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P A Edwards

Publications and source records attributed to P A Edwards.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of a 6-base pair element involved in the sterol-mediated transcriptional regulation of farnesyl diphosphate synthase.

Previous studies identified a 115-base pair (bp) region of the farnesyl diphosphate (FPP) synthase promoter which is involved in the transcriptional regulation of this gene by sterols (Spear, D. H., Kutsunai, S. Y., Correll, C. C., and Edwards, P. A. (1992) J. Biol. Chem. 267, 14462-14469). In the current study we fused a 117-bp fragment, containing this region of interest, upstream of the heterologous minimal promoter of the herpes simplex virus thymidine kinase gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Chinese hamster ovary (CHO) cells were stably transfected with this fusion gene and incubated in the absence or presence of sterols. Analysis of CAT mRNA by primer extension indicated that transcription of the fusion gene was under sterol-mediated control. Thus, when cellular sterols were present, the CAT mRNA levels were reduced 2-4-fold. To further localize the FPP synthase sterol-responsive element(s), additional promoter-reporter gene constructs containing either deletions or mutations were constructed and transfected into CHO or CV-1 cells. These studies localized a 6-bp region (ATTGGC) that is required for both transcriptional induction in the absence of sterols and transcriptional repression in the presence of sterols. Gel shift and footprinting analyses demonstrated that nuclear proteins isolated from CHO cells bound to six distinct regions of the promoter between nucleotides -293 to -47. Taken together, these results further define both the cis-acting elements controlling normal transcription of the FPP synthase gene and identify a novel sequence involved in sterol regulation.

Alkyl and Aryl Transferases↗
PubMed1994

Identification of farnesol as the non-sterol derivative of mevalonic acid required for the accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

The degradation of the microsomal enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is highly regulated and is dependent on both a sterol and non-sterol derivative of mevalonic acid (MVA). We recently proposed that the non-sterol component is derived from farnesyl diphosphate (FPP), presqualene pyrophosphate, or squalene (Correll, C. C. and Edwards, P. A. (1994) J. Biol. Chem. 269, 633-638). In the current study, we have used digitonin-permeabilized cells to further define this MVA-derived non-sterol component required for the regulated degradation of HMG-CoA reductase. The addition of either FPP or farnesol to digitonin-permeabilized cells resulted in a rapid and dose-dependent degradation of HMG-CoA reductase. The effect of FPP, but not farnesol, was blocked by the phosphatase inhibitor sodium fluoride. The enhanced degradation of HMG-CoA reductase in permeabilized cells specifically required farnesol, since the addition of any of the structurally related isoprenoids geraniol, geranyl diphosphate, geranylgeranyl diphosphate, nerolidol, or all-cis-farnesol, or of the non-sterol squalene to the permeabilized cells did not stimulate enzyme degradation. The present studies demonstrate for the first time that the accelerated degradation of HMG-CoA reductase can be initiated in vitro. Further, since farnesol is shown to be specifically required for the enhanced degradation of the enzyme in vitro, we propose that this isoprenoid alcohol is important in this process in intact cells.

Animals↗
PubMed1994

Farnesyl-diphosphate synthase is localized in peroxisomes.

In this study, we have investigated the subcellular localization of farnesyl-diphosphate synthase (FPP synthase). FPP synthase produces FPP, which is utilized in the synthesis of squalene, cholesterol, farnesylated and geranylgeranylated proteins, dolichols, coenzyme Q, and the isoprenoid moiety of heme a. This enzyme is found in the 100,000 x g supernatant fraction of cells or tissues and has been considered to be a cytoplasmic protein. In this study, analysis of FPP synthase activity and protein in fractionated rat liver together with immunofluorescent and immunoelectron microscopy studies demonstrated unequivocally that FPP synthase is largely localized in peroxisomes. These data, in combination with the previous observation that mevalonate kinase is predominantly localized in peroxisomes, suggest that peroxisomes are the major site of synthesis of FPP from mevalonate. We also demonstrate that in liver tissue obtained from patients with peroxisomal deficiency diseases (Zellweger syndrome and neonatal adrenoleukodystrophy), the activities of five enzymes involved in isoprenoid synthesis, namely mevalonate kinase, phosphomevalonate kinase, mevalonate-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, and FPP synthase, are significantly reduced, consistent with a peroxisomal localization of these enzymes.

Adrenoleukodystrophy↗
PubMed1994

Mevalonic acid-dependent degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo and in vitro.

The microsomal enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to rapid degradation when cells are incubated with sterols or mevalonic acid (MVA). It has been shown that this rapid degradation is dependent upon both a sterol and another MVA-derived metabolite (Nakanishi, M., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 258, 8929-8937). In the current study, inhibitors of the isoprene biosynthetic pathway were used to define further this mevalonic acid derivative involved in the accelerated degradation of HMG-CoA reductase. The accelerated degradation of HMG-CoA reductase in met-18b-2 cells, which is induced by the addition of MVA, was inhibited by the presence of the squalene synthase inhibitor, zaragozic acid/squalestatin, or the squalene epoxidase inhibitor, NB-598. Accelerated degradation of HMG-CoA reductase was observed when NB-598-treated cells were incubated with both MVA and sterols. In contrast, the addition of MVA and sterols to zaragozic acid/squalestatin-treated cells did not result in rapid enzyme degradation. This MVA- and sterol-dependent degradation of HMG-CoA reductase persisted in cells permeabilized with reduced streptolysin O. Finally, the selective degradation of HMG-CoA reductase was also observed in rat hepatic microsomes incubated in vitro in the absence of ATP and cytosol. We conclude that the MVA-derived component that is required for the accelerated degradation of HMG-CoA reductase is derived from farnesyl disphosphate and/or squalene in the isoprenoid biosynthetic pathway. We propose that this component has a permissive effect and does not, by itself, induce the degradation of HMG-CoA reductase. We also conclude that the degradation of HMG-CoA occurs in the endoplasmic reticulum, and, once the degradation of HMG-CoA reductase has been initiated by MVA and sterols, all necessary components for the continued degradation of HMG-CoA reductase reside in the endoplasmic reticulum.

Animals↗
PubMed1994

Using available resources to implement research.

Nurses practicing in hospital settings are in a unique position to become involved in research. Day-to-day contact with a variety of patients offers the ideal opportunity to launch an investigation based on actual or potential nursing problems. Often these nurses are not aware of the resources that could help them implement a study that would provide valuable data to staff and others who work with a similar patient population. The purpose of this article is to aid staff nurses in initiating research by discussing resources that may be available within their immediate geographical area.

Consultants↗
PubMed1994

Developing care plans for psychosocial nursing diagnoses.

In 1992, the Nursing Department Documentation Committee at Spaulding Rehabilitation Hospital decided to revise the process of and forms for documenting nursing diagnoses. A sub-committee developed a format for standardized nursing care plans and piloted its use on two patient care units. The unit nursing staffs selected a number of common nursing diagnoses and, using the new format, developed standardized nursing care plans for them. After refining the process based on this pilot, the development of care plans was extended to all patient care units. A collaborative partnership was then formed between interested Primary Nurses, the Psychiatric Clinical Nurse Specialist and the Nursing Practice Coordinator to focus on developing psychosocially-oriented care plans as well as physically-oriented plans. Although psychosocial diagnoses are less frequently identified in the assessment process and included in nursing care planning, they are still very important for providing comprehensive care. This article describes the process of developing and implementing psychosocial care plans. This article also describes the components of the standardized nursing care plans developed for four psychosocial nursing diagnoses.

Humans↗
PubMed1994

Alterations of the growth characteristics of the fibroblast cell line C3H 10T1/2 by members of the Wnt gene family.

Retroviral vectors were used to introduce members of the Wnt gene family into the embryonic fibroblast cell line C3H 10T1/2. In contrast to previous reports where no effect of Wnt genes on fibroblasts was seen, we found that the expression of Wnt-1, Wnt-6 and Wnt-7b altered the appearance of the cells when maintained at confluence. The cells were smaller and grew to a higher density than cells containing a control retrovirus or cells expressing Wnt-4 or Wnt-5b. Detailed analysis of growth characteristics of polyclonally infected cultures demonstrated that the Wnt-1, Wnt-6 and Wnt-7b expressing cells grew to a higher density in 5% fetal calf serum than control or Wnt-5b expressing cells. Wnt-4 expressing cells grew to a marginally higher density than control cells. In 0.5% serum, growth of the Wnt-1, Wnt-6 and Wnt-7b containing cells appeared to be inhibited. No growth in soft agar was observed for either control cells or Wnt expressing cells. We conclude that at least some mesenchymal cells can respond to Wnt gene products. Our results are also the first report of biological effects of Wnt-6 and Wnt-7b.

Animals↗
PubMed1993

Effect of site-directed mutagenesis of conserved aspartate and arginine residues upon farnesyl diphosphate synthase activity.

All polyprenyl synthases catalyze the condensation of the allylic substrate, isopentenyl diphosphate, with a specific homoallylic diphosphate substrate. Polyprenyl synthases from Homo sapiens, Ratus rattus, Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and Erwinia herbicola contain two conserved "aspartate-rich domains" (Ashby, M.N., and Edwards, P.A. (1992) J. Biol. Chem. 267, 4128-4136). In order to determine the importance of these domains in catalysis, the conserved aspartates or arginines in domains I and II of rat farnesyl diphosphate synthase were individually mutated to glutamate or lysine, respectively. The putative "active site" arginine (Brems, D.N., Breunger, E., and Rilling, H. C. (1981) Biochemistry 20, 3711-3718) was mutated to lysine. Each mutant enzyme was overexpressed in E. coli and purified to apparent homogeneity. Detailed kinetic analyses of the wild type and mutant enzymes indicated that mutagenesis of Asp104, Asp107, Arg112, Arg113, and Asp243 resulted in a decreased Vmax of approximately 1000-fold compared to wild type. However, no significant change in the Km values for either the isopentenyl diphosphate or geranyl diphosphate substrate were observed. The results strongly suggest that these amino acids, and to a lesser extent Asp244, are involved in either the condensation of isopentenyl diphosphate and geranyl diphosphate to form farnesyl diphosphate and/or the release of the farnesyl diphosphate product from farnesyl diphosphate synthase. The conservation of these amino acid residues in different enzymes from several species suggests that these domains play a similar role in other polyprenyl synthases.

Alkyl and Aryl Transferases↗
PubMed1993

Minimum views required to characterize cataracts when using the Scheimpflug camera.

We performed Scheimpflug slit lamp photography and computerized image analysis on 20 normal and 25 cataractous lenses using 18 slit images for each lens taken 10 degrees apart. The data gathered from the normals served as the reference to estimate the accuracy of representation of the cataracts by the least number of views (18 and less) using a Fourier interpolative algorithm. Using the error obtained with one view for the normals, our study suggests that the minimum number of views necessary for adequate characterization is two for cortical cataracts, two for nuclear cataracts, and six for posterior subcapsular cataracts. This information will be useful in longitudinal studies of cataracts, since most researchers presently use only one view, which may be adequate for normals but not for cataractous lenses. We found the Fourier interpolative algorithm useful in estimating the minimum views required for the current method of analyzing Scheimpflug images, and it can be easily applied to other similar images.

Algorithms↗
PubMed1993

Induction of epithelial abnormalities that resemble human breast lesions by the expression of the neu/erbB-2 oncogene in reconstituted mouse mammary gland.

We have developed a transplantation system that allows us to introduce oncogenes into mouse mammary epithelial cells in culture and then to reconstitute an epithelial tree in vivo from the genetically altered cells. Introduction of the neu oncogene, a transforming homologue of the human proto-oncogene c-erbB-2, produced a variety of abnormal patterns of epithelial growth, many of which resembled lesions found in human breasts. In four of 43 oncogene-bearing glands, areas of ductal carcinoma in situ were found, an abnormality previously observed in transgenic neu-bearing mice. Six glands developed localized areas of dense stroma containing excess ductal structures comprised of mildly hyperplastic epithelium. These areas resembled the human breast lesion termed sclerosing adenosis. Other glands developed hyperplastic epithelium, sometimes with multilayering of the cells and/or atypical changes such as abnormally large nuclei. In human breasts such lesions would be termed mild or atypical hyperplasia. In all the abnormal areas examined, levels of neu protein above background level were detected by immunohistochemistry. Some staining was localized to membranes (as observed in ductal carcinoma in situ in humans) but cytoplasmic staining was also common in the lesions induced in mice by the neu oncogene. The range of abnormalities seen in the reconstituted glands carrying the neu oncogene suggests that the matching lesions in the human breast may be stages on one pathway to tumour development.

3T3 Cells↗
PubMed1993

Tissue reconstitution models of breast cancer.

Oncogenes have been expressed in mammary epithelium by reconstituting epithelium in vivo from mammary cells. Genetically manipulated primary cultures are transplanted into a mammary fat pad from which the natural epithelium has been removed, where they reform an epithelium in which a few cells express the oncogene. Genes can be expressed in other tissues in a similar way. A wide variety of oncogenes have a clearly observable effect on the pattern of growth of mammary epithelium. Expression of individual oncogenes usually produces stable, characteristic patterns of abnormal growth that can be regarded as preneoplastic states. Different oncogenes produce a very diverse variety of such growth patterns, by altering branching pattern, inducing formation of alveoli, causing epithelium to multilayer and/or altering hormone dependence. Myc and wnt1 seem to enable cells to overgrow neighbouring normal cells, suggesting that they promote clonal expansion, whereas others give focal lesions. Oncogene co-operation can be studied by introducing further oncogenes into preneoplastic epithelium, for example the introduction of ras into epithelium that already expresses activated myc gives tumours. The effects of both neu/c-erbB2 and myc on mouse mammary epithelium may mimic events in human breast, encouraging the hope that this will prove a way to model human breast cancer. The tissue reconstitution approach promises to reconstruct tumour development in more detail than the transgenic systems are able to, showing the development of focal lesions, the restraining effects of normal on transformed cells and the expansion of clones of hyperplastic cells at the expense of their normal neighbours.

Animals↗
PubMed1993

Aging studies on normal lens using the Scheimpflug slit-lamp camera.

PURPOSE: To study the changes in density and thickness in normal lenses related to aging, and to study changes in anterior chamber depth related to aging. METHODS: Eighty nine normal volunteers (ages 9-80 yr) were examined and their eyes were photographed to obtain Scheimpflug photographs. The images were digitized and linear densitometry was performed, dividing the lens into five areas: posterior capsular (area 1), posterior cortical (area 2), nuclear (area 3), anterior cortical (area 4), and anterior capsular (area 5). Total lens thickness and anterior chamber depth were similarly measured for 90 normal eyes from the densitometry profiles. These were correlated with age. RESULTS: There was a strong positive correlation between increasing age and the density in all lens areas (area 2: r = 0.805; P < 0.0001; area 3: r = 0.836, P < 0.0001; area 4: r = 0.767, P < 0.0001; and area 5: r = 0.319, P < 0.0023), except the posterior capsular area, where correlation was negative (area 1: r = -0.426; P < 0.0001). In addition, there was a significant correlation between age and overall lens thickness (r = 0.756; P < 0.0001), thickness of nucleus (r = 0.543; P < 0.0001), and cortex (r = 0.632; P < 0.0001), and a negative correlation with anterior chamber depth (r = -0.513, P < 0.0001). CONCLUSION: This report shows human lens changes in density and thickness correlated with aging using Scheimpflug photography and image analysis techniques. The results will aid future development of systems for automated detection, classification, and monitoring of human cataracts, as well as other anterior segment disorders.

Adolescent↗
PubMed1992

Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity.

Comparison of the farnesyl diphosphate (FPP) synthase amino acid sequences from four species with amino acid sequences from the related enzymes hexaprenyl diphosphate synthase and geranylgeranyl diphosphate synthase show the presence of two aspartate rich highly conserved domains. The aspartate motif ((I, L, or V)XDDXXD) of the second of those domains has homology with at least 9 prenyl transfer enzymes that utilize an allylic prenyl diphosphate as one substrate. In order to investigate the role of this second aspartate-rich domain in rat FPP synthase, we mutated the first or third aspartate to glutamate, expressed the wild-type and mutant enzymes in Escherichia coli, and purified them to apparent homogeneity using a single chromatographic step. Approximately 12 mg of homogeneous protein was isolated from 120 mg of crude bacterial extract. The kinetic parameters of the purified wild-type recombinant FPP synthase containing the DDYLD motif were as follows: Vmax = 0.84 mumol/min/mg; GPP Km = 1.0 microM; isopentenyl diphosphate (IPP) Km = 2.7 microM. Substitution of glutamate for the first aspartate (EDYLD) decreased the Vmax by over 90-fold. The Km for IPP increased, whereas the Km for GPP remained the same in this D243E mutant. Substitution of glutamate for the third aspartate (DDYLE) did not result in altered enzyme kinetics in the D247E mutant. These results suggest that the first aspartate in the second domain is involved in the catalysis by FPP synthase.

Alkyl and Aryl Transferases↗
PubMed1992

Molecular cloning and promoter analysis of the rat liver farnesyl diphosphate synthase gene.

The isolation and characterization of the rat genomic clone encoding the cholesterogenic enzyme farnesyl diphosphate (FPP) synthase is reported. The gene is localized on a 15-kilobase (kb) genomic fragment, spans approximately 12 kb and contains eight exons. Sequences containing from 3.9 kb to 132 base pairs (bp) of the putative promoter were joined to the coding region of the bacterial reporter gene chloramphenicol acetyltransferase (CAT). The CAT activities or CAT mRNA levels of the hybrid genes were determined following either transient transfections into human hepatoma HepG2 cells or stable transfections into Chinese hamster ovary cells. The transient transfections identified a 319-bp fragment that was required for a 4-fold induction in the absence of sterols. Sequence analysis of this region showed it contained five potential copies of the sterol regulatory element (SRE-1) (Smith, J.R., Osborne, T.F., Brown, M.S., Goldstein, J.L., and Gil, G. (1988) J. Biol. Chem. 263, 18480-18487) previously identified in the promoters of the 3-hydroxy-3-methyl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein receptor genes. Further mutational and deletion analysis of the FPP synthase promoter-CAT constructs followed by stable transfection and primer extension of the CAT mRNA levels indicated that these potential SRE-1 regulatory elements were not involved in the sterol-mediated transcriptional regulation of the gene. Our analyses have identified a 115-bp region that is required for the transcriptional induction of FPP synthase in the absence of sterols. These results suggest that the FPP synthase gene may be regulated at the transcriptional level by a different mechanism than other sterol regulated genes.

Alkyl and Aryl Transferases↗
PubMed1992

COQ2 is a candidate for the structural gene encoding para-hydroxybenzoate:polyprenyltransferase.

Coenzyme Q functions as a lipid-soluble electron carrier in eukaryotes. In Saccharomyces cerevisiae, the enzymes responsible for the assembly of the polyisoprenoid side chain and subsequent transfer to para-hydroxybenzoate (PHB) are encoded by the nuclear genes COQ1 and COQ2, respectively. Yeast mutants defective in coenzyme Q biosynthesis are respiratory defective and provide a useful tool to study this non-sterol branch of the isoprenoid biosynthetic pathway. We isolated a 5.5-kilobase genomic DNA fragment that was able to functionally complement a coq2 strain. Additional complementation analyses located the COQ2 gene within a 2.1-kilobase HindIII-BglII restriction fragment. Sequence analyses revealed the presence of a 1,116-base pair open reading frame coding for a predicted protein of 372 amino acids and a molecular mass of 41,001 daltons. The amino acid sequence exhibits a typical amino-terminal mitochondrial leader sequence and six potential membrane-spanning domains. Primer extension and Northern analyses indicate the gene is transcriptionally active. Transformation of a coq2 strain with the 2.1-kilobase HindIII-BglII genomic restriction fragment on a multicopy plasmid restores PHB:polyprenyltransferase activity to wild-type levels. Disruption of the chromosomal COQ2 gene indicates the gene is not essential for viability, yet is required for PHB:polyprenyltransferase activity and respiratory function. In addition, the deduced amino acid sequence of PHB:polyprenyltransferase contains a putative allylic polyprenyl diphosphate-binding site. The presence of this aspartate-rich domain in a number of functionally distinct proteins which utilize polyprenyl diphosphate substrates is reported.

Alkyl and Aryl Transferases↗
PubMed1992

Calcium ionophore treatment impairs the sterol-mediated suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, and farnesyl diphosphate synthetase.

We report that the sterol-mediated suppression of the mRNA levels of three cholesterogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and farnesyl diphosphate (FPP) synthetase is partially overcome by the calcium ionophore A23187. Addition of A23187 to the human monocytic leukemia cell line THP-1 in the presence of fetal calf serum led to rapid increases in mRNA concentration of up to 40-fold for HMG-CoA synthase and 15-fold for HMG-CoA reductase with little or no change in FPP synthetase mRNA levels. Treatment of HepG2 cells with A23187 resulted in approximately 2-4-fold increases in the mRNA levels for these three enzymes. The increases in HMG-CoA synthase and HMG-CoA reductase mRNAs were maximal after treatment of THP-1 cells with 10 micrograms/ml A23187 for 3 h. The stimulation was blocked by actinomycin D but not by cycloheximide treatment. Ionophore treatment had no effect on the half-lives of the mRNAs for HMG-CoA reductase and HMG-CoA synthase. Surprisingly, the addition of A23187 to THP-1 cells incubated in the presence of 25-hydroxycholesterol and mevalonic acid also led to significant increases in the mRNA levels for HMG-CoA reductase and HMG-CoA synthase. Finally, the stimulation of these mRNA levels by A23187 was reduced in cells in which protein kinase C had been inactivated by preincubation of the cells with a phorbol ester. Taken together, these data suggest that A23187 treatment results in increased transcription of HMG-CoA reductase, HMG-CoA synthase, and, in some cell types, FPP synthetase by a mechanism that does not involve de novo protein synthesis. We speculate that A23187 treatment results in the modification of a trans-acting factor(s) which is common for the transcription of all these genes.

Alkyl and Aryl Transferases↗
PubMed1992

Mechanisms controlling competence gene expression in murine fibroblasts stimulated with minimally modified LDL.

Mildly oxidized low density lipoprotein (minimally modified low density lipoprotein [MM-LDL] is capable of inducing gene expression in cells of the artery wall. In this study, we investigated the mechanisms that control the mRNA expression of JE, KC, c-myc, and c-fos in quiescent mouse L-cell fibroblasts stimulated with MM-LDL. The data demonstrate that MM-LDL induces increases greater than or equal to 20-fold in the levels of transcripts of these genes within 15-60 minutes. Of the four genes examined, JE and KC mRNA showed the greatest response to MM-LDL. The pattern of induction by MM-LDL is distinct from that observed in fibroblasts stimulated with serum, a known inducer of these genes. Treatment with cycloheximide (10 micrograms/ml) did not block the MM-LDL-induced increase in the mRNA levels of these genes. The increase of JE and KC mRNA levels in response to MM-LDL could be blocked by treatment with actinomycin D (5 micrograms/ml). In nuclear runoff studies, MM-LDL increased the transcription rate of JE and KC at 4 hours by 13-fold and fivefold, respectively. Small but reproducible stimulations of c-fos and c-myc transcription by MM-LDL were also observed. In addition, the half-life of JE mRNA was increased after addition of MM-LDL to fibroblasts, suggesting that the MM-LDL-induced accumulation of these mRNAs might be accomplished by both transcriptional and posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗