PubMed Health⌕ Search

Biomedical subjects

C Roncero

Publications and source records attributed to C Roncero.

At least 37 records · Page 2Linked to original sources
PubMed1998

Transforming growth factor-beta (TGF-beta) and EGF promote cord-like structures that indicate terminal differentiation of fetal hepatocytes in primary culture.

When fetal hepatocytes were cultured in the presence of transforming growth factor-beta (TGF-beta 1) and epidermal growth factor (EGF), some morphological changes were observed. Under these conditions, cells migrated, from typical clusters that hepatocytes adopt in culture, to form elongated, cord-like structures similar to the hepatic acinus organization. Immunocytochemical analysis of these cells revealed high levels of albumin and cytokeratin 18, phenotypic markers of parenchymal hepatocytes. Although some of the cells in the cord-like structures presented a cortical ring distribution of F-actin filaments, the cord also presented thick peripheral bundles and cells of the tips showed thin stress fibers oriented to the cell edges, typical of a migratory phenotype. In addition to these morphological effects, flow cytometric analysis of the cells revealed a larger size, granularity and intracellular lipid content (as a parameter related to liver metabolic function), in TGF-beta + EGF-treated hepatocytes. Western blot analysis of the albumin levels revealed that both expression and secretion of albumin were increased in EGF + TGF-beta-treated cells. Finally, all these changes were coincident with an enhancement in the DNA-binding activity for hepatocyte nuclear factors (HNF1, HNF3, and HNF4), as revealed in gel-shift experiments with nuclear extracts. We conclude that a cooperative action between TGF-beta and EGF might modulate terminal maturation of fetal hepatocytes.

Actin Cytoskeleton↗
PubMed1998

Phorbol esters down-regulate alpha-fetoprotein gene expression without affecting growth in fetal hepatocytes in primary culture.

The effects of phorbol esters (phorbol-12,13-dibutyrate, PDB) on alpha-fetoprotein expression and cell growth were assayed by using fetal hepatocytes in primary culture. PDB acts synergistically with epidermal growth factor (EGF) to specifically decrease alpha-fetoprotein (AFP) mRNA levels, without affecting the expression of other genes of the same family, such as albumin and Vitamin D-binding protein (DBP). This effect is PDB-dose dependent, maximal effects being at 10 ng/ml. The implication of protein kinase C (PKC) in this effect seems clear since bisindolylmaleimide (BIS), a specific PKC inhibitor, completely blocks the PDB effect on AFP expression. Nuclear run-on experiments show that the decrease in AFP mRNA levels is mainly due to an inhibition in the transcription rate of the gene. Determination of PKC activities shows that fetal hepatocytes contain mainly Ca(2+)-independent isoenzymes, which patterns of activation was not modified by EGF plus PDB treatment with respect to PDB treatment. We have found that MAPK and JNK activities, c-jun and c-fos mRNA levels and AP-1 binding activity are notably increased when cells are incubated with both EGF and PDB, PDB does not stimulate growth of fetal hepatocytes, measured either as [3H]-thymidine incorporation into DNA or by cell cycle analysis using flow cytometry. All these results suggest that activation of PKC may affect liver gene expression rather than cell growth in fetal hepatocytes.

Albumins↗
PubMed1997

Characterization of CHS4 (CAL2), a gene of Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4.

We have cloned CHS4, a gene that complements the resistance to Calcofluor of the Saccharomyces cerevisiae cal2 mutant. We show that CHS4 is allelic to the previously described SKT5 and CSD4 genes. CHS4 encodes a 696 amino acids protein with no potential transmembrane domain. chs4-null mutants are resistant to Calcofluor white and exhibit a considerable reduction in cell wall chitin and in chitin synthase III (CSIII) activity. Biochemical characterization of chitin synthase III from these null mutants indicates that the defect is due to a reduced V(max) of the enzyme. This defect can be overcome in vitro by trypsin treatment of the membrane preparations. Chs4p does not act as a transcriptional or translational regulator of CHS3, the gene coding for the catalytic subunit of CSIII activity, and we therefore propose that Chs4p would be an essential component of the CSIII complex, acting as a post-translational regulator of this activity. In addition to the chitin defect, the chs4 mutant shows a severe defect in mating.

Alleles↗
PubMed1996

Triiodothyronine induces the transcription of the uncoupling protein gene and stabilizes its mRNA in fetal rat brown adipocyte primary cultures.

Confluent fetal rat brown adipocytes in primary culture showed an almost undetectable level of uncoupling protein (UCP) mRNA and a low mitochondrial content of functional UCP. Treatment of confluent cells with 10 nM triiodothyronine in a serum-free medium, in the absence of noradrenergic stimulation, increased the amount of UCP mRNA in a time-dependent manner. This effect was due to an increased UCP gene transcription rate and UCP mRNA stabilization, resulting in a higher content of immunoreactive mitochondrial UCP and functional UCP (detected by its ability to bind GDP). Thus, triiodothyronine might play a significant physiological role in the UCP expression throughout fetal development, when brown adipose tissue starts to differentiate and UCP is primarily expressed.

Adipose Tissue, Brown↗
PubMed1996

Nutritional and hormonal regulation of expression of the gene for malic enzyme.

We have provided a historical and personal description of the analysis of physiological and molecular mechanisms by which diet and hormones regulate the activity of hepatic malic enzyme. For the most part, our analyses have been reductionist in approach, striving for increasingly simpler systems in which we can ask more direct questions about the molecular nature of the signaling pathways that regulate the activity of malic enzyme. The reductionist approaches that were so successful at analyzing molecular mechanisms in cells in culture may now provide the means to analyze more definitively questions about the physiological mechanisms involved in nutritional regulation of gene expression. In addition to physiological questions, however, there are still many aspects of the molecular mechanisms that have not been elucidated. Despite considerable effort from many laboratories, the molecular mechanisms by which T3 regulates transcription are not clear. Similarly, the molecular details for the mechanisms by which glucagon, insulin, glucocorticoids, and fatty acids regulate gene expression remain to be determined. The role of fatty acids is particularly interesting because it may provide a model for mechanisms by which genes are regulated by metabolic intermediates; this is a form of transcriptional regulation widely used by prokaryotic organisms and extensively analyzed in prokaryotic systems, but poorly understood in higher eukaryotes. At any specific time, there is, of course, only one rate of transcription for each copy of the malic-enzyme gene in a cell. Our long-term objective is to understand how signals from all of the relevant regulatory pathways are integrated to bring about that rate.

Animals↗
PubMed1996

Malic enzyme gene in chick embryo hepatocytes in culture: clofibrate regulates responsiveness to triiodothyronine.

In chick embryo hepatocytes, triiodothyronine (T3) causes a 30- to 40-fold increase in malic enzyme activity when added between 1 and 3 days, but has no effect when added between 5 and 7 days in culture. This transcription-mediated decline in T3 responsiveness is partially reversed by corticosterone (Roncero, C. and A. G. Goodridge, 1992. Arch. Biochem. Biophys. 295: 258-267). Clofibrate also reversed the decline in responsiveness to T3, and did so in the absence of an increase in binding of T3 to nuclear receptors. The effects of clofibrate and corticosterone were additive, suggesting different mechanisms. The responsiveness of a gene to a specific agent depends on specific regulatory sequences of DNA in that gene. When 5.8 kb of the 5'-flanking DNA of the malic enzyme gene was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into hepatocytes, T3 stimulated CAT activity. Responsiveness of CAT activity to T3 decreased with time, and this decrease was partially reversed by clofibrate. The T3 responses of cells transfected with various chimeric DNAs that contained T3 response elements (T3REs) of the malic enzyme gene or synthetic consensus T3REs also were increased by clofibrate. The results suggest that clofibrate regulates expression of a metabolite or a protein factor which, in turn, influences function of the T3 receptor.

Animals↗
PubMed1995

Regulation of gene expression by interleukin-6 in fetal rat hepatocyte primary cultures: role of epidermal growth factor and dexamethasone.

Fetal rat hepatocytes incubated in the absence of hormonal signals, or under proliferative (presence of epidermal growth factor [EGF]) or differentiative (presence of dexamethasone) culture conditions, showed responsiveness to interleukin-6 (IL-6). Northern blotting analysis for some typical acute phase genes such as haptoglobin and other proteins not previously identified as acute-phase reactants, such as alpha-fetoprotein, beta 2-microglobulin, and fibronectin, showed a positive modulation by IL-6, in a dose-dependent manner. However, a well-characterized negative acute-phase reactant such as albumin was not responsive to IL-6. The well-established synergism between glucocorticoids and IL-6 on inducing transcription is absent in fetal hepatocytes. Conversely, the combination of IL-6 and EGF produced different patterns of expression, depending on the messenger RNA (mRNA) analyzed. Thus, EGF abolished the increased mRNA levels of haptoglobin caused by IL-6 but had no effect on other genes such as alpha-fetoprotein and fibronectin.

Albumins↗
PubMed1994

Noradrenergic modulation of albumin expression in growth-stimulated adult rat hepatocytes in primary culture.

Serum albumin is the most abundant protein synthesized by liver cells, and its production is a reliable indicator of the differentiated state of hepatocytes. We have recently shown that fetal rat hepatocytes cultured under proliferative conditions, i.e., in the presence of EGF, responded to glucagon and noradrenaline increasing albumin protein and mRNA levels (de Juan et al., 1992. J. Cell. Physiol., 152:95-101). This effect was mimicked by agents that increase cyclic AMP levels. In this report, we show that in regenerating liver, noradrenaline modulation of albumin expression seems to be different. Hepatocytes from hepatectomized rats were cultured at low cell density and in the presence of EGF. Under these conditions, noradrenaline, which acted synergistically with EGF increasing DNA synthesis (de Juan et al., 1992. Exp. Cell. Res., 202:495-500), produced a decrease in albumin mRNA levels. This effect was dose-dependent, being maximum at 1 microM noradrenaline. Noradrenergic effect seemed to be mediated by alpha 1-receptors, because it was blocked by prazosin, but not by propranolol. Other Ca(2+)-increasing agents, as vasopressin, angiotensin II, or ATP, did not produce any effect. However, albumin mRNA levels decreased when the cells were incubated in the presence of tetradecanoyl phorbol-13-acetate (TPA). In addition, noradrenergic modulation of albumin expression was blocked by staurosporine, a protein kinase inhibitor with relative specificity for protein kinase C. Thus we can conclude that the role of noradrenaline on the regulation of liver growth and differentiation changes from fetal to adult life. This change is probably due to its action on different receptors: beta-receptors in fetal hepatocytes and alpha 1-receptors in the adult liver.

Adenosine Triphosphate↗
PubMed1994

Triiodothyronine induces the expression of the uncoupling protein in long term fetal rat brown adipocyte primary cultures: role of nuclear thyroid hormone receptor expression.

Fetal rat brown adipocytes at the beginning of culture showed a minimal T3 nuclear binding capacity and very low expression of the c-erbA genes, with low steady state levels of the mRNA forms beta-type and 5.5- and 2.6-kilobase (kb) alpha-types. The levels of these mRNA species increased after 7 days in culture in the presence of thyroid hormone-depleted serum; however, no significant increase in nuclear T3-binding capacity was observed. The addition of T3 incremented the abundance of the c-erbA beta-mRNA, induced the appearance of the 6.6-kb c-erbA alpha-mRNA and increased the nuclear T3-binding capacity by 30-fold. Parallel analysis of the uncoupling protein (UCP) mRNA, immunoreactive UCP, and functional UCP (detected by its ability to bind GDP) demonstrated that T3 induced the expression of the UCP gene in long term treated fetal brown adipocytes in culture. The presence of noradrenaline, a positive control for UCP expression, increased only the level of expression of the 5.5-kb c-erbA alpha-mRNA, but values for nuclear T3-binding capacity similar to those obtained in T3-treated cells were observed. Simultaneous addition of both hormones gave a pattern of expression of the c-erbA-mRNA forms similar to those obtained with these agents individually, and no further increment in their separate effects was observed on the nuclear T3-binding capacity and UCP expression.

Adipose Tissue, Brown↗
PubMed1992

Hexanoate and octanoate inhibit transcription of the malic enzyme and fatty acid synthase genes in chick embryo hepatocytes in culture.

Hexanoate and octanoate inhibit the triiodothyronine (T3)-induced increases in the activities of malic enzyme and fatty acid synthase in chick embryo hepatocytes in culture. Butanoate was less effective as an inhibitor, and palmitate, stearate, and oleate had no effect or small stimulatory effects. Hexanoate and octanoate inhibited the lipogenic enzyme activities at a transcriptional step, and did so within 30 min of addition. Incubation for 2 h in the absence of fatty acid reversed the inhibition of transcription caused by hexanoate. The inhibitory effect of hexanoate was selective because DNA content and transcription of the glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes were not inhibited. Hexanoate-mediated inhibition of transcription rates of the lipogenic genes was not correlated with an inhibition of binding of T3 to its nuclear receptor. 2-Bromooctanoate and carnitine stimulated the T3-induced accumulation of the mRNAs for malic enzyme and fatty acid synthase. The presence of hexanoate stimulated by 2- to 3-fold the increase caused by carnitine, suggesting that hexanoate and carnitine may regulate lipogenic gene expression by a common pathway. Hexanedioate, acetoacetate, beta-hydroxybutyrate, branched chain fatty acids, and branched chain keto acids had little or no effect on abundance of the lipogenic mRNAs. We suggest that the active inhibitor is a metabolite derived from hexanoate or octanoate, possibly an intermediate derived from an acyl-CoA derivative.

Actins↗
PubMed1992

Regulation of the malic enzyme and fatty acid synthase genes in chick embryo hepatocytes in culture: corticosterone and carnitine regulate responsiveness to triiodothyronine.

Triiodothyronine (T3) added to chick embryo hepatocytes between 20 and 68 h of culture caused a 30- to 40-fold increase in malic enzyme activity. This T3 response decreased as a function of time; after 1 week in culture, a 48-h incubation with T3 had no effect on hepatocyte malic enzyme activity. Neither corticosterone nor carnitine had a significant effect on malic enzyme activity in the absence of T3 at any time or on the response of malic enzyme to T3 during the first 68 h of culture; both stimulated responsiveness to T3 subsequent to 68 h. The effects of corticosterone and carnitine on malic enzyme activity were additive, suggesting different mechanisms. Corticosterone and carnitine regulated abundance of malic enzyme mRNA. For corticosterone, at least, this effect was due to regulation of transcription. Abundance of fatty acid synthase mRNA was also stimulated by T3 in chick embryo hepatocytes in culture, and its responsiveness to T3 decreased with time. Corticosterone and carnitine stimulated responsiveness to T3 at times subsequent to 68 h. Corticosterone had no effect on binding of T3 to nuclear receptors. Intracellular accumulation of long-chain fatty acids or long-chain acyl-CoAs probably did not cause the loss of responsiveness to T3 or the stimulation of that responsiveness by corticosterone or carnitine because adding serum albumin (0.5%) or long-chain fatty acids (0.25-0.5 mM) to the medium was without effect. Corticosterone and carnitine may control the levels of other metabolic intermediates or protein factors which, in turn, regulate the transcriptional response of the lipogenic genes to T3.

Actins↗
PubMed1992

Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus.

The region of the Escherichia coli K-12 chromosome encoding the enzymes responsible for the synthesis of responsible for the synthesis of the lipopolysaccharide (LPS) core has been cloned in vivo by using a mini-Mu vector. This region, formerly known as the rfa locus, comprises 18 kb of DNA between the markers tdh and rpmBG. Results of in vitro mutagenesis of this region with MudII1734 indicate the presence of at least 17 open reading frames or genes, a number considerably higher than expected on the basis of genetic and biochemical studies. Specific insertions in different genes have been recombined into the chromosome, and the mutations have been phenotypically characterized. Complementation analysis indicates that these genes are arranged in three different operons transcribed in opposite directions. A detailed physical map of this region has been constructed on the basis of complementation analysis, fusion protein data, and phenotypic characterizations. Additionally, the role of some genes in the synthesis of LPS has been defined by complementation analysis with known Salmonella typhimurium LPS mutants. The genetic organization of this locus seems to be identical in E. coli K-12 and S. typhimurium.

Chromosome Mapping↗
PubMed1991

Characterization of a Schizosaccharomyces pombe morphological mutant altered in the galactomannan content.

In a search for Schizosaccharomyces pombe mutants resistant to the antifungal agent papulacandin B, a morphological mutant was isolated. The mutant is round shaped in contrast to the rod shaped parental strain. This morphological defect segregated as a recessive Mendelian character and was not observed in other papulacandin B resistant mutants belonging to the same complementation group. The mutation mapped in the right arm of S. pombe chromosome III very close to pap1 marker. Mutant cell walls were more susceptible to alkali extraction and Novozyme degradation than those from the wild-type. A specific reduction in the cell wall galactomannan fraction was the only significant difference detected as compared to the wild-type strain. Levels of beta (1,3)-glucan and mannan synthases as well as other enzymic periplasmic mannoproteins were very similar in wild type and mutant strains.

Acid Phosphatase↗
PubMed1991

Analysis of the host ranges of transposon bacteriophages Mu, MuhP1, and D108 by use of lipopolysaccharide mutants of Salmonella typhimurium LT2.

The lipopolysaccharide receptors for the mutator bacteriophages Mu, MuhP1, and D108 were investigated with lipopolysaccharide mutants of Salmonella typhimurium LT2. Mu adsorbed only to mutants lacking the terminal O antigen but retaining the main chain sugars of the core; the side chain N-acetylglucosamine was not required. MuhP1 and D108 adsorbed partially to cells with the same receptors but adsorbed well only to cells with shorter lipopolysaccharides of the Rc and Rd1 chemotypes.

Acetylglucosamine↗
PubMed1990

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption.

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 were found to require pili for infection. Seventy mutants of P. aeruginosa PAO selected by resistance to D3112 or B3 were also resistant to the phage not used in the selection and suggested that the receptors of these two phages are identical. Of five resistant mutants examined, all were defective in the production of pili and did not adsorb either phage. P. aeruginosa PAK strains altered in pilus expression, such as hyperpiliated or nonpiliated mutants, adsorbed the phage but were not productively infected, implying that an additional host function was required for infection. The cell-associated lipopolysaccharide was not required for D3112 or B3 infection, since mutants deficient in O side-chain and core biosynthesis were still capable of adsorption and productive infection. This is in contrast to Escherichia coli mutator phages Mu and D108, which are dependent on lipopolysaccharide for adsorption. The P. aeruginosa phages adsorbed only to cells grown on solid media or in liquid media supplemented with agents that increase the macroviscosity, such as polyvinylpyrrolidone. Adsorption time course studies of D3112 and B3 using cells grown in solid media revealed similar but not identical adsorption patterns. These studies suggested that expression of the D3112 and B3 cell receptor is induced by growth on solid media.

Adsorption↗
PubMed1989

Rates of lipogenesis in fetal hepatocytes in suspension and in primary culture: hormonal effects.

Lipogenesis in isolated fetal hepatocytes in suspension for 3 h was modulated by insulin depending on substrates utilization, but was inhibited by glucagon and noradrenaline from all substrates studied. After primary culture for 5 days in the presence of glucose, the lipogenic response to insulin increased, the glucagon response decreased and noradrenaline produced the same degree of inhibition at 3 h. At 24 h, insulin produced an even higher increase on lipogenesis parallel to an increase in fatty acid synthase activity. Dexamethasone increased lipogenesis, but progesterone had no effect. Both hormones, in the presence of insulin, increased lipogenesis and fatty acid synthase activity. Triiodothyronine, alone or in the presence of insulin, increased lipogenesis and fatty acid synthase activity.

Animals↗
PubMed1988

Hormonal regulation of rat foetal lipogenesis in brown-adipocyte primary cultures.

Insulin stimulates lipogenesis by 100% for 5 h by a covalent modulation of acetyl-CoA carboxylase, and by 200% for 24 h by increasing malic enzyme and fatty acid synthase enzymic activities in brown-adipocyte primary cultures. At short times, noradrenaline and isoprenaline decrease lipogenesis. However, phenylephrine and glucagon have no effect. At long times, dexamethasone inhibits lipogenesis. This effect is precluded in the presence of insulin. Progesterone and tri-iodothyronine, alone or in the presence of insulin, produce a stimulation of the rates of lipogenesis.

Adipose Tissue, Brown↗