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R Bertolotti

Publications and source records attributed to R Bertolotti.

At least 19 recordsLinked to original sources
PubMed2006

Investigating the estrogenic risk along the river Po and its intermediate section.

Different endpoints have been used to investigate the occurrence of estrogenic risk along the Po River, particularly its middle section. An in vitro assay based on recombinant yeast could not detect estrogenic activity in bed sediments of the Italian river or in bile samples of five Cyprinid species, with the only exception being one carp (Cyprinus carpio) captured downstream of the River Lambro, a polluted tributary of the middle River Po. Chemical analyses of fish bile and water samples from the same middle section showed diffuse contamination by moderately low levels of estrogenic chemicals (estrone [E1], 17beta-estradiol, estriol [E3], 17alpha-ethinylestradiol, 4-nonylphenol [NP], 4-tert-octylphenol [tOP], 4-n-octylphenol, and bisphenol A) but they were of limited help in understanding the risk present in the downstream area where intersex barbel were previously found. In contrast, the analyses of River Lambro waters showed that this tributary is a source to the middle River Po of all eight estrogens investigated. Analyses of bed sediments and macroinvertebrates from the same area consistently showed at least two levels of contamination, with the downstream stretch showing higher concentrations of natural steroids (E1 and E3) and xenoestrogens (NP and tOP). Accordingly, new histologic examinations undertaken on young barbel (Barbus sp.) showed intersex gonads only in the individuals captured in the downstream stretch, thereby confirming previous results. Present findings confirm the occurrence of disrupting conditions in the middle River Po and provide the first suggestions of cause-effect relationships.

Animals↗
PubMed2005

In vivo and in vitro exposures of carp and carp tissues to graded concentrations of endocrine disrupting chemicals.

The objective of this study was to correlate morphological and biochemical parameters of reproductive activity in fish exposed to graded concentrations of endocrine disrupting chemicals. Two-year-old carps were exposed for two weeks to graded concentrations of ethynyl-estradiol, tamoxifen, and flutamide. For each chemical, morphological alterations of the gonads and liver were described according to biomarker and sex steroid levels. In parallel, isolated follicular cells (FCs) and testis fragments were incubated in the presence or absence of carp pituitary homogenate and 25-hydroxycholesterol. Media were analyzed for sex steroid level evaluations.

Animals↗
PubMed2005

Fish community characterization in two stretches upstream and downstream of the Lambro River confluence with the Po River.

This study aimed to characterize the fish community in two stretches of the Po River, upstream and downstream of the Lambro River confluence, to verify the environmental effects of the presence of endocrine disrupters (EDs). More than 5000 specimens were captured, identified at the species level, and recorded for biometric parameters. Plasma, liver, and gonad fragments were sampled for histological and biochemical analyses. Few specimens belonging to carp and barbel showed gonads with the concomitant presence of male and female tissues and altered plasma levels of vitellogenin and sex steroids; thus, these species will be considered for successive environmental studies.

Animals↗
PubMed1995

Glucocorticoids upregulate high-affinity, high-density lipoprotein binding sites in rat hepatocytes.

Glucocorticoid hormones (GL) regulate high-density lipoprotein (HDL) plasma concentrations by increasing synthesis and secretion of HDL by the liver. However, little is known about the effect of GL on the uptake and processing of HDL by hepatocytes (HEP). To investigate this question, we studied the effects of dexamethasone (DEX) on the expression of high-affinity HDL-binding sites via the specific binding and internalization of iodine-labeled apolipoprotein E (apo E)-free HDL3 in a culture of rat HEP. Specific binding and internalization of HDL3 decreased by 60% in cells cultured in the absence of DEX for 48 hours. At concentrations of 10(-7) and 10(-5) mol/L, DEX prevented the decrease, maintaining specific binding and internalization versus the control level (at 24 hours). HDL-binding sites with a Kd of 20 micrograms/mL were revealed on the surface of cultured HEP. HEP demonstrated a greater binding capacity in the presence of DEX at concentrations of 10(-7) and 10(-5) mol/L (125 v 45 ng/mg cell protein). The effect of the hormone has demonstrated to be dose-dependent at concentrations between 10(-9) and 10(-7) mol/L, leveling off at 10(-7). Higher concentrations did not induce a further increase in specific binding and internalization. Withdrawal of the hormone from culture medium was associated with a decrease in specific binding of the ligand by 60% in the following 24 hours. To investigate the effect of glucocorticoid deficiency on liver uptake of HDL in vivo, specific binding and internalization were studied in a culture of HEP isolated from adrenalectomized rats (AER) at 2 hours after seeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1995

Liver fructose-1,6-bisphosphatase cDNA: trans-complementation of fission yeast and characterization of two human transcripts.

The SV40 early promoter is active both in mammalian cells and in the fission yeast Schizosaccharomyces pombe, and is used to drive full-length cDNA in polyvalent pcD-libraries. Two such liver libraries, of human and rat origin, were used to trans-complement a S. pombe mutant deficient in fructose-1,6-bisphosphatase (Fru-1,6-Pase) activity, a key gluconeogenic enzyme restricted to liver, kidney and intestine in mammals. A rat liver Fru-1,6-Pase cDNA was readily cloned and sequenced. Complementary PCR experiments revealed full-length Fru-1,6-Pase cDNA also present in the human liver library, however at a low abundance. Two human liver transcripts were thus characterized. Contrary to expectation, they were not differentially spliced products. They both encoded the same protein and were generated by a polyadenylation choice mechanism. The longest transcript comprised two polyadenylation signals and a consensus GT-rich element for the 3' processing of the upstream site. Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) analysis of 3' ends from hepatic, renal and intestinal mRNA disclosed that both Fru-1,6-Pase transcripts are expressed in the three main gluconeogenic cell types and are subject to insulin differential modulation. On the other hand, overcoming liver cell heterogeneity problems, sequence analysis of 16 independent clones of 3' end-cDNA demonstrated that, in addition to a monocytic type corresponding to a previously described lambda gt11 clone, human liver does not contain a hepatic type Fru-1,6-Pase comprising a liver-specific carboxyl-terminal extension like its rat counterpart. This liver-specific extension is involved in enzyme up-regulation and appears to give a conclusive advantage to the rat hepatic enzyme over the human one when trans-complementing mutant yeast.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Expression of mRNA for atrial natriuretic peptide receptor-A in human liver: detection using RT-PCR.

cDNA preparations obtained from human liver poly A+ RNA by reverse transcription were subjected to polymerase chain reaction. Primers used in the reaction were designed from the reported cDNA sequence for human placental atrial natriuretic peptide receptor-A (ANPRA). Sequence analysis of the amplified product (approximately 700 bp) showed 100% identity to the 2476-3189 bp region of the reported cDNA sequence of human placental ANPRA. Northern blot analysis of human liver poly A+ RNA fractions showed a hybridization signal at approximately 4.4 kb, identical to the signal obtained from the poly A+ RNA fractions of human placenta. These results indicate the possible mRNA expression for atrial natriuretic peptide receptor-A in human liver.

Atrial Natriuretic Factor↗
PubMed1992

Selection of variant hepatoma cells in liver-specific growth media: regulation at the mRNA level.

Three liver-specific growth media, respectively free of arginine (Arg-), tyrosine (Tyr-) and glucose (G-), have been used to characterize cells of the rat H4IIEC3, human HepG2 and mouse BW hepatoma lines. Cells of clone FaO, a derivative of line H4IIEC3, freely grew in Tyr- and G- media, and gave rise to stable variants in Arg- conditions. Cells of line HepG2 and clone BWTG3, a derivative of line BW, degenerated in all three media. Arg and tyr variants were however derived from HepG2 cells; their genesis appeared to be pathway specific, illustrating the complexity of the regulatory loops that are implicated in the control of the differentiated state. No variant was ever obtained with BWTG3 cells, demonstrating the stability of their deficiency in the post-natal hepatic functions that are involved in Arg-, Tyr- and G- selections. Variant clones of HepG2 and FaO cells that have been isolated in Arg- medium were characterized in details for liver-specific urea-cycle enzyme activities and mRNA. These variants were shown to be controlled at the mRNA level, most likely at transcription. Isolation of stable FaO and HepG2 variant clones as well as the converse demonstration of the stable deficiency of BWTG3 cells in post-natal hepatic functions were aimed at expression cloning. Our results are thus discussed in terms of transfection with full-length cDNA expression libraries and cloning of regulatory genes that could activate or extinguish liver specific genes.

Animals↗
PubMed1989

Construction of an EBNA-producing line of well-differentiated human hepatoma cells and of appropriate Epstein-Barr virus-based shuttle vectors.

Using cloned Epstein-Barr nuclear antigen 1 (EBNA) and oriP elements from the Epstein-Barr virus (EBV) in conjunction with liver-specific growth media, we have constructed an EBNA-producing line of well-differentiated human hepatoma cells (Hep-EBNA-2) and appropriate EBV-oriP vectors. These vectors, pBEDC1 and pBEUG1, were maintained as free extrachromosomal elements only in cells that expressed the trans-acting EBNA protein. They were readily rescued from transfected Hep-EBNA-2 cells upon transformation of recA- Escherichia coli with cellular low-Mr DNA. They are true shuttle vectors in that they can propagate as free closed circular elements in both human Hep-EBNA-2 cells and E. coli. Finally, we have demonstrated the vector capability of our shuttle system by inserting into the SV40 expression cassette of pBEUG1 a large full-length cDNA encoding coagulation factor VIII. Our data clearly show that EBV-oriP episomes are able to stably propagate in an hepatic background and that neither high levels of EBNA protein nor multiple copy episomes significantly interfere with the expression of the set of hepatic functions that have been analyzed. These results are discussed in terms of gene amplification and cloning of genes that program liver differentiation.

Antigens, Viral↗
PubMed1989

KIN, a mammalian nuclear protein immunologically related to E. coli RecA protein.

A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.

Animals↗
PubMed1987

Plasmodium ovale: in vitro development of hepatic stages.

Primary cultures of human hepatocytes, a culture-derived clone from the human hepatoma Hep G2 line, and cultured rat hepatocytes were inoculated in vitro with Plasmodium ovale sporozoites extracted from Anopheles stephensi, An. gambiae, and An. dirus mosquitoes. Penetration and differentiation of P. ovale sporozoites into trophozoite stage parasites occurred in all three cell types, but with a lower transformation rate in the Hep G2 cell line than in the primary cultured hepatocytes. Further maturation was obtained only in the human hepatocytes, in which the parasites were uninucleate until the third day after infection, before development to 60 micron in length by the eighth day. Additionally, this culture system was used to assess the ability of an anti-P. ovale sporozoite monoclonal antibody to inhibit penetration of sporozoites into hepatocytes and to detect sporozoite determinants in the maturing liver stage parasites.

Animals↗
PubMed1986

A polyoma-derived plasmid vector maintained episomally in both E. coli and mouse hepatoma cells.

We describe a recombinant plasmid, pBBPY1, containing polyoma virus sequences which persists episomally in mouse hepatoma (MH) cells and can be shuttled between these cells and bacteria. This plasmid is composed of a subgenomic fragment of a polyoma virus mutant that includes two origins of replication; sequences of plasmid pML2; the xanthine-guanine phosphoribosyltransferase gene of Escherichia coli (Ecogpt) under the control of SV40 early-region promoter and RNA processing signals, providing a dominant selectable marker for mammalian transfection. MH cells from colonies growing in HAT medium (hypoxanthine, aminopterin and thymidine) were found to contain vector DNA molecules in an episomal state, the majority of them unrearranged. When HAT-selective pressure was applied for only 3 days, the resulting cells contained up to 50-100 copies of intact plasmid, i.e. 20-fold more than cells grown under standard selection conditions with continuous HAT-selective pressure. Contrary to standard conditions, transient selection does not alter the epithelial morphology nor ability of transfected hepatoma cells to produce albumin.

Albumins↗
PubMed1985

Shuttling of integrated vectors from mammalian cells to E. coli is mediated by head-to-tail multimeric inserts.

With the aim of producing nonviral shuttle vectors for mammalian cells, we have constructed mouse mitochondrial DNA derivatives comprising the xanthine-guanine phosphoribosyltransferase gene as a selectable marker. Complete or subcomplete mitochondrial genomes were inserted into the plasmid pBB3 and transferred into hepatoma cells in order to generate, in vivo, new recombinant molecules. A second- and a third-generation vector, p12.2b and p delta respectively, were thus isolated for their ability to shuttle from mammalian cells to recA+ E. coli. Transfection of rodent fibroblasts and hepatoma cells showed that, contrary to our expectations, p12.2b and p delta are not self-replicating episomes; their shuttling from mammalian cells to recA+ E. coli is mediated by tandem integrated copies. The relevant property of p12.2b and p delta is a ubiquitous propensity to form head-to-tail multimeric structures when they integrate into mammalian host chromosomes. This ability is missing in pBB3 and appears only following the insertion of various mitochondrial or nuclear DNA fragments into the plasmid. These data are discussed in terms of homologous recombination and shuttling of integrated vectors.

Animals↗
PubMed1981

Short-lived cytoplasmic regulators of gene expression in cell cybrids.

Somatic cell hybridization is a valuable tool for investigation the control of gene expression in eukaryotic cells. Studies of hybrid cells, heterokaryons, reconstructed cells and cybrids (cytoplasmic hybrids) have suggested that cytoplasmic factors may be involved in this regulatory process. Unfortunately, studies of this kind usually require that hybrid or modified cells be maintained for some time in a selective environment during which chromosomal losses or other changes may modify the genetic functions of the cells and thus vitiate conclusions about the mechanism of gene regulation. We report here the preparation of cybrids between enucleated mouse fibroblasts (Cl-1-D) and differentiated rat hepatoma cells (Fao) and the use of a combination of histological techniques to identify these modified cells early after fusion without the use of selective media. We found that albumin production in most cybrids was suppressed (extinguished) at 12-20 h after fusion but was restored by 48 h. These results suggest that there is a cytoplasmic factor in the fibroblast which exerts negative control over expression of the albumin gene, but which in the absence of the fibroblast nucleus, is not renewed and therefore short-lived.

Albumins↗
PubMed1980

A cell surface molecule involved in aggregation of embryonic liver cells.

Aggragation of chicken enbryo hepatocytes can be inhibited by Fab' fragments of antibodies prepared against the cells. An aqueous extract of liver cell membranes contained antigens that neutralized the adhesion-blocking properties of the Fab' fragments. This neutralization activity was associated with a polypeptide of Mr68,000 in NaDodSO4; the polypeptide was distinct from serum albumin. Specific antibodies prepared against the 80-fold purified active fraction inhibited liver cell adhesion and immunoprecipitated the 68,000 Mr polypeptide from active fractions as well as from a detergent extract of liver cell membranes. In hepatocyte cultures, Fab' fragments of antibodies against the liver molecule prevented both colony formation and appearance of histotypic patterns. Liver cell adhesion was compared at the cellular and molecular levels to that of embryonic neural retina cells. Antibodies against the cell adhesion molecule from neural tissue inhibited retinal but not liver cell aggregation; conversely, antibodies against the liver polypeptide inhibited liver but not retinal cell aggregation. By means of antibody absorption and immunoprecipitation, it was confirmed that the two cell adhesion molecules are antigenically unrelated.

Animals↗
PubMed1977

Expression of differentiated functions in hepatoma cell hybrids: selection in glucose-free media of segregated hybrid cells which reexpress gluconeogenic enzymes.

Selective glucose-free media have been used to study the reexpression of liver-specific gluconeogenic enzymes in rat hepatoma X mouse lymphoblastoma somatic hybrids. The utilization for gluconeogenesis of dihydroxyacetone or oxaloacetate requires two enzymes: fructose diphosphatase as well as either triokinase for the former or phosphoenolpyruvate carboxykinase for the latter. By sequential selection with these substrates, the reexpression of the three gluconeogenic enzymes has been dissociated. The reexpression of these enzymes is correlated with the loss of mouse chromosomes. In addition, the characterization of the parental forms of aldolase B, another liver-specific enzyme, shows that reexpression corresponds to the simultaneous production of the rat and mouse enzymes. These results demonstrate the chromosomal origin of extinction and suggest that activation of mouse silent genes which accompanies reexpression can occur without loss of the parental determinations. The hypothesis that determination involves regulatory rather than structural genes is discussed.

Alcohol Oxidoreductases↗
PubMed1977

A selective system for hepatoma cells producing gluconeogenic enzymes.

Gluconeogenesis is a liver-specific pathway which permits the synthesis of phosphorylated sugars from oxaloacetate, pyruvate, amino acids, or trioses. The absolute requirement for glucose or an alternative hexose which characterizes most mammalian cells probably reflects an inablility to perform gluconeogenesis rather than to generate sufficient energy by respiration alone. Cells of diverse histogenetic origins have been tested in glucose-free medium, supplemented with oxaloacetate or with dihydroxyacetone. The only cells able to grow are well-differentiated hepatoma cells which produce the relevant gluconeogenic enzymes: phosphoenolpyruvate carboxykinase, fructose diphosphatase, and triokinase. Reconstruction experiments demonstrate that glucose-free media permit the selective growth of cells producing gluconeogenic enzymes. These media should be useful for analysis of reexpression of differentiated functions in somatic cell hybrids and for the isolation of mutants.

Carcinoma, Hepatocellular↗