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F Cimino

Publications and source records attributed to F Cimino.

At least 37 records · Page 2Linked to original sources
PubMed1995

Differentially expressed mRNAs as a consequence of oxidative stress in intact cells.

Intracellular redox conditions influence the activity of several transcription factors leading to a modulation of the expression of the genes controlled by these factors. We examined the changes in cell transcription patterns after oxidative stress induced by diethylmaleate (DEM). Using the differential display technique we identified several differentially expressed sequence tags, four of which are identical or highly homologous to sequences contained in the human cDNAs encoding vimentin, c-fos, cytochrome oxidase IV and ribosomal protein L4; another one corresponds to a transcript of the mitochondrial genome of unknown function. The remaining five cDNAs are not recorded in any sequence data bank. One of these, named Rox3, lights up two mRNA species of approximately 3400 and 3600 bp, significantly increased after treatment with DEM or with other oxidizing agents. This increase appears precociously after exposure to DEM and it is completely prevented by pretreatment with N-acetylcysteine. The Rox3 fragment was used to screen a cDNA library; one fully sequenced clone showed 100% homology with the putative human guanine nucleotide regulatory protein nep1.

Cell Line↗
PubMed1995

DNA binding activity of the glucocorticoid receptor is sensitive to redox changes in intact cells.

The effect of changes of redox conditions on glucocorticoid receptor (GR) activity in intact cells has been studied using two approaches. One was to evaluate the GR-DNA binding in extracts of COS2 cells transiently overexpressing GR and in which reactive oxygen intermediates (ROI) accumulate as a consequence of glutathione (GSH) depletion. GR-DNA binding was significantly decreased in COS2 cells treated with diethylmaleate (DEM), which causes GSH depletion by forming GSH-DEM complexes. A similar effect was observed for Sp1, another Zn-finger transcription factor, whereas no difference was observed for the C/EBP transcription factor, which is known to be unaffected by redox changes in vitro. N-Acetylcysteine (NAC), which counteracts the effects of DEM by increasing GSH biosynthesis, prevents the decrease of GR-DNA binding in cells treated with DEM. The GR-DNA binding efficiency was similarly decreased using extracts from H2O2-treated COS2 cells and from COS2 cells treated with buthionine sulphoximine, which causes GSH depletion via a mechanism different from that of DEM. The other approach was to evaluate the efficiency of a GR-regulated promoter under different redox conditions. In HeLa cells, transfected with a plasmid containing the CAT gene under the control of the glucocorticoid responsive element (GRE) within the mouse mammary tumor virus promoter, and treated with dexamethasone to activate GR, exposure to DEM significantly impaired the activation of CAT gene expression induced by dexamethasone. Also in this case NAC treatment inhibited the effects of DEM.

Base Sequence↗
PubMed1995

Low flow anaesthesia reduces occupational exposure to inhalation anaesthetics. Environmental and biological measurements in operating room personnel.

In the present study we evaluated the occupational exposure to N2O and isoflurane during open circuit (OC) (fresh gas flow > or = minute volume) and low flow (LF) (fresh gas flow = 1.5 l/min) anaesthesia. The effects of active scavenging and of a charcoal filter positioned on the exhausting branch of the ventilator on environmental and urinary concentrations of inhalation anaesthetics were also investigated. The study was carried out in the same operating room provided with non-recirculating air changes (10 per hour). It involved anaesthetists and nurses during routine activity. N2O and isoflurane concentrations (time-weighted average) were measured after 3-hour continuous exposure: 1) in the environment at the level of the personnel's breathing zone (Ci); 2) in the environment at the ventilator zone (C); 3) in urine (Cu). During OC anaesthesia without active scavenging the breathing zone concentration of both N2O and isoflurane was very high (194.6 +/- 15.2 and 5.0 +/- 0.4 ppm, respectively). The activation of the scavenging greatly reduced the breathing zone concentration of N2O (31.6 +/- 4.1 ppm) and isoflurane (1.7 +/- 0.2 ppm). LF anaesthesia (with active scavenging) significantly reduced the environmental concentration of both anaesthetics (Ci N2O and isoflurane 22.7 +/- 1.8 and 0.6 +/- 0.04 ppm, respectively). During LF anaesthesia the breathing zone concentration of N2O remained low, even without scavenging (22.7 +/- 1.8 ppm). Similar results were obtained by measuring N2O and isoflurane concentrations at the ventilator zone and in urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Air Pollutants, Occupational↗
PubMed1994

The DNA sequence encompassing the transcription start site of a TATA-less promoter contains enough information to drive neuron-specific transcription.

The FE65 gene encodes a nuclear protein of unknown function that is expressed in several areas of the rat nervous system during development and in the adult animal, particularly in somatic and visceral ganglia. FE65 mRNA is abundant in neuronal cell lines, whereas it is barely detectable in non-neuronal cells. We identified the two transcription start sites of the FE65 gene and we isolated the rat genomic fragment containing one of these two transcriptional start sites. We demonstrate that this fragment contains a promoter able to direct an efficient transcription of a reporter gene in PC12 cells and in NTERA2 cells upon their differentiation with retinoic acid, whereas it functions poorly in non-neuronal cells, such as Rat2 fibroblasts and BRL hepatocytes. This promoter is composed of two regions. The first includes a cis-element whose removal greatly decreases the transcriptional efficiency in all cells examined and which forms similar complexes with proteins from PC12 and Rat2 cells. This cis-element binds Sp1 or another GC-binding factor. The second cis-element encompasses the transcription start site and is still able to direct transcription only in neuronal cells. The DNA-protein complexes formed by this cis-element in neuronal cells differ from those formed in non-neuronal cells. The analysis of point mutations in this region indicates that the proteins that bind to this cis-element interact with both overlapping and distinct nucleotide sequences.

Amino Acid Sequence↗
PubMed1994

The DNA-binding efficiency of Sp1 is affected by redox changes.

We have previously demonstrated that the DNA-binding efficiency of Sp1 is greatly decreased in nuclear extracts from 30-month-old rat tissues compared to those from young ones, although its gene appears to be normally expressed. As reactive oxygen intermediates are known to accumulate in aged animals, we investigated the effect of oxidation on the Sp1 DNA-binding activity. Electrophoretic mobility shift assays and DNase I footprintings showed that high concentrations of dithiothreitol, added to the aged tissue extracts, fully restore the Sp1 DNA-binding efficiency. However, in young nuclear extracts hydrogen peroxide treatment strongly decreases the Sp1 DNA-binding activity that is restored by the treatment with high dithiothreitol concentrations. To ascertain whether the oxidative stress is directed toward the Sp1 molecule alone, or whether it acts on unknown Sp1 cofactor(s) necessary for DNA binding, we purified Sp1 from young rat liver and demonstrated that when the purified protein is added to aged nuclear extracts, it efficiently binds to its DNA cis-element. Moreover, purified Sp1 treated with hydrogen peroxide lost its ability to bind its cis-element and the DNA-binding efficiency was fully restored after incubation with dithiothreitol.

Aging↗
PubMed1994

Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion.

We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12-myristate 13-acetate-induced activation of the transcription factors AP-1 and Egr-1, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins.

Antigens, CD↗
PubMed1994

Transcriptional regulation of the human H ferritin-encoding gene (FERH) in G418-treated cells: role of the B-box-binding factor.

We have analysed the molecular basis underlying the increase in ferritin heavy-chain mRNA (FERH) levels in cells exposed to the antibiotic Geneticin (G418). Transient transfection experiments demonstrate that this increase is paralleled by an enhanced transcription driven by the promoter (pFERH) for the human FERH gene, in which the most proximal promoter element (B-box) appears to play a key role. This region is conserved in human and rat, and binds an unknown factor. The DNA-protein complex composed of B-box-binding factor and its cis-element becomes more abundant in the G418-treated cells, as compared with the untreated ones.

Animals↗
PubMed1994

PCR analysis of the H ferritin multigene family reveals the existence of two classes of processed pseudogenes.

The human gene coding for the apoferritin H subunit belongs to a complex multigene family constituted by the expressed gene and by an undefined number of pseudogenes. We have used a strategy based on PCR to amplify specifically the H pseudogenes from a sample of human genomic DNA. With this approach, three new H pseudogenes have been cloned and characterized by DNA sequence analysis. In addition, we have identified a new type of pseudogene, the size of which (700 bp) is caused by multiple detection events in the putative coding region.

Base Sequence↗
PubMed1994

Expression of the neuron-specific FE65 gene marks the development of embryo ganglionic derivatives.

The major transcript of the FE65 gene is a neuron-specific mRNA that encodes a nuclear protein whose aminoterminal domain strongly activates the transcription of a reporter gene when fused to a heterologous DNA-binding domain. FE65 gene expression is regulated during neuronal differentiation of the NTERA-2 cell line, and it is temporally and spatially restricted during mouse embryo development. It is first detected around day 10 of gestation in the basal plate of the neural tube, and then, at the subsequent stages of development and in the newborn animals, it is found solely in neural structures. Its expression is most abundant in the neural crest derivatives (e.g. spinal and encephalic ganglia), ganglionic structures of sense organs (ganglionic layer of the retina and olfactory epithelium), as well as the ganglionic structures of the autonomic nervous system. Thus FE65 gene expression can be considered a marker of the development of embryo ganglionic derivatives.

Animals↗
PubMed1994

[Electric cardioversion in pregnant patients with obstructive hypertrophic cardiomyopathy. A clinical case].

A case of atrial fibrillation during pregnancy in a woman at 30 weeks of gestation is described. She was affected by obstructive hypertrophic myocardiopathy, a rare disease usually non interfering with the normal course of pregnancy. The mortality rate is of 2-4% per year. The most frequent cause is "cardiac sudden death". High frequency atrial fibrillation can lead to right and left cardiac failure. The treatment of atrial fibrillation is to be chosen between a pharmacological or electrical therapy. However, during pregnancy, pharmacological cardioversion, although not excluded, could cause damaging side effects to the fetus. Conversely, according to literature, electrical cardioversion is effective on the mother and safer for the fetus. At admission the patient showed dyspnea, palpitations, tachycardia and hypotensions; ECG showed an atrial fibrillation at high frequency that required an immediate therapeutic decision. In the case we studied, we reported the recovery of cardiac rhythm with a single synchronized electric shock. Non alteration of fetal cardiac rhythm or uterine contractility was observed. Pregnancy went on without accidents until the 36th week, when a cesarean section was performed under general anaesthesia. We described our experience reviewing previous literature on the account of the rareness of the disease, its association to a pregnancy and the serious alterations of cardiac rhythm at the admission, which led us to take an immediate decision, safeguarding both the mother and the fetus.

Adult↗
PubMed1993

Immortalization of a cell line showing some characteristics of the oligodendrocyte phenotype.

We have used the polyoma middle T oncogene to immortalize cells from rat embryo encephalon. Immunostaining experiments with monoclonal antibodies demonstrated that the cells of one of the obtained lines, named CEINGE CL3, are stained by anti-vimentin and anti-S100 antibodies, are not stained by anti-neurofilaments (NF) or anti-glial fibrillary acidic-protein (GFAP) antibodies. Only a subset of the CEINGE CL3 cells (20-30%) is stained by an anti-galactocerebroside antibody. Northern blot analysis demonstrated that these cells express low levels of proteolipid protein mRNA, whereas polymerase chain reaction (PCR) amplification failed to evidentiate the presence of both NF and GFAP mRNAs. Either retinoic acid or forskolin treatments or a combination of them are able to induce morphological changes that are accompanied by a complete growth arrest.

Animals↗
PubMed1993

Isolation and structural characterization of the rat gene encoding the brain specific snRNP-associated polypeptide "N".

We isolated the gene encoding the brain specific snRNP-associated polypeptide "N" from a rat genomic library. Some intronless genes have previously been reported for this polypeptide in the rat. In contrast, our gene consists of at least nine exons (the last exon(s) encoding the 3'-end of the mRNA has not been isolated). The second intron of the gene is probably so long as to hamper the cloning of the complete gene into a single phage vector. Therefore, we used polymerase chain reaction amplification of the rat genome with oligonucleotides designed on the basis of known cDNA sequences that allowed us to isolate and clone the first two exons at the 5'-end of the gene. Primer extension studies revealed multiple transcription start sites, all of them contained within the first exon. The intron/exon organization coincides with the alternative splicing events suggested by the sequences of the various cDNA species isolated so far. As also reported by others, genomic Southern analysis suggests the presence of other genomic regions containing sequences strongly hybridizing to the cDNA. We isolated and characterized two of these regions. They contain sequences very similar to the cDNA, not interrupted by introns and flanked by a polyA tail at their 3'-ends; hence they are considered two different pseudogenes.

Amino Acid Sequence↗
PubMed1992

Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues.

To explore the role of transcriptional factors in the genesis of the senescent phenotype, nuclear extracts from 4- and 30-month-old rat brains were analyzed for the presence of DNA-binding proteins able to interact with double-stranded oligonucleotides containing recognition sites for sequence-specific DNA-binding factors. Gel shift assays revealed that the DNA-binding efficiency of Sp1 is significantly reduced in aged animals compared to young ones, whereas CTF/NF1 and AP1 from young and old rat nuclear extracts bind their DNA targets with the same efficiency. The quantitative analysis of Sp1 by immunoblotting indicated that equivalent quantities and degrees of heterogeneity of Sp1 protein are present in both nuclear extracts, suggesting that the observed difference is not due to a different expression of this transcriptional factor. DNase I footprinting of the heavy chain ferritin gene promoter, which contains a Sp1 binding site, demonstrated that the nuclear extract from 30-month-old rat brain does not protect the region involved in the regulation of the H ferritin gene by Sp1. This results in a reduction of about 50% of the expression of the H ferritin mRNA in aged rat brains. Furthermore, the Sp1 binding sites present in the SV40 early promoter are not protected in a DNase I footprinting assay where a nuclear extract from 30-month-old rat brain was used as a source of DNA binding proteins. Liver nuclear extracts prepared from young and aged rats demonstrated that a decrease of Sp1 binding efficiency is similarly present in this tissue.

Aging↗
PubMed1992

Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization.

We conducted a functional analysis of the promoter for the human ferritin heavy chain-encoding gene (pFERH) in HepG2 and HeLa cells. The activity of pFERH is equivalent in both cell types, despite their different ferritin (Fer) isotypes. Transfections of a series of 5'-deletion mutants indicate that pFERH activity is essentially dependent on two motifs. One of them, accounting for about 50% of the total transcriptional activity, is recognized by the RNA polymerase II transcription factor, Sp1, and the other by a low-affinity factor present in both the cell types analyzed.

Base Sequence↗
PubMed1991

A rat brain mRNA encoding a transcriptional activator homologous to the DNA binding domain of retroviral integrases.

We have isolated a rat cDNA, named FE65, hybridizing to an mRNA of about 2,300 nucleotides present in rat brain, undetectable in rat liver and very poorly represented in other tissues. An mRNA of the same size is present in human neuroblastoma cells and is absent from other human cell lines. The FE65 cDNA contains an open reading frame (ORF) coding for a polypeptide of 499 amino acids in which 143 residues can be aligned with the DNA binding domain of the integrases encoded by mammalian immunodeficiency viruses. The remaining part of the FE65 ORF is not homologous with the correspondent regions of the integrases; the first 206 residues of the FE65 ORF show numerous negative charges and a short sequence not dispensable for the function of the transactivating acidic domain of the jun family transcriptional factors. A plasmid which expresses FE65 amino acids 1-232 fused to the yeast GAL4 DNA binding domain was co-transfected with a plasmid containing five GAL4 binding sites upstream of a minimal Adenovirus promoter controlling the expression of the CAT gene. This experiment showed that the fused protein GAL4-FE65 is able to obtain a 30-40 fold increase of the CAT gene expression compared to the expression observed in the presence of the GAL4 DNA binding domain alone. Two types of FE65 mRNA are present in rat brain, differing only for six nucleotides. We demonstrate that this is the consequence of a neuron-specific alternative splicing of a six-nucleotide miniexon, which is also present in the human genome, in an intron/exon context very similar to that of the rat FE65 gene.

Amino Acid Sequence↗