrotestosterone, catalyzed by the enzyme steroid 5 alpha-reductase, is required for the differentiation of male external genitalia. Here, we report the isolation of cDNA clones encoding the rat steroid 5 alpha-reductase using expression cloning in Xenopus oocytes. DNA sequence analysis demonstrates that the liver and ventral prostate forms of steroid 5 alpha-reductases are identical hydrophobic proteins of 29 kDa. The amount of steroid 5 alpha-reductase mRNA in liver increased in response to castration, but remained unchanged in the prostate. Testosterone administration to castrates induced expression of mRNA in the prostate but had no effect on liver. The data suggest that the steroid 5 alpha-reductase gene is differentially regulated by testosterone in androgen-responsive versus non-responsive tissues.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1988

A field evaluation of air sampling methods for TNT and RDX.

A comparative field study was conducted at an ammunition plant to evaluate the use of several sampling techniques for 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The objective was to compare the currently recommended combination Tenax-plus-filter tubes with the older, colorimetric diethylaminoethanol bubbler method which was in use in July 1950 when the American Conference of Governmental Industrial Hygienists (ACGIH) published their first Threshold Limit Values (TLVs). These values included a TLV of 1.5 mg/m3 for TNT. Regular Tenax tubes and 37-mm glass fiber filters also were tested since they also had been used by the United States Army for a number of years. The survey results showed fair agreement between the bubbler and tube methods in those instances where sufficient TNT was present to produce a measurable color in the diethylaminoethanol (DEAE). The glass fiber filters were not satisfactory for TNT retention, but the two types of tubes both were effective. The combination tubes were superior for RDX and appeared to be the best overall sampling medium.

Air↗
PubMed1986

Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit.

The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal with familial hypercholesterolemia, produces a mutant receptor for plasma low-density lipoprotein (LDL) that is not transported to the cell surface at a normal rate. Cloning and sequencing of complementary DNA's from normal and WHHL rabbits, shows that this defect arises from an in-frame deletion of 12 nucleotides that eliminates four amino acids from the cysteine-rich ligand binding domain of the LDL receptor. A similar mutation, detected by S1 nuclease mapping of LDL receptor messenger RNA, occurred in a patient with familial hypercholesterolemia whose receptor also fails to be transported to the cell surface. These findings suggest that animal cells may have fail-safe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues.

Amino Acid Sequence↗
PubMed1986

Evaluation of a new ammonia sampling and analytical procedure.

A new monitoring procedure is described for the collection of ammonia in the workplace environment. Sampling is conducted using air sampling tubes packed with beaded activated carbon coated with sulfuric acid. The low cationic background of this packing permits the analysis of samples to be performed by ion chromatography, a technique which offers considerable advantage over current methods used to analyze industrial hygiene samples for ammonia. Excellent precision and accuracy are demonstrated on samples collected from atmospheres containing 0.5, 1 and 2 times TLV levels of ammonia. The analytical detection limit is such that a concentration 0.05 of TLV may be detected in a 3-L air sample with less than one percent breakthrough found in a 24-L sample at TLV.

Air Pollutants, Occupational↗
PubMed1983

A gas chromatographic procedure for the determination of airborne MDI and TDI.

A method is described for the analysis of airborne diphenylmethane diisocyanate (MDI) and toluene diisocyanate (TDI) using gas-liquid chromatography. The diisocyanates are collected in acidic absorbing solution where they undergo hydrolysis, converted to the free diamines with caustic, and extracted into toluene. The diamines are derivatized in the toluene with heptafluorobutyric anhydride (HFBA). These derivatives are analyzed by GLC using an electron capture detector. The method has the advantages of specificity for the two diisocyanates, while demonstrating excellent accuracy and precision. A value one-tenth of the NIOSH recommended Occupational Health Standard for either compound may easily be detected in a 40 liter air sample.

Air↗