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D A Brenner

Publications and source records attributed to D A Brenner.

At least 145 records · Page 8Linked to original sources
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↗
PubMed1990

Expression of collagen genes in the liver.

Hepatic production of type I collagen is markedly increased in liver cirrhosis. Previous studies using primary liver cell cultures have demonstrated that hepatocytes, lipocytes and endothelial cells are all capable of producing collagen. In this study in situ hybridization and hepatic cell sorting have been used to identify which cells are expressing the type I collagen gene, alpha 1(I), in normal rat liver. Northern blotting of mRNAs from purified hepatic cell populations demonstrated that both hepatocytes and several types of non-parenchymal cells express the collagen alpha 1(I) gene. Calculations based on cell numbers, yields of mRNA, and cellular mRNA concentration demonstrated that the majority of collagen alpha 1(I) mRNA originates from the hepatocytes in the normal liver. Localization of a collagen alpha 1(I) mRNA by in situ hybridization confirmed that both hepatocytes and non-parenchymal cells express this gene. Furthermore, collagen alpha 1(I) gene expression in hepatocytes was obtained by transfecting a reporter gene driven by the collagen alpha (I) 5' regulatory segment in primary liver cell cultures. Future experiments will further characterize the regulation of collagen alpha 1(I) gene expression in the liver.

Animals↗
PubMed1989

Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation?

Ascorbic acid stimulates collagen gene expression in cultural fibroblasts (Lyons, B. L., and Schwartz, R. L. (1984) Nucleic Acids Res. 12, 2569-2579), but the 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. Since another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts (Brenner, D. A., and Chojkier, M. (1987) J. Biol. Chem 262, 17690-17696), 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 mM) induced lipid peroxidation in cultured human fibroblasts judging by the production of thiobarbituric acid-reactive substances and carbonyl groups, and by the presence of malondialdehyde- and 4-hydroxynonenal-protein adducts. Ascorbic acid stimulated (2-3-fold) the net production of collagen relative to total proteins, the levels of procollagen alpha 1 (I) mRNA and the transcription of this gene. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 microM) or methylene blue (10 microM) prevented the stimulation of collagen gene expression. The addition of malondialdehyde (200 microM), a product of lipid peroxidation, to cultured human fibroblasts also increased 2-3-fold 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 the stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.

Ascorbic Acid↗
PubMed1989

Analysis of the collagen alpha 1(I) promoter.

The collagen alpha 1(I) gene is regulated at a developmental and tissue specific level. We have previously demonstrated that only 220bp of the promoter region of the collagen alpha 1(I) gene are required for efficient expression in NIH 3T3 cells. DNAse I protection assays demonstrated 4 footprinted segments in the promoter region. Deletional analysis revealed that the 3 most proximal footprints were required for maximal expression. The most proximal footprint contains a CCAAT sequence and a 12bp segment that forms a direct repeat with the preceding footprint. Ligation of the proximal footprint sequence to a heterologous promoter enhanced transcription of the reporter gene. These studies, therefore, identify and characterize elements in the promoter region of the collagen alpha 1(I) gene that interact with DNA binding proteins and are required for efficient expression.

Animals↗
PubMed1989

Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes.

The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts (Flaherty, M., and Chojkier, M. (1986) J. Biol. Chem. 261, 12060-12065). In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+ (Thomas, A.P., Marks, J.S., Coll, K.E., and Williamson, J. R. (1983) J. Biol. Chem. 258, 5716-5725). However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with [5-3H]proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic [8-arg]vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with [35S]methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific [32P]cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment. Vasopressin did not affect collagen production in quiescent cultures of mouse Swiss 3T3, human myofibroblast or rat smooth muscle cells; and hepatocyte collagen production was unaffected by treatment with glucagon or dibutyryl cAMP. Thus, accelerated Ca2+ fluxes induced by vasopressin are associated with decreased production of hepatocyte collagen and albumin in primary cultures that simulate quiescent adult rat liver.

Aging↗
PubMed1989

Prolonged activation of jun and collagenase genes by tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF-alpha) is secreted by macrophages in response to inflammation, infection and cancer. Sublethal doses of recombinant TNF-alpha to rats causes cachexia, anaemia and inflammation. TNF-alpha plays a major part in tissue inflammation and remodelling by stimulating production of collagenase. Cellular responses to TNF-alpha are initiated by binding to high-affinity cell surface receptors. TNF-alpha then profoundly affects gene regulation, stimulating the fos, myc, interleukin-1 and interleukin-6 genes and inhibiting the type I collagen gene. Here we demonstrate that TNF-alpha also stimulates collagenase gene transcription; this stimulation is mediated by an element of the gene that is responsive to the transcription factor AP-1, the major component of which (jun/AP-1) is encoded by the jun gene; and that TNF-alpha stimulates prolonged activation of jun gene expression. This prolonged induction of jun contrasts with its transient activation by the phorbol ester TPA and provides a physiological example of the ability of jun/AP-1 to stimulate its own transcription. This may be a key mechanism for mediating at least some of the biological effects of TNF-alpha.

Blotting, Northern↗
PubMed1989

Transforming growth factor-alpha stimulates proto-oncogene c-jun expression and a mitogenic program in primary cultures of adult rat hepatocytes.

Human transforming growth factor-alpha (TGF-alpha, MW 5547) initiates a mitogenic program in "quiescent" 11-to 13-day-old primary cultures of adult rat hepatocytes. Using validated growth reinitiation assays and chemically defined conditions (Koch and Leffert, 1979a) that simulate proto-oncogene expression in regenerating liver (Kruijer et al., 1986), we find that 5.4 nM TGF-alpha stimulates: (i) increases in rates of amiloride-sensitive 22Na+ uptake; (ii) a transient induction in steady-state mRNA levels of proto-oncogene c-jun; (iii) specific increases in hepatocyte nuclear [3H]dT labeling indices, augmented synergistically by insulin and glucagon; and (iv) increases in rates of S-phase entry. Comparative studies indicate that TGF-alpha is a more effective hepatocyte growth promoter than mouse epidermal growth factor. These observations, and published reports linking normal and cancerous liver as biosynthetic sources of TGF-alpha, suggest an autocrine or paracrine role for TGF-alpha in the control of hepatic growth, regeneration, and gene expression.

Animals↗
PubMed1989

Regulatory elements in the 5'-flanking region and the first intron contribute to transcriptional control of the mouse alpha 1 type I collagen gene.

We have identified two blocks of regulatory sequences located in the 5'-flanking region and the first intron of the mouse alpha 1 type I collagen (COL1A1) gene. Both blocks were found to contain positive as well as negative regulatory elements. Sequences located within 222 base pairs upstream of the transcription start site showed a strong stimulatory effect on the COL1A1 promoter and were sufficient for tissue-specific regulation of the COL1A1 gene. The combined upstream and intron regulatory sequences showed a marked inhibition of COL1A1 promoter activity in fibroblasts. This finding suggests that additional, more remote regulatory sequences may be required for establishing the high level of activity of the endogenous COL1A1 gene in fibroblastoid cells.

Animals↗
PubMed1989

Analysis of RNA transcripts for HLA class II genes in human small intestinal biopsies.

Studies of the expression of selected genes within the intestinal mucosa will provide important new information about physiologic pathological processes that effect mucosal growth, differentiation, and function. To study gene expression in the gut, we developed a method to obtain sufficient undegraded RNA from human endoscopic intestinal biopsy specimens for Northern and slot blot analysis. To verify the method, we examined the differential expression of HLA class II genes in small intestinal mucosa. Levels of RNA transcripts for HLA-DR, -DP, and -DQ alpha and beta chains were assessed in freshly isolated endoscopic intestinal mucosal biopsy specimens and compared with levels in Epstein-Barr virus transformed B cells from the same individuals. Sufficient undegraded cellular RNA with distinct 28S and 18S ribosomal bands could be obtained from as few as two 2-3 mm endoscopic biopsies. Using chain and locus specific cDNA probes, HLA-DR, -DP, and -DQ subregion genes were shown to be expressed in intestinal mucosa, with the relative magnitude of RNA transcripts being DR greater than DP greater than DQ. The same hierarchy of expression was seen for EBV-transformed B cell lines. This method, in conjunction with the polymerase chain reaction for amplifying specific RNA transcripts and in situ hybridisation methods for the cellular localisation of RNA transcripts, will enable studies on the regulation of gene expression in the intestinal mucosa.

Biopsy↗
PubMed1988

Tumor necrosis factor alpha inhibits collagen gene transcription and collagen synthesis in cultured human fibroblasts.

The effects of recombinant human tumor necrosis factor alpha (TNF alpha) on collagen production and gene expression in cultured fibroblasts were studied. Cells were labeled with [3H]proline, and the radioactivity of collagenase-sensitive and -resistant proteins were used to calculate the rates of protein production. The net production of collagen relative to total proteins was inhibited by TNF alpha (0-1.2 nM) in a dose- and time-related manner. The specific activities of the free [3H]proline pool, which were similar in control and TNF alpha-treated cells, were used to calculate the absolute rates of protein production. The absolute rate of collagen production was decreased by 50% in the presence of 1.2 nM TNF alpha during 24-h incubations (851 +/- 104 versus 426 +/- 39 pmol/micrograms of DNA/h; p less than 0.01), whereas noncollagen protein production and the rate of procollagen secretion were unchanged. We found no evidence of cellular toxicity in cultured cells treated with TNF alpha. In addition, TNF alpha did not affect cell proliferation as determined by [6-3H]thymidine incorporation into DNA. Most of the collagen produced by the cultured fibroblasts was type I. Using hybridization with specific DNA probes there was an approximately 50% decrease in the quantity of procollagen alpha 1(I) mRNA, without changes in the quantity of alpha tubulin mRNA or the size of the transcripts, in cells incubated with TNF alpha. Interleukin-1 (2.5 ng/ml) also decreased the levels of procollagen alpha 1(I) mRNA by approximately 50%. Cycloheximide (0.1 mM), an inhibitor of protein synthesis, blocked the inhibitory effect of both TNF alpha and interleukin-1 on procollagen alpha 1(I) mRNA. Nuclear run-off assays demonstrated that TNF alpha decreased procollagen alpha 1(I) transcriptional activity by 50% and had no effects on alpha tubulin gene transcription. Thus, TNF alpha decreases collagen gene transcription, collagen mRNA levels, and collagen production in cultured fibroblasts.

Cell Division↗
PubMed1987

Acetaldehyde increases collagen gene transcription in cultured human fibroblasts.

Acetaldehyde, the first metabolite of ethanol, mediates many of the biological effects of ethanol. We have previously shown that acetaldehyde, but not ethanol, stimulates collagen production in cultured human fibroblasts (Holt, K., Bennett, M., and Chojkier, M. (1984) Hepatology 4, 843-848). Here, we examined the effects of acetaldehyde on collagen gene expression. Confluent human fetal fibroblasts were incubated for up to 4 h in the presence of ascorbate (0.2 mM) alone or with the addition of either ethanol (12 mM) or acetaldehyde (200 microM). Acetaldehyde induced the production of collagen (up to 2.5-fold) and had a small inhibitory effect on procollagen secretion (-20%). The steady-state levels of mRNAs were measured by hybridizing total cellular RNA to specific cDNA probes at high stringency. Acetaldehyde increased the steady-state level of collagen alpha 1(I) and collagen alpha 2(I) mRNAs about 3-fold and had small effects on beta-actin mRNA (+50%) and collagenase mRNA (-50%). Northern blots revealed that the RNAs were intact and that acetaldehyde preferentially increased the abundance of the longer of the two collagen alpha 1(I) transcripts. Acetaldehyde increased both collagen alpha 1(I) and collagen alpha 1(III) transcriptional activity by 2.5-fold and had small effects on beta-actin and collagenase gene transcription. The increase in both collagen production and collagen mRNA levels induced by acetaldehyde was blocked by methylene blue, a scavenger of reducing equivalents. These data indicate that reducing equivalents, which enhance the formation and stability of acetaldehyde-protein adducts, may be required for acetaldehyde-stimulated collagen production. Thus, this study suggests that acetaldehyde increases collagen production by increasing collagen gene transcription in cultured human fibroblasts.

Acetaldehyde↗
PubMed1986

Double-strand gap repair results in homologous recombination in mouse L cells.

Previous studies have demonstrated that the presence of double-strand breaks or double-strand gaps increases the frequency of homologous recombination between two cotransferred DNAs when they are introduced into cultured mammalian cells. Here we demonstrate that the repair of these double-strand gaps is a major mechanism for homologous recombination between exogenous DNAs. In particular, when a plasmid DNA containing a 104-base-pair (bp) gap in its tk gene (herpes simplex virus gene for thymidine kinase) undergoes recombination in mouse L cells to generate an intact gene, the majority of events result from direct repair of the double-strand gap using a cotransferred DNA as the template. We analyzed the recombination events by comparing the frequency of tk+ colonies, Southern blotting of cloned tk+ cell lines, and cloning recombined functional tk genes by plasmid rescue. In addition, by creating double-strand breaks within or adjacent to heterologous insertions in a mutant tk gene, we estimate that the L cell can generate a double-strand gap of between 152 and 248 bp and then can repair the gap to create a functional tk gene. We conclude that double-strand breaks and double-strand gaps are recombinogenic in transferred plasmid DNAs because they serve as intermediates in homologous recombination by double-strand gap repair, a nonreciprocal exchange of DNA or gene conversion event.

Animals↗
PubMed1985

Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.

We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells. A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1 thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk- cells by the calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion mutant of greater than or equal to 24 bp in the same manner did not increase the frequency. We show how our data are consistent with models that postulate at least two phases in the recombination process: homologous pairing and heteroduplex formation.

Animals↗
PubMed1980

The enzymatic defect in variegate prophyria. Studies with human cultured skin fibroblasts.

Fecal protoporphyrin is increased in patients with variegate porphyria, even during clinical remission, suggesting an enzymatic defect in the terminal portion of the heme biosynthetic pathway. We measured the activities of protoporphyrinogen oxidase, which catalyzes the oxidation of protoporphyrinogen to protoporphyrin, and heme synthase, which catalyzes the chelation of iron to protoporphyrins, in cultured skin fibroblasts from five normal controls and five patients with variegate porphyria. Heme synthase activity was shown to be normal in variegate porphyria cells by direct assay in cell sonicates and indirect assay in intact cells. Protoporphyrinogen oxidase activity, however, was reduced to 43 per cent of normal in sonicates of variegate porphyria cells (0.90 +/- 0.13 vs. 2.12 +/- 0.25 nmol of protoporphyrin per milligram of protein per hour [mean +/- S.E.M.] [P less than 0.005]). We conclude that protoporphyrinogen oxidase activity is deficient in variegate porphyria. Fecal protoporphyrin may increase because an excess amount of protoporphyrinogen is excreted into bile and subsequently auto-oxidized to protoporphyrin.

Adult↗