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P A Edwards

Publications and source records attributed to P A Edwards.

At least 91 records · Page 5Linked to original sources
PubMed1992

Genetic and dietary interactions in the regulation of HMG-CoA reductase gene expression.

Inbred strains of mice exhibit large genetic variations in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. A tissue-specific genetic variation between the strains BALB/c and C57BL/6, resulting in about 5-fold higher levels in hepatic reductase activity in strain C57BL/6, was examined in detail. The activity difference between these two strains could be explained entirely by differences in hepatic reductase mRNA levels. In genetic crosses, the variation segregated as a single major Mendelian element. Surprisingly, the mode of inheritance was recessive since F1 mice exhibited the BALB/c levels of enzyme activity. Despite the fact that the rates of hepatic sterol synthesis also differed between the strains by a factor of about five, the altered hepatic reductase expression did not significantly influence plasma lipoprotein levels. The response to a high cholesterol, high fat diet between the strains was remarkably different. Thus, in BALB/c mice, both hepatic reductase activity and mRNA levels were affected only slightly, if at all, by cholesterol feeding, while in strain C57BL/6 mice both were reduced more than 10-fold by cholesterol feeding. Several lines of evidence, including analysis of cis-acting regulatory elements, the nonadditive mode of inheritance, and genetic studies of the HMG-CoA reductase gene locus on mouse chromosome 13, support the possibility that the variation in reductase expression is not due to a mutation of the structural gene but, rather, is determined by a trans-acting factor controlling reductase mRNA levels. The variation provides a striking example, at the molecular level, of the importance of dietary-genetic interactions in the control of cholesterol metabolism.

Animals↗
PubMed1992

Hyperplasia of mouse mammary epithelium induced by expression of the Wnt-1 (int-1) oncogene in reconstituted mammary gland.

We have expressed the Wnt-1 (formerly int-1) oncogene in Balb/c mouse mammary epithelium in vivo, using a tissue reconstitution method in which primary cultures of mammary epithelial cells are infected with a retrovirus vector and then transplanted into mouse mammary fat pads from which the natural epithelium has been removed. Transplants carrying the Wnt-1 gene grew in a hyperplastic pattern, the duct epithelium showing abundant fine side-branches, but without development of clusters of alveoli. The hyperplasias were similar, but not identical, to transplants of normal epithelium in a mid-pregnant host. Transplants of epithelium that expressed Wnt-1 into mammary fat pads of male or ovariectomized females grew to form a similar three-dimensional pattern, but the extent of growth, and so presumably the rate of growth, was slower than in intact females, and there were no terminal end buds at the edges of the outgrowths. Thus, although Wnt-1 may enhance growth of epithelium in the male or ovariectomized-female environment, it does not restore the major mode of growth in the intact female, the extension of major ducts from terminal end buds. Normal epithelium showed no change in morphology when in close proximity to hyperplasia induced by Wnt-1, confirming the limited range of diffusion of Wnt-1 protein in vivo. Our results are consistent with the hypothesis that Wnt-1 acts principally by mimicking the signal that causes ducts to develop side-branches in pregnancy.

Animals↗
PubMed1991

Induction of mouse mammary tumours in a transplantation system by the sequential introduction of the myc and ras oncogenes.

A novel helper-free defective retrovirus containing the v-Ha-ras oncogene has been constructed and used to introduce the gene into primary mouse mammary epithelial cells already containing the v-myc oncogene. Transplantation of such doubly altered cells into cleared mammary fat pads led to the formation of mammary tumors within 6 to 8 weeks of transplantation. In a separate experiment, both oncogenes were simultaneously introduced to normal epithelium and once again tumours were formed. Neither oncogene alone gave a significant rate of tumour formation, although myc alone gave a reproducible hyperplasia as previously reported and ras alone gave occasional dysplastic or alveolar lesions. The results presented here demonstrate the progression of a normal cell through a hyperplastic intermediate to a tumour-forming cell in a versatile in vivo transplantation model.

Animals↗
PubMed1991

In vitro identification of a soluble protein:geranylgeranyl transferase from rat tissues.

The gamma subunit of mammalian trimeric G proteins has been shown previously to be modified in vivo on a cysteine residue situated at the carboxyl-terminal sequence-Cys-Ala-Ile-Leu-COOH by a 20-carbon prenyl moiety geranylgeranyl (Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S., and Sternweis, P. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5873-5877; Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K-K., Clarke, S., Gelb, M. H., and Glomset, J. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5866-5872). A biotinylated peptide acceptor comprising the eight carboxyl-terminal amino acids of the gamma subunit and tritiated geranylgeranyl diphosphate were utilized to monitor a protein:prenyl transferase activity in rat organs of varying age. The transferase activity was dependent upon the presence of divalent metal ions and maximal activity was achieved with either 1 mM ZnCl2 or 20 mM MgCl2. Activity was shown to be linear with respect to time, protein concentration, substrate concentration, and the pH optimum was 7.5. Protein:geranylgeranyl transferase activity was detected in all rat organs studied with the highest specific activity in brain S100. No activity was detected in the membrane fraction. The specific activity in brain, liver, kidney, and heart increased with age. Radioactivity incorporated into the peptide acceptor from both [1-3H]geranylgeranyl diphosphate and [5-3H]mevalonate by 21-day-old rat brain S100 was released by treatment with methyl iodide, and in both cases, analysis of the cleavage products by reversed phase high performance liquid chromatography showed a peak of radioactivity co-eluting with a geranylgeraniol standard which was well resolved from a farnesol standard. This indicated that the rat brain S100 contained not only the protein:geranylgeranyl transferase but also geranylgeranyl synthetase activity and that the peptide acceptor was specific for geranylgeranyl under the conditions tested.

Age Factors↗
PubMed1991

Transformation of a mammary epithelial cell line by the v-raf and v-mos oncogenes.

Defective retroviruses were used to investigate the effects of the oncogenes v-raf and v-mos on the behaviour of mammary epithelial cells. Cultures of the mammary epithelial cell lines #43 and NMuMG infected with either virus showed obvious areas of overgrowing cells, which were not seen when cells were infected with control viruses not containing an oncogene. Several clonal cell lines were derived from the areas of overgrowth on #43 cells. Most of these were tumorigenic on subcutaneous inoculation. In culture, several of the cloned lines showed marked morphological abnormalities, had altered rates of growth and saturation densities, and were able to clone in soft agar. Unexpectedly, one raf-containing and one mos-containing line grew more slowly in culture than did control or uninfected cells. Other lines showed only some of these changes. In the mos-containing lines the level of viral transcript corresponded approximately to the extent of transformation, whereas the raf-containing lines showed no such correlation.

Animals↗
PubMed1991

Testis-specific transcripts of rat farnesyl pyrophosphate synthetase are developmentally regulated and localized to haploid germ cells.

A rat farnesyl pyrophosphate (FPP) synthetase cDNA was used to study the expression of FPP synthetase mRNA levels during spermatogenesis in the rat. RNA blot analysis showed an increase in the level of FPP synthetase transcripts during postnatal development of rat testes. The increase is due to the appearance of longer testis-specific FPP synthetase transcripts as assayed by primer extension mapping. In situ hybridization analysis of adult rat testis sections with an FPP synthetase antisense probe showed that FPP synthetase mRNA levels are greatly enriched in the haploid round spermatidis at stages 7 to 8 of the seminiferous epithelium. These results show that FPP synthetase transcripts in testis are expressed at high levels in haploid male germ cells in a stage-specific manner.

Animals↗
PubMed1990

Quantitative structure-retention relationship studies of odor-active aliphatic compounds with oxygen-containing functional groups.

High-quality regression equations (R greater than 0.996) modeling the gas chromatographic retention indices of 115 odor-active compounds on stationary phases of different polarity are generated by using the ADAPT software system. Multiple linear regression techniques are used to describe the statistical relationship between the Kováts retention indices and structure-based molecular descriptors. These descriptors encode topological, geometrical, and electronic features of the molecules. The utility of several new descriptors encoding functions of partial atomic charge and solvent accessible surface area is demonstrated. Quantitative predictions of odor threshold values for a subset of these compounds containing the alcohol functional group are also calculated by using a similar methodology.

Hydrocarbons↗
PubMed1990

Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase.

The assembly of a polyisoprenoid side chain and its transfer to para-hydroxybenzoate are the first two steps of coenzyme Q biosynthesis. In yeast these reactions are catalyzed by hexaprenyl pyrophosphate synthetase and PHB:polyprenyltransferase, respectively. We have screened nine complementation groups of yeast coenzyme Q mutants for the activities of these two enzymes and found two strains deficient in either activity. The strain deficient in hexaprenyl pyrophosphate synthetase activity, C296-LH3, is complemented by the plasmid pG3/T1. When C296-LH3 was transformed with a shuttle vector containing a 2,187-base pair fragment from the genomic insert of pG3/T1, both glycerol growth and hexaprenyl pyrophosphate synthetase activity were restored. The activity of the latter enzyme was higher than that seen in wild-type yeast. The increase in activity could be attributed to a gene dosage effect of the multi-copy plasmid. A 1,419-base pair open reading frame encoding a 52,560-dalton protein was found on the genomic fragment. The size of the RNA transcript and the location of transcriptional initiation indicate that the entire open reading frame is contained within the mRNA. Comparison of the hexaprenyl pyrophosphate synthetase amino acid sequence with amino acid sequences from the related enzyme farnesyl pyrophosphate synthetase show the presence of three highly conserved domains. Within two of the domains is an aspartate-rich motif found invariantly in the amino acid sequences of farnesyl pyrophosphate synthetase from three species and the hexaprenyl pyrophosphate synthetase amino acid sequence reported here. These aspartic acid motifs may comprise binding sites for the allylic and homoallylic substrates. The hydrophobicity profiles of the hexaprenyl pyrophosphate synthetase sequence and the farnesyl pyrophosphate synthetase sequence from rat appear similar. Furthermore, the hydrophobicity correlation coefficient of the comparison of these two sequences indicate with a high degree of confidence (p less than 0.001) that the two proteins will fold into similar three-dimensional structures.

Alkyl and Aryl Transferases↗
PubMed1990

Isolation and sequence of the human farnesyl pyrophosphate synthetase cDNA. Coordinate regulation of the mRNAs for farnesyl pyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and 3-hydroxy-3-methylglutaryl coenzyme A synthase by phorbol ester.

We report the isolation and nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA, an enzyme in the cholesterogenic pathway. Partial cDNAs for the human farnesyl pyrophosphate synthetase were isolated by screening human hepatoma (HepG2) and placental cDNA libraries with the rat liver cDNA for farnesyl pyrophosphate synthetase as a probe. Anchored polymerase chain reaction was used to isolate the 5'-end of the cDNA. The nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA has high identity (86%) to the rat liver cDNA. Treatment of the human monocytic leukemia cell line THP-1 with phorbol esters led to 2--7-fold increases in mRNA concentrations for the three cholesterogenic enzymes, farnesyl pyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and HMG-CoA synthase within 5 h. Immunoprecipitation of radiolabeled cells demonstrated that there was a corresponding increase in the rate of synthesis of all three proteins. The addition of cycloheximide to cells also led to increases in the mRNA concentrations of the three enzymes. Treatment of cells with phorbol esters and cycloheximide resulted in superinduction of all three mRNAs; HMG-CoA synthase mRNA levels increased 35-fold, farnesyl pyrophosphate synthetase 17-fold, and HMG-CoA reductase 16-fold 5 h after treatment. The mRNA levels returned to pretreatment levels by 20 h. Cells were also preincubated in the presence of a lipoprotein-deficient fraction of serum plus mevinolin to induce the levels of the three mRNAs. Addition of phorbol esters and cycloheximide to these derepressed cells led to further increases in the mRNA levels for all three enzymes. These results are consistent with the hypothesis that THP-1 cells contain a short-lived negative transcription factor which regulates transcription of the FPP synthetase, HMG-CoA reductase, and HMG-CoA synthase genes. Phorbol esters also regulate these same genes, presumably by modifying a common negative transcription factor and/or by inducing a positive transcription factor(s).

Amino Acid Sequence↗
PubMed1990

Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA.

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

Amino Acid Sequence↗
PubMed1990

Computerized cataract detection and classification.

156 eyes of patients and normal volunteers were classified at the slit lamp into the following pure groups: normal (n = 50), nuclear (n = 39), cortical (n = 33) and PSC (n = 34). The eyes were photographed with the Topcon SL-45 Scheimpflug camera and the images scanned and processed to obtain one dimensional profiles through a 40 x 440 micron axial window. Of the 156 profiles, 90 were used as reference samples and were processed to obtain average profiles. The remaining 66 "unknown" profiles and each of the reference profiles, were classified into the four groups based on their distance from the average profiles in Euclidean space. The system was found to be very sensitive (98%) in detecting the presence of cataracts and specific (100%) in identifying normal, i.e. cataract negative lenses. In classifying pure cataracts into the various classes 98% of answers were correct.

Adult↗
PubMed1989

Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells.

Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.

Cell Line↗
PubMed1989

Developmental changes in the expression of genes involved in cholesterol biosynthesis and lipid transport in human and rat fetal and neonatal livers.

Cloned cDNAs encoding a number of enzymes involved in cholesterol biosynthesis as well as extracellular and intracellular lipid transport were used to compare the developmental maturation of these biologic functions in the fetal and neonatal rat and human liver. The results of RNA blot hybridization analyses indicate that steady-state levels of rat HMG-CoA synthase, HMG-CoA reductase and prenyl transferase mRNAs are highest in late fetal life and undergo precipitous (up to 80-fold) co-ordinate reductions immediately after parturition. These changes reflect the ability of the fetal rat liver to produce large quantities of cholesterol as well as the repression of this function during the suckling period in response to exogenous dietary cholesterol. Striking co-ordinate patterns of HMG-CoA synthase, reductase and prenyl-transferase mRNA accumulation were also observed in four extrahepatic rat tissues (brain, lung, intestine and kidney) during the perinatal period. The concentrations of all three mRNAs in the 8-week-old human fetal liver are similar to those observed throughout subsequent intrauterine development with less than 2-fold changes noted between the 8th through 25th weeks of gestation. Analysis of the levels of human apo AI, apo AII, apo B and liver fatty acid binding protein mRNAs during this period and in newborn liver specimens also indicated less than 2-3-fold changes. These observations suggest that the 8-week human liver has achieved a high degree of biochemical differentiation with respect to functions involved in lipid metabolism/transport which may be comparable to that present in 19-21 day fetal rat liver. Further analysis of human and rat fetal liver RNAs using cloned cDNAs should permit construction of a developmental time scale correlating hepatic biochemical differentiation to be constructed between these two mammalian species.

Animals↗
PubMed1989

Identification and regulation of a rat liver cDNA encoding farnesyl pyrophosphate synthetase.

CR39 is a cholesterol-repressible rat liver cDNA previously isolated by differential hybridization (Clarke, C.F., Tanaka, R.D., Svenson, K., Wamsley, M., Fogelman, A.M., and Edwards, P.A. (1987) Mol. Cell. Biol. 7, 3138-3146). To precisely identify the function of CR39 a fusion protein was constructed that contained the amino-terminal region of the bacterial protein anthranilate synthetase fused to the full length CR39 polypeptide. Affinity purified antisera directed against the fusion protein inactivated rat liver cytosolic prenyltransferase activity in vitro. In addition, affinity purified antisera made to purified chicken prenyltransferase cross-reacted with the fusion protein containing CR39. Rat hepatic prenyltransferase activity and enzyme mass were quantitated in animals fed diets or drugs known to alter endogenous cholesterol biosynthesis. Rats fed a diet supplemented with cholestyramine and mevinolin showed a 3.5-fold increase in activity and a 5.0-fold increase in mass of cytosolic prenyltransferase. A diet supplemented with cholesterol resulted in approximately a 4.0-fold decrease in hepatic enzyme activity and a 10-fold decrease in enzyme mass. Under these same dietary regimens the mass of prenyltransferase in the testes remained unchanged. We conclude that CR39 encodes the prenyltransferase of cholesterol biosynthesis, farnesyl pyrophosphate synthetase. Furthermore, in the liver this enzyme shows coordinate regulation with two other enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and 3-hydroxy-3-methylglutaryl-CoA synthase, in response to cholesterol feeding and hypocholesterolemic drugs.

Amino Acid Sequence↗
PubMed1989

The association of posterior capsular lens opacities with bilateral acoustic neuromas in patients with neurofibromatosis type 2.

This study of 47 patients from 11 families with neurofibromatosis type 2 (NF 2) confirms our previously reported association between posterior capsular cataract and NF 2. A highly significant statistical association was found between the presence of posterior capsular lens opacities and the presence of NF 2 as determined by magnetic resonance imaging or pathologic diagnosis. This association was not present for other types of lens opacities that could be the result of age-related or nonspecific changes. When considering the diagnosis of NF 2, this finding now makes it essential to use a careful dilated biomicroscopic examination of the lens to evaluate known, suspected, or at-risk individuals for this potentially early associated manifestation.

Cataract↗
PubMed1989

Dispersed family of human genes with sequence similarity to farnesyl pyrophosphate synthetase.

Prenyltransferases are a group of enzymes involved in the biosynthesis of both sterol and nonsterol isoprene compounds. Somatic cell hybrid studies and in situ hybridization show that the human genome contains five distinct loci that hybridize to the cDNA for the enzyme farnesyl pyrophosphate synthetase (FPS), a prenyltransferase that catalyzes the synthesis of an intermediate common to both the sterol and the nonsterol branches of the isoprene biosynthetic pathway. The loci identified in this report may correspond to unique prenyltransferase genes related to FPS or to pseudogenes. The loci mapped have been identified as farnesyl pyrophosphate synthetase-"like"-1 (FPSL-1) on chromosome 1q24-31, FPSL-2 on chromosome 7, FPSL-3 on chromosome 14, FPSL-4 on chromosome 15q14-q21, and FPSL-5 on chromosome Xq21-22. Multiple copies of sequences similar to those of FPS are also present in both the mouse and the rat.

Animals↗
PubMed1989

A new monoclonal antibody to a cell-surface antigen that distinguishes luminal epithelial and myoepithelial cells in the rat mammary gland.

A monoclonal antibody (25.5) has been produced that recognises luminal epithelial cells of the rat mammary gland. This antibody together with monoclonal anti-CALLA antibodies, which react with mammary myoepithelial cells, has been used in biochemical, immunocytochemical and flow cytometric studies. Antibody 25.5 bound to proteins of molecular weight 70K and 25K (K = 10(3) Mr) in both the rat milk fat globule membrane and in single cell suspensions prepared from the virgin adult rat mammary gland. Anti-CALLA antibody (J5), recognised a 93-100K protein in the gland extracts, which co-electrophoresed with the CALLA/CD-10 antigen from NALM-6 acute lymphoblastic leukaemia cell line. Antibody 25.5 bound to the luminal surface of rat mammary epithelial cells at all stages of development from neonatal through to pregnancy, lactation and involution. CALLA immunoreactive staining has previously been shown on basally located presumptive myoepithelial cells at all stages of development. Flow cytometric analyses demonstrated that 25.5 and anti-CALLA antibodies stained independent cell populations in suspensions of single cells prepared from purified epithelial elements from the mammary gland of adult virgin rat.

Animals↗