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Biomedical subjects

M Vidal

Publications and source records attributed to M Vidal.

At least 127 records · Page 7Linked to original sources
PubMed1995

E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle.

The E2F transcription factors play a role in regulating the expression of genes required for cell proliferation. Their activity appears to be regulated by association with the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130. In vivo, pRb is found in complex with a subset of E2F components--namely, E2F-1, E2F-2, and E2F-3. Here we describe the characterization of cDNAs encoding two unusual E2Fs, E2F-4 and E2F-5, each identified by the ability of their gene product to interact with p130 in a yeast two-hybrid system. E2F-4 and -5 share common sequences with E2F-1, E2F-2, and E2F-3 and, like these other E2Fs, the ability to heterodimerize with DP-1, thereby acquiring the ability to bind an E2F DNA recognition sequence with high affinity. However, in contrast to E2F-1, E2F-4 and E2F-5 fail to bind pRb in a two-hybrid assay. Moreover, they show a unique pattern of expression in synchronized human keratinocytes: E2F-4 and E2F-5 mRNA expression is maximal in mid-G1 phase before E2F-1 expression is detectable. These findings suggest that E2F-4 and E2F-5 may contribute to the regulation of early G1 events including the G0/G1 transition.

Amino Acid Sequence↗
PubMed1995

A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene.

The GTPase-activating protein related domain of the human neurofibromatosis type 1 protein (NF1GRD) can down-regulate RAS in Saccharomyces cerevisiae. Using a technique termed the FASAY method, for Functional Analysis of Separated Alleles in Yeast, we designed a rapid method for detection of heterozygous NF1GRD loss-of-function mutations. In our method, PCR amplified NF1GRD cDNA is directly cloned into a centromeric vector by homologous recombination in a cdc25 temperature-sensitive mutant strain expressing human Ha-ras. This strain is dependent on the Ha-ras for growth, allowing a simple growth assay for NF1GRD loss-of-function mutations. In a test of our method, two alternatively spliced NF1GRD cDNAs (type I and II) inhibited yeast growth whereas four mutants with amino acid substitutions at highly conserved residues did not. This simple method thus permits the rapid screening for heterozygous germline or somatic NF1GRD mutations. In an initial application of this method, no mutations disrupting NF1GRD function were detected in lymphoblasts from 11 previously untested neurofibromatosis type 1 patients.

Base Sequence↗
PubMed1995

Characterization of phospholipase A2 activity in reticulocyte endocytic vesicles.

Electron spin resonance spectroscopy was used to investigate the presence of phospholipase A2 activity in endocytic vesicles prepared from reticulocytes and to define some of its characteristics. Using spin-labeled phospholipid analogues, we measured the hydrolysis rate of the ester bond at position 2 during incubation with reticulocyte endocytic vesicles. We have shown that this phospholipase A2 activity was membrane-associated, enriched in endocytic vesicles as compared to cytosol and plasma membrane. Enzymic activity was also observed in exosomes, vesicles coming from the endocytic compartment and released by reticulocytes during their maturation in erythrocytes. Neither the hydrolytic activity nor the membrane association was found to be Ca(2+)-dependent. Spin-labeled phospholipids with choline and serine polar heads were better substrates than glycerophosphoethanolamine analogues. Optimal pH was found to be close to neutral. 5,5'-Dithiobis(2-nitrobenzoic acid) and diisopropyl fluorophosphate very efficiently inhibited spin-labeled phospholipid hydrolysis. This phospholipase A2 activity was confirmed using a radioactive assay, although with much lower sensitivity. (E)-6-(Bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a specific mechanism-based inhibitor of calcium-independent phospholipases A2, was found to abolish the enzymic activity present in endocytic vesicles.

Animals↗
PubMed1995

Effects of an individual intensive educational control program for insulin-dependent diabetic subjects with poor metabolic control.

The aim of our study was to evaluate the efficiency of an individual intensive educational control program on improving the metabolic control of insulin-dependent diabetic patients at short- and long-term follow-up. Fifteen insulin-dependent diabetic subjects with poor metabolic control (hemoglobin A1c > 9%) were included. At entry, their knowledge of diabetes (DKQ2 test), total energy intake and its distribution, insulin schedule, technical skill for insulin administration and self monitoring of blood glucose were evaluated. According to the initial evaluation, individual goals were stipulated and monitored in weekly visits. Individual life-style was particularly kept in mind. Thereafter, patients were switched to our ambulatory clinic for outpatients. At 1, 6, 12 and 24 months of follow-up, the items analyzed at the beginning were reevaluated. After 1 month, the program produced a significant decrease in hemoglobin A1c and an increase in knowledge of diabetes. The same beneficial effects were present at 6, 12 and 24 months evaluation compared to those values recorded at entry. There were neither major changes in dietary intake nor insulin schedule nor any increase in the frequency of hypoglycemic episodes. In conclusion, our program (5.2 +/- 0.8 weekly visits) significantly reduced and sustained hemoglobin A1c values close to those levels recommended by multicenter controlled trials. We consider that our program produced two major changes: a long-lasting improvement in knowledge of diabetes and an increase in self-monitoring blood glucose which provided the key for optimal self-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Tissue-specific and efficient expression of the human simple epithelial keratin 8 gene in transgenic mice.

Keratin 8 is a type II intermediate filament protein found in simple epithelia. We have introduced a 12 kb DNA fragment of the human K8 locus into the germ line of mice. The transgene, containing 1.1 kb of 5' flanking sequences, 7.7 kb corresponding to the body of the gene and 3.2 kb of 3' flanking sequences, was expressed in all six lines obtained. Immunolocalization and RNA analysis of adult tissues showed that the tissue-specific expression pattern of the transgene was almost indistinguishable from that of the endogenous gene. This pattern was found in organs containing single epithelial cell types, such as trachea, lung, stomach, intestine, liver, kidney, thymus and glands. The highest expressing line, however, also produced human K8 in tissues such as stratified epithelia, where it formed part of the pre-existing keratin cytoskeleton of basal cells. Steady state levels of human K8 RNA were proportional to the copy number of the transgene, but transgene expression was less efficient, per gene copy, than that of the endogenous gene. When in the 12 kb DNA fragment the exons and introns of the gene were replaced by the Escherichia coli lacZ gene, the resulting construct showed no expression in transgenic mice. This suggests that 5' and 3' flanking sequences, in the absence of intragenic sequences, are not sufficient for K8 expression and that important control elements are located in the body of the K8 gene.

Animals↗
PubMed1995

A point mutation in the RDS-peripherin gene in a Spanish family with central areolar choroidal dystrophy.

The RDS-peripherin gene encodes a photoreceptor-specific protein that is localized in the outer segment disc membranes of both rods and cones. We screened a Spanish family with central areolar choroidal dystrophy for mutations in candidate genes. A base substitution was identified in the RDS-peripherin gene of one patient and DNA sequencing revealed a C-to-T transition in codon 172, arginine being substituted by tryptophan. The mutation was also detected in two asymptomatic family members who showed irregular pigmentation in the retinal pigment epithelium (RPE). The phenotype is similar to other macular dystrophies caused by mutation in the RDS-peripherin gene.

Adult↗
PubMed1995

A GTP-binding protein modulates a Ca2+ pump present in reticulocyte endocytic vesicles.

Rat reticulocytes were used to prepare endocytic vesicles to study calcium fluxes across the endosomal membrane. We used 45Ca2+, and found that endocytic vesicles from reticulocytes present a Ca(2+)-ATPase that pumps Ca2+ into the lumen of vesicles. This activity was sensitive to vanadate and the calmodulin antagonists: trifluoperazine and calmidazolium. Western blot analysis using a monoclonal antibody evidenced that Ca(2+)-ATPase present in reticulocyte endocytic vesicles is probably the same as the erythrocyte Ca2+ pump. Ca2+ pump activity was shown to be partially inhibited by GTP gamma S. Moreover, mastoparan and benzalkonium chloride, both activators of heterotrimeric G proteins, were found to decrease 45Ca2+ uptake by endocytic vesicles. These results suggest the involvement of a trimeric G protein in the modulation of Ca(2+)-ATPase.

Animals↗
PubMed1995

A rapid redistribution of transferrin receptors to the cell surface of L2C lymphocytes upon fixation of holoform transferrin.

The internalization and recycling kinetics of transferrin receptors in leukemic lymphocytes (L2C) differed in the absence or presence of ligand. At 37 degrees C in the absence of ligand, transferrin receptors were mainly distributed internaly. We demonstrated, using a sepharose-bead-Tf complex, the rapid recycling of unoccupied internal transferrin receptors was correlated with ligand binding to surface receptors. The recycling amplitude was related to the occupancy of iron ligated transferrin to plasma membrane surface receptors. Contrary to the results obtained with other cells, redistribution of Tf receptors was not triggered by binding of other ligands to their receptors.

Animals↗
PubMed1994

Selectable marker replacement in Saccharomyces cerevisiae.

Selectable markers integrated by the 'gamma' deletion method (Sikorski and Hieter, 1989) can be efficiently replaced in vivo with other markers by transformation with homologous plasmids. Transformation frequencies in experiments designed to replace original selectable markers with an alternate marker were high and molecular analysis confirmed that all transformants that exhibited the expected phenotypes (loss of the original prototrophy and gain of the alternate prototrophy) resulted from homologous recombination between plasmid sequences at the target locus. This technique involves no plasmid construction and greatly facilitates the generation of yeast cells containing multiple gene disruptions.

Genes, Fungal↗
PubMed1994

Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development.

Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes. When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface. As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed. To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice. In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5. Proximal sequences from -1300 bp to +138 bp were inactive. We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5. Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages. This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene. These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult.

Aging↗
PubMed1994

Impaired left ventricular filling rate induced by treatment with recombinant interleukin 2 for advanced cancer.

BACKGROUND: Immunotherapy with recombinant interleukin 2 (rIL 2) has been extensively used to treat cancer but its use has been hampered by serious side effects including severe hypotension, arrhythmias, and myocardial infarction. OBJECTIVE: To assess the effects of rIL 2 on human left ventricular function. METHODS: Left ventricular (LV) function was monitored in 22 patients (9 women, 13 men) (mean (SD) age 53 (10) years) undergoing a 120 h continuous intravenous infusion of rIL 2 (18 x 10(6) IU/m2/day) for melanoma (4), renal cell (16), ovarian (1), and colon cancer (1). Radionuclide ventriculography was performed before and 1 h after the end of treatment. Ejection fraction (EF), peak emptying rate (PER), peak filling rate (PFR), and regional left ventricular wall motion were analysed. Heart rate (HR), central venous pressure (CVP), systolic (SBP) and diastolic blood pressures (DBP), the electrocardiogram, and myocardial enzyme concentrations were monitored throughout the study. RESULTS: All variables (mean (SD)) were normal before rIL 2 was given. After rIL 2 administration HR increased significantly from 84 (11) to 125 (18) beats/min (p < 0.0001), SBP fell from 128 (11) to 100 (9) mmHg (p < 0.001) and DBP from 76 (9) to 65 (7) mmHg (p < 0.0001). CVP decreased from 3.70 (3.2) to 1.30 (0.45) cm H2O (p < 0.001). EF (65 (7) to 64 (8%) and PER (3.56 (0.60) to 3.86 (0.83) EDV/s) did not change significantly. PFR decreased significantly at the end of the rIL 2 infusion from 2.68 (0.46) to 2.37 (0.43) EDV/s (p < 0.01). Left ventricular segmental hypokinesia developed in 6 patients. Myocardial enzyme concentrations remained normal throughout the study. CONCLUSIONS: The results of this study confirmed that rIL 2 produces important haemodynamic changes, predominantly related to decreased systemic resistance. However, the observed reduction in PFR in most patients suggested that rIL 2 might exert its action at the level of the heart muscle itself. The localised systolic dysfunction in some patients suggested that rIL 2 might also adversely affect myocardial perfusion.

Blood Pressure↗
PubMed1994

The endothelium and vascular effects of the ACE inhibitor trandolaprilat.

Experiments were designed to study the effects of trandolaprilat on endothelium-dependent responses in isolated blood vessels. Rings of either femoral or left circumflex coronary arteries of the dog or thoracic aortas of normotensive rats were suspended in organ chambers for isometric tension recording. During contractions induced by prostaglandin F2 alpha, trandolaprilat did not cause direct endothelium-dependent or -independent relaxation. However, when given to preparations incubated with angiotensin I or bradykinin, the compound evoked significant endothelium-dependent relaxation. By contrast, trandolaprilat failed to cause any change in tension when given in the presence of acetylcholine (ACh). In rings of femoral arteries, trandolaprilat potentiated the endothelium-dependent relaxation evoked by bradykinin and adenosine diphosphate; it did not modify the endothelium-dependent relaxations induced by ACh, substance P, or thrombin. In rings of femoral arteries without endothelium, trandolaprilat augmented relaxation induced by adenosine diphosphate (ADP) but not by adenosine. In perfused coronary arteries with but not those without endothelium, trandolaprilat caused relaxation in the absence of exogenous bradykinin (or ADP). These experiments suggest that trandolaprilat does not directly release endothelium-derived relaxing factor from the endothelial cells, does not interfere with the ability of the endothelium to release endothelium-derived relaxing factor, augments the endothelium-dependent responses to bradykinin (given exogenously or produced locally) and angiotensin I by direct interaction with converting enzyme, and potentiates the relaxation induced by ADP by augmenting its direct effect on vascular smooth muscle.

Adenosine Diphosphate↗
PubMed1993

Functional analysis of the human neurofilament light chain gene promoter.

We have carried out a structural and functional analysis on the human NF-L (H-NF-L) gene. It contains a methylation-free island, spanning the 5' flanking sequences and the first exon and a number of neuronal-specific DNase I hypersensitive sites have been identified in the upstream region as well as within the body of the gene. Analysis in cell lines and transgenic mice using a combination of these sites has revealed the presence of a conserved element(s) between -300bp and -190bp which is required for neuronal-specific expression.

Animals↗
PubMed1993

Involvement of CD56/N-CAM molecule in the adhesion of human solid tumor cell lines to endothelial cells.

Tumor-endothelial cell adhesion has been investigated, as this event might represent one of the first steps in the generation of tumor metastasis. We focused our attention on the N-CAM homologous CD56 (besides the CD44, CD15, and CDw49d antigens), as we have recently reported that it can mediate lymphocyte-endothelial cell adhesion [11]. Herein we show that CD56 is involved in tumor cell-endothelial cell binding. This phenomenon is independent of Ca2+ and unlikely to be influenced by the degree of CD56 sialylation.

Antigens, CD↗
PubMed1993

Uses of beta-galactosidase tag in on-line monitoring production of fusion proteins and gene expression in Escherichia coli.

A simple method for monitoring and quantifying automatically the production by fermentation of beta-galactosidase fusion proteins, making use of the remaining activity of the beta-galactosidase part, is considered. A hybrid protein carrying the major antigenic domain of foot-and-mouth disease virus C1 joined at the N-terminus of beta-galactosidase has been expressed in Escherichia coli. The yield of the chimeric protein has been monitored by flow injection analysis (FIA) during batch fermentations at laboratory scale, and a high correlation between values of product concentration from FIA and from immunological quantizations has been obtained. Because of the possibility of employing FIA in large-scale experiments, and the high sampling frequency, versatility, and reproducibility offered by this method, we propose FIA as a general, simple, quick, flexible, and reliable instrument for both monitoring the yield of recombinant proteins produced industrially, and performing basic research at laboratory scale.

Aphthovirus↗
PubMed1993

Enhanced production of pL-controlled recombinant proteins and plasmid stability in Escherichia coli RecA+ strains.

Overexpression of pL-controlled foot-and-mouth disease virus recombinant proteins was studied in Escherichia coli RecA+ strains and in a recA mutant. Higher protein yield and extractable plasmid DNA amounts were found in wild type cells, in absence of detectable RecA proteolytic activity. Minor but still significant differences in pBR322 DNA amounts were also detected between RecA+ and its recA13 and lexA1 derivatives. These data should be seriously considered to select expression systems and to design production processes for recombinant proteins, specially if they are expected to be toxic for Escherichia coli cells.

Aphthovirus↗
PubMed1993

Screening patients for heterozygous p53 mutations using a functional assay in yeast.

Inherited mutations of the p53 gene significantly increase the risk of developing diverse malignancies, and germline p53 mutations can be detected by assaying the transcriptional activity of the p53 protein in mammalian cells. Here we describe a method starting with lymphocytes that allows detection of germline p53 mutations by 'functional' analysis of p53 protein expressed in Saccharomyces cerevisiae. The p53 PCR products are directly cloned into yeast expression vectors in vivo and subsequently tested for transcriptional activity in a simple growth assay. This technique, functional analysis of separated alleles in yeast (FASAY), requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.

Animals↗