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D A Brenner

Publications and source records attributed to D A Brenner.

At least 127 records · Page 7Linked to original sources
PubMed1993

Identification of c-fos-responsive elements downstream of TAR in the long terminal repeat of human immunodeficiency virus type-1.

Activation of HIV-1 requires the binding of host cell transcription factors to cis elements in the proviral long terminal repeat (LTR). This study identifies c-fos-responsive sequence motifs in the U5 transcribed noncoding leader sequences downstream of the viral transactivator responsive (TAR) element. These DNA sequence motifs are the most downstream regulatory elements described thus far in the HIV-1 LTR. Functional studies, using human colon epithelial cell lines, demonstrate that the downstream elements are transactivated by expression of the c-fos protooncogene and can transmit PMA and TNF alpha activation signals to the viral LTR. Moreover, the c-fos-responsive elements mediate HIV-1 LTR transcription independent of Tat and the NF kappa B-binding enhancer element. Nuclear extracts of colon epithelial cells form distinct gel mobility shift complexes with the c-fos-responsive elements. These complexes comigrate with a gel shift complex formed on a classical CRE oligonucleotide and are competed by CRE oligonucleotides. These data indicate that the HIV-1 LTR contains previously unrecognized functional DNA cis-regulatory elements downstream of TAR in the transcribed noncoding 5' leader sequence and suggest that early response genes such as c-fos play a role in the activation of HIV-1 gene expression.

Base Sequence↗
PubMed1993

Mithramycin selectively inhibits collagen-alpha 1(I) gene expression in human fibroblast.

The products of the collagen-alpha 1(I) and -alpha 2(I) genes form the triple helical molecule collagen type I, which constitutes the major ECM protein in tissue fibrosis. The collagen-alpha 1(I) gene is mainly transcriptionally regulated, and its promoter activity depends on the interaction of the transcription factors NF-I and Sp1 with a tandem repeat of evolutionary conserved NF-I/Sp1 switch elements. An increased affinity of Sp1 to these elements has been observed in experimental liver fibrosis. Here, we demonstrate that the DNA binding drug mithramycin displays a high affinity binding to the GC-rich elements in the collagen-alpha 1(I) promoter as measured by DNAse I protection and gel retardation assays. Mithramycin interferes with Sp1 but not with NF-I binding to these sites. At a concentration of 100 nM, mithramycin efficiently reduces basal and TGF-beta-stimulated alpha 1(I) gene expression in human primary fibroblasts. The transcriptional activity and mRNA steady state levels of other genes, including the collagenase gene, as well as the growth rate of fibroblasts remained unchanged on exposure to this drug. Taken together, our results indicate that the transcriptional activity of the type I collagen gene highly depends on its GC-rich regulatory elements, and further, that these elements can be differentially blocked, thereby changing the balance between ECM structural and degrading gene activities in human fibroblasts.

Blotting, Northern↗
PubMed1993

Stimulation of the collagen alpha 1 (I) endogenous gene and transgene in carbon tetrachloride-induced hepatic fibrosis.

Cirrhosis is characterized by a marked increase in the deposition of type I collagen and in the expression of the type I collagen genes alpha 1(I) and alpha 2(I). Although alpha 1(I) gene regulation has been extensively studied in cultured cells, these results may not be applicable to hepatic fibrogenesis in vivo. Therefore the regulation of the alpha 1(I) endogenous gene and an alpha 1(I) transgene was studied in a transgenic mouse model that has a single copy of a human alpha 1(I) gene segment containing the structural gene and 1.6 Kb of 5' DNA and 20 Kb of 3' DNA. To initiate hepatic fibrogenesis, we treated mice with the hepatotoxin carbon tetrachloride, either in a single dose or in biweekly doses for a period of 3 to 8 wk. Subsequently, hepatic alpha 1(I) messenger RNA levels were determined by a species-specific RNase protection assay. Carbon tetrachloride injections coordinately increased the messenger RNA levels of the alpha 1(I) endogenous gene and the transgene, both immediately and after 8 wk. These experiments demonstrate that this alpha 1(I) transgene fragment contains information sufficient for appropriate basal and carbon tetrachloride-stimulated hepatic expression. They further demonstrate that sufficient homology exists between the human and mouse regulatory elements for the recognition of human cis-acting elements by mouse trans-acting factors. Thus transgenic mice provide a unique model in which to characterize the collagen alpha 1(I) regulatory elements that are required in vivo for pathophysiological responses.

Animals↗
PubMed1992

Differential expression of the transfected liver-specific alpha 1-inhibitor III gene in normal hepatocytes and hepatoma cells in culture.

Normal and malignant hepatocytes were transfected during log phase culture with a nested series of DNA plasmids containing 5'-flanking regions of the rat liver-specific acute phase plasma proteinase alpha 1-inhibitor III (alpha 1 I3) gene. Under these conditions, luciferase reporter gene expression in primary adult rat and mouse hepatocytes was 10-fold higher than luciferase expression in hepatoma lines (human HepG2 and Hep3B; rat FAZA). Optimal expression in primary rat hepatocytes required regions stretching 2214 bp 5'-upstream of the transcription start site. Shorter 5'-flanking sequences were optimal for expression in hepatoma cells (-1025 and -186 bp for rat and human lines, respectively) and primary mouse hepatocytes (-225 bp). In contrast, regions from -186 to -225 bp drove luciferase expression in primary rat hepatocytes, but only 20-75% of optimal levels. Qualitative differences were unaccounted for by non-equivalent uptake of plasmid DNA, suggesting that tissue specific gene expression is regulated differently in normal and malignant cells, and with apparent species specificity.

Acute-Phase Proteins↗
PubMed1992

A molecular defect in human protoporphyria.

Protoporphyria is generally an autosomal dominant disease that is characterized clinically by photosensitivity and hepatobiliary disease and that is characterized biochemically by elevated protoporphyrin levels. The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is deficient in all tissues of patients with protoporphyria. In this study, sequencing of ferrochelatase cDNAs from a patient with protoporphyria revealed a single point mutation in the cDNAs resulting in the conversion of a Phe(TTC) to a Ser(TCC) in the carboxy-terminal end of the protein, F417S. Further, the human ferrochelatase gene was mapped to chromosome 18q21.3 by chromosomal in situ suppression hybridization. Finally, expression of recombinant ferrochelatase in Escherichia coli demonstrated a marked deficiency in activity of the mutant ferrochelatase protein and of mouse-human mutant ferrochelatase chimeric proteins. Therefore, a point mutation in the coding region of the ferrochelatase gene is the genetic defect in some patients with protoporphyria.

Amino Acid Sequence↗
PubMed1991

Cloning of murine ferrochelatase.

Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme. The activity of ferrochelatase is deficient in the inherited disease protoporphyria. In this study, murine ferrochelatase cDNAs were obtained by screening cDNA libraries with an oligonucleotide probe. The derived amino acid sequence of murine ferrochelatase has 47% identity with the recently cloned Saccharomyces cerevisiae ferrochelatase, but it is not significantly similar to other published sequences. Results of Southern blotting are consistent with a single murine ferrochelatase gene, while Northern blotting demonstrates two ferrochelatase transcripts in all tissues examined. The ferrochelatase protein and mRNAs have different relative concentrations in different tissues. The cloning of murine ferrochelatase cDNAs provides the basis for future studies on ferrochelatase gene expression and on the identification of the molecular defect in protoporphyria.

Amino Acid Sequence↗
PubMed1991

Ascorbic acid stimulation of collagen biosynthesis independent of hydroxylation.

Ascorbic acid stimulates collagen gene expression in cultured fibroblasts but mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Because another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts, we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mmol/L) induced lipid peroxidation and stimulated collagen alpha 1(I) gene transcription in cultured human fibroblasts. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 mumol/L) or methylene blue (10 mumol/L) prevented the stimulation of collagen gene expression. Addition of malondialdehyde (200 mumol/L), a product of lipid peroxidation, to cultured human fibroblasts also increased two- to threefold collagen production and procollagen alpha 1(I) mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes, and this step may be necessary for stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.

Ascorbic Acid↗
PubMed1991

HIV-1 infection and expression in human colonic cells: infection and expression in CD4+ and CD4- cell lines.

Three human colonic epithelial cell lines, SW620, HT29, and T84, were characterized with respect to HIV-1 infection and gene expression. SW620 and HT29, but not T84, could be infected with HIV-1. CD4 messenger RNA and its protein product were identified in SW620 cells but not in HT29 or T84 cells. Anti-CD4 antibody blocked infection of SW620 cells but had no effect on infection of HT29 cells. In SW620 and HT29 cells transfected with the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, an intact HIV-1 enhancer element was required for stimulation of CAT activity by tumor necrosis factor alpha (TNF alpha) and phorbol ester. T84 was not able to mediate a TNF alpha or phorbol ester response. These studies provide further evidence that HIV-1 can infect cells by mechanisms other than those mediated by the CD4 receptor and describe complementary models for analyzing HIV-1 infection and expression in colonic epithelial cells.

CD4 Antigens↗
PubMed1991

Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter.

The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1.

Animals↗
PubMed1991

d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation.

Ascorbic acid stimulates collagen gene transcription in cultured fibroblasts, and this effect is mediated through the induction of lipid peroxidation by ascorbic acid. Quiescent cultured fibroblasts in the absence of ascorbic acid have a high constitutive level of collagen production, but the mechanisms of collagen gene regulation in this unstimulated state are not known. Because lipid peroxidation also occurs in normal cells, we wondered if lipid peroxidation plays a role in the regulation of basal collagen gene expression. Inhibition of lipid peroxidation in cultured human fibroblasts with d-alpha-tocopherol or methylene blue decreased the synthesis of collagen, the steady-state levels of procollagen alpha 1(I) mRNA and the transcription of the procollagen alpha 1(I) gene. This effect on collagen gene expression was selective and not associated with cellular toxicity. Thus, these experiments suggest a role for lipid peroxidation in the modulation of constitutive collagen gene expression.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1990

Transient induction of c-jun during hepatic regeneration.

The cellular oncogene c-jun is transiently expressed in cultured cells stimulated to proliferate but has not been identified in normal liver. Because partial hepatectomy results in coordinated cell proliferation in the remaining liver, we investigated c-jun expression after partial hepatectomy in mice. Northern analysis of whole liver mRNA demonstrated a transient increased expression of c-jun within half an hour of the operation. The related gene junB increased only 50%, whereas c-jun expression increased 13-fold compared with sham-operated controls. To determine the cell of origin of the c-jun transcript, both in situ hybridization and Northern analysis of mRNAs from parenchymal and nonparenchymal cell fractions were performed 2 hr after partial hepatectomy. C-jun expression was found in both cell populations. To investigate the mechanism of increased c-jun expression, cycloheximide was given to some animals preoperatively. C-jun induction occurred with cycloheximide alone, but partial hepatectomy further increased c-jun expression, indicating that new protein synthesis was not required for this effect. Furthermore, run-on transcriptional assay demonstrated a twofold increase in c-jun expression. Thus c-jun expression increases after hepatectomy by transcriptional and posttranscriptional mechanisms. Because the extracellular matrix-degrading enzyme transin, which bears the recognition site for jun/AP-1, showed sustained induction after hepatectomy, we speculate that an important function of c-jun expression could be the remodeling of extracellular matrices to accommodate cell proliferation.

Animals↗
PubMed1990

Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels.

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.

Amino Acid Sequence↗
PubMed1990

Therapy for hepatic fibrosis.

Although there is no established therapy for the fibrogenesis of hepatic cirrhosis, many potential therapies are now emerging. The requirements for the "perfect therapy" for hepatic fibrosis can be listed: (1) the pharmacologic agent should be active only in the liver; (2) its effect should be specific for collagen (or another critical extracellular matrix component); and (3) it should not be toxic. To date no agents fulfill these criteria. Of the agents we reviewed, only colchicine appears sufficiently safe for use outside of controlled clinical trials for cirrhotic patients whose underlying disease is not otherwise treatable. However, confirmation of the efficacy of colchicine in additional well-controlled clinical trials is still required. Agents such as collagen propeptides require extensive in vitro development, while trials in animal models are required for prolyl 4-hydroxylase inhibitors, proline analogues, and prostaglandins. For more developed agents, such as malotilate and gamma-interferon, there is now a need for well-designed long-term clinical trials.

Colchicine↗