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Biomedical subjects

D A Brenner

Publications and source records attributed to D A Brenner.

At least 19 recordsLinked to original sources
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↗
PubMed1990

A simplified method for the preparation of transcriptionally active liver nuclear extracts.

We have developed a simplified method for the preparation of liver nuclear extracts to study gene regulation and protein-DNA interactions. This protocol uses conventional laboratory equipment and standard reagents. The liver tissue is homogenized in a low-salt solution at physiological molarity with subsequent adjustment of the molarity and purification of nuclei by density sedimentation. The nuclear extracts are transcriptionally active in a validated cell-free transcription assay and contain functional DNA-binding proteins. This protocol results in the rapid preparation of highly reproducible and active liver nuclear extracts.

Animals↗
PubMed1990

DNA-mediated gene transfer into adult rat hepatocytes in primary culture.

Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity. Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C. Chen and H. Okayama (Mol. Cell. Biol. 7:2745-2752, 1987). Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells). The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration [optimal at 2.4 micrograms of DNA per ml] and duration of exposure [optimal between 5 and 10 h]), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha. Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation. These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.

Animals↗
PubMed1990

Tumor necrosis factor-alpha inhibits albumin gene expression in a murine model of cachexia.

The mechanisms responsible for decreased serum albumin levels in patients with cachexia-associated infection, inflammation, and cancer are unknown. Since tumor necrosis factor-alpha (TNF alpha) is elevated in cachexia-associated diseases, and chronic administration of TNF alpha induces cachexia in animal models, we assessed the regulation of albumin gene expression by TNF alpha in vivo. In this animal model of cachexia, Chinese hamster ovary cells transfected with the functional gene for human TNF alpha were inoculated into nude mice (TNF alpha mice). TNF alpha mice became cachectic and manifested decreased serum albumin levels, albumin synthesis, and albumin mRNA levels. However, even before the TNF alpha mice lost weight, their albumin mRNA steady-state levels were decreased approximately 90%, and in situ hybridization revealed a low level of albumin gene expression throughout the hepatic lobule. The mRNA levels of several other genes were unchanged. Hepatic nuclei from TNF alpha mice before the onset of weight loss were markedly less active in transcribing the albumin gene than hepatic nuclei from control mice. Therefore, TNF alpha selectively inhibits the genetic expression of albumin in this model before weight loss.

Animals↗