Search PubMed⌕ Search

Biomedical subjects

M Vidal

Publications and source records attributed to M Vidal.

At least 19 recordsLinked to original sources
PubMed1995

In vitro fusion of reticulocyte endocytic vesicles with liposomes.

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Animals↗
PubMed1995

A 5'-upstream region of a bovine keratin 6 gene confers tissue-specific expression and hyperproliferation-related induction in transgenic mice.

Keratins, the constituents of epithelial intermediate filaments, are precisely regulated in a tissue- and development-specific manner, although little is known about the molecular mechanisms underlying this regulation. The expression pattern of keratin 6 is particularly complex, since besides being constitutively expressed in hair follicles and in suprabasal cells of a variety of internal stratified epithelia, it is induced in epidermis in both natural and artificially caused hyperproliferative situations. Therefore, the regulatory sequences controlling keratin 6 gene activity are particularly suitable for target gene expression in a tissue-specific manner. More interestingly, they can be skin-induced in transgenic animals or in gene therapy protocols, particularly those addressing epidermal hyperproliferative disorders. To delimit the regions containing these regulatory elements, different parts of the bovine keratin 6 gene linked to a beta-galactosidase reporter gene have been assayed in transgenic mice. A 9-kbp fragment from the 5' upstream region was able to provide both suprabasal tissue-specific and inducible reporter expression.

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↗
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

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

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

Endothelium-dependent effects of converting-enzyme inhibitors.

Angiotensin-converting enzyme (ACE) inhibitors were designed to prevent the vasoconstrictor influence of the activated renin-angiotensin system. However, it has long been suspected that the vasodilator actions of these compounds are not entirely related to inhibition of the generation of angiotensin II. Bradykinin, which is rapidly degraded by ACE, stimulates the release of endothelium-derived vasodilator mediators, including nitric oxide, endothelium-derived hyperpolarizing factor, and prostacyclin. These mediators do not contribute to the vasodilator effect of bradykinin in every arterial bed. However, the prevention by ACE inhibitors of the degradation of bradykinin-induces an augmentation of the production of these substances and thus potentiates the dilatation evoked by the peptide. The existence of a local kallikrein-kinin system in the vascular wall has been demonstrated, and locally generated kinins contribute to the acute vasodilator actions of ACE inhibitors. ACE inhibitors can potentiate endothelium-dependent dilatations evoked by neurohumoral mediators that are not substrates for ACE. Thus, the vasodilator properties of ACE inhibitors not only reflect inhibition of the renin-angiotensin system but also depend on the enhanced production of endothelium-derived mediators.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1992

A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F.

The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3, was identified by the ability of its gene product to interact with pRB. RBP3 bound to pRB both in vitro and in vivo, and this binding was competed by viral proteins known to disrupt pRB-E2F association. RBP3 bound to E2F recognition sequences in a sequence-specific manner. Furthermore, transient expression of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein.

Amino Acid Sequence↗
PubMed1991

The influence of transferrin binding to L2C guinea pig leukemic lymphocytes on the endocytosis cycle kinetics of its receptor.

The parameters regulating the internalization and recycling of transferrin-specific receptors were determined in guinea pig leukemic B lymphocytes, in the absence or presence of ligand. We show that after the cells were purified, 45-56% of the total receptors were on the cell surface. In the absence of transferrin, unoccupied receptors are quickly internalized (rate constant, 0.12 min-1) whereas their recycling is much slower (rate constant, 0.026 min-1). This difference between endocytosis and recycling rates leads to a balanced receptor distribution with only 22% of the total receptors outside after incubation of the cells for 20-30 min at 37 degrees C. The internalization rate of occupied receptors, measured in the presence of transferrin is faster (rate constant, 0.21 min-1) than that of unoccupied receptors calculated in the absence of transferrin (0.12 min-1; see above). On the other hand, mere binding of transferrin to its receptor, without internalization, arrested by cytoplasm acidification, is sufficient to induce a large increase (by a factor of seven) in the recycling rate of unoccupied internal receptors from 0.026 min-1 to 0.17 min-1. Thus, in these lymphocytes, transferrin mobilizes internal receptors by modifying the kinetic rates of internalization and recycling, leading to a new equilibrium between external and internal receptors.

Animals↗
PubMed1991

Molecular cloning and expression of the VP1 gene of foot-and-mouth disease virus C1 in E. coli: effect on bacterial cell viability.

The VP1 gene of foot-and-mouth disease virus (serotype C1) has been cloned in Escherichia coli Clts cells, under the control of the bacteriophage lambda pL promoter. The expressed VP1 protein was complete and non-fused, and its molecular weight was indistinguishable from that of the VP1 obtained from virions. Cells harbouring the recombinant vectors exhibited symptoms of plasmid instability and toxicity and died in a few weeks even when never exposed to inducing conditions. A new plasmid clone in which a segment of the VP1 gene was fused with contiguous genes of the viral genome was very stable. The expressed partial VP1 protein contains the two major immunogenic domains of the virion. This system can be used as a tool to design an immunogenic VP1, and to explore possible synthetic vaccines against foot-and-mouth disease.

Animals↗
PubMed1991

RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes.

We show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is dependent on RPD1 (and RPD3 [M. Vidal and R. F. Gaber, Mol. Cell. Biol. 11:6317-6327, 1991]). Genes regulated by stimuli as diverse as external signals (PHO5), cell differentiation processes (SPO11 and SPO13), cell type (RME1, FUS1, HO, TY2, STE6, STE3, and BAR1), and genes whose regulatory signals remain unknown (TRK2) depend on RPD1 to achieve maximal states of transcriptional regulation. RPD1 enhances both positive and negative regulation of these genes: in rpd1 delta mutants, higher levels of expression are observed under repression conditions and lower levels are observed under activation conditions. We show that several independent genetic screens, designed to identify yeast transcriptional regulators, have detected the RPD1 locus (also known as SIN3, SD11, and UME4). The inferred RPD1 protein contains four regions predicted to take on helix-loop-helix-like secondary structures and three regions (acidic, glutamine rich, and proline rich) reminiscent of the activating domains of transcriptional activators.

Alleles↗
PubMed1991

RPD3 encodes a second factor required to achieve maximum positive and negative transcriptional states in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, TRK1 and TRK2 encode the high- and low-affinity K+ transporters, respectively. In cells containing a deletion of TRK1, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels of K+ (Trk- phenotype). Recessive mutations in RPD1 and RPD3 suppress the TRK2, conferring an approximately fourfold increase in transcription. rpd3 mutations confer pleiotropic phenotypes, including (i) mating defects, (ii) hypersensitivity to cycloheximide, (iii) inability to sporulate as homozygous diploids, and (iv) constitutive derepression of acid phosphatase. RPD3 was cloned and is predicted to encode a 48-kDa protein with no extensive similarity to proteins contained in current data bases. Deletion of RPD3 is not lethal but confers phenotypes identical to those caused by spontaneous mutations. RPD3 is required for both full repression and full activation of transcription of target genes including PHO5, STE6, and TY2. RPD3 is the second gene required for this function, since RPD1 is also required. The effects of mutations in RPD1 and RPD3 are not additive, suggesting that these genes are involved in the same transcriptional regulatory function or pathway.

Amino Acid Sequence↗
PubMed1991

Dissociation between endothelium-dependent relaxations and increases in cGMP in systemic veins.

In canine systemic veins, in contrast to what is observed in mammalian systemic arteries, endothelium-dependent relaxations to the calcium ionophore A23187 are not diminished by the inhibitor of soluble guanylate cyclase, methylene blue. Therefore, experiments were designed to determine whether these relaxations in the veins are associated with the accumulation of guanosine 3',5'-cyclic monophosphate (cGMP). Rings of canine femoral arteries and veins with and without endothelium were suspended for the measurement of isometric force in organ chambers; cGMP was measured by radioimmunoassay. In arteries and veins contracted with norepinephrine, the tissue content of cGMP was greater in rings with than without endothelium. This difference was decreased by methylene blue (10(-5) M). A23187 (3 X 10(-7) M, for 1 min) increased the accumulation of cGMP, which was temporally related with the onset of relaxation in tissues with endothelium. Methylene blue inhibited the accumulation of cGMP in both blood vessels but inhibited the relaxations only in the arteries. In rings without endothelium, sodium nitroprusside (3 X 10(-7) and 10(-5) M) initiated increases in cGMP, which followed the onset of relaxation. Neither response to sodium nitroprusside was reduced by methylene blue. These results suggest that in canine femoral arteries and veins, relaxation of the smooth muscle to sodium nitroprusside are mediated by a mechanism distinct from changes in cGMP. Likewise, in canine systemic veins, endothelium-derived factor(s) released in response to A23187 also can initiate relaxation of the smooth muscle by a mechanism distinct from changes in cGMP.

Animals↗
PubMed1991

Activation of T lymphocytes by cross-linking of glycophospholipid-anchored Thy-1 mobilizes separate pools of intracellular second messengers to those induced by the antigen-receptor/CD3 complex.

T cells can be activated, not only by the conventional (antigen-receptor/CD3 complex) route, but also by cross-linking any one of their lipid-anchored surface glycoproteins. We have compared early transmembrane signalling events mediated through CD3 with those mediated through Thy-1, a lipid-linked surface glycoprotein, on the human lymphoid cell line Jurkat and transfectants expressing higher levels of Thy-1. Cross-linking of Thy-1 causes immediate phosphatidylinositol (PI) turnover and an influx of extracellular Ca2+, while releasing very little Ca2+ from intracellular stores. CD3 activation, on the other hand, causes PI turnover which releases intracellular Ca2+, and only secondarily induces an influx of extracellular ions. The Thy-1 response is detectable at very low levels of surface Thy-1, and is not mimicked by enzymatic removal of lipid-linked proteins from the cell surface. The Thy-1-induced Ca2+ influx is more sensitive to L channel blockers than the CD3-mediated flux. These results indicate that the initial stages of Thy-1-mediated activation involve the rapid and extensive mobilization of the intracellular second messengers, PI and Ca2+, by mechanisms separate to those activated by the antigen-receptor/CD3 complex.

Antigens, CD↗