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J Cervera

Publications and source records attributed to J Cervera.

At least 55 records · Page 3Linked to original sources
PubMed1995

Phosphorylation of the Goodpasture antigen by type A protein kinases.

Collagen IV is the major component of basement membranes. The human alpha 3 chain of collagen IV contains an antigenic domain called the Goodpasture antigen that is the target for the circulating immunopathogenic antibodies present in patients with Goodpasture syndrome. Characteristically, the gene region encoding the Goodpasture antigen generates multiple alternative products that retain the antigen amino-terminal region with a five-residue motif (KRGDS). The serine therein appears to be the major in vitro cAMP-dependent protein kinase phosphorylation site in the isolated antigen and can be phosphorylated in vitro by two protein kinases of approximately 50 and 41 kDa associated with human kidney plasma membrane, suggesting that it can also be phosphorylated in vivo. Consistent with this, the Goodpasture antigen is isolated from human kidney in phosphorylated and non-phosphorylated forms and only the non-phosphorylated form is susceptible to phosphorylation in vitro. Since this motif is exclusive to the human alpha 3(IV) chain and includes the RGD cell adhesion motif, its phosphorylation might play a role in pathogenesis and influence cell attachment to basement membrane.

Amino Acid Sequence↗
PubMed1995

Characterization and expression of multiple alternatively spliced transcripts of the Goodpasture antigen gene region. Goodpasture antibodies recognize recombinant proteins representing the autoantigen and one of its alternative forms.

Collagen IV, the major component of basement membranes, is composed of six distinct alpha chains (alpha 1-alpha 6). Atypically among the collagen IV genes, the exons encoding the carboxyl-terminal region of the human alpha 3(IV) chain undergo alternative splicing. This region has been designated as the Goodpasture antigen because of its reactivity in the kidney and lung with the pathogenic autoantibodies causing Goodpasture syndrome. The data presented in this report demonstrate that, in human kidney, the gene region encompassing the Goodpasture antigen generates at least six alternatively spliced transcripts predicting five distinct proteins that differ in their carboxyl-terminus and retain, except in one case, the exon that harbors the characteristic amino-terminus of the antigen. Goodpasture antibodies specifically recognize recombinant proteins representing the antigen and the alternative form that retains the amino-half of the antigen, suggesting that this moiety could be involved in the in vivo binding of the pathogenic antibodies. Furthermore, the sera of control individuals contain autoantibodies against the antigen that can be differentiated from those causing the syndrome based on their specific reactivities, suggesting that the binding of the pathogenic autoantibodies to a specific determinant likely trigger a distinct and unique cascade of events causing the disease.

Alternative Splicing↗
PubMed1993

The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry.

Differential scanning calorimetry of Escherichia coli carbamoyl-phosphate synthetase and its isolated large and small subunits reveals in each case an irreversible, kinetically controlled transition, at a temperature 14 degrees C higher for the holoenzyme than for the subunits, indicating dramatic stabilization of the subunits in the heterodimer. The deletion of the COOH-terminal 171 (mutant CarB'2373) or 385 (mutant CarB2177) residues of the large subunit results in more asymmetric transitions at a temperature 7 degrees C lower than for the wild type. The allosteric effectors IMP, UMP, and ornithine induce small reversible transitions at low temperature in the endotherm for the wild-type enzyme, but not for CarB'2373, as expected if the effectors bind in the 171-residue, COOH-terminal region. In contrast, two ligands that bind outside the deleted region, Ap5A (a ligand of both ATP sites) and glycine (an analog of glutamine) decrease and increase, respectively, the stability of the two mutants and of the wild type. The stabilization by glycine requires that the subunits are associated. The results support the implication of the 20-kDa COOH-terminal domain of the large subunit in the allosteric modulation by all the effectors and are consistent with the folding of the large subunit as a pseudohomodimer of its two homologous halves.

Allosteric Regulation↗
PubMed1993

Chronic pain in the spinal cord injured: statistical approach and pharmacological treatment.

We include in this article the results of a postal inquiry into chronic pain in SCI patients in Valencia (Spain), and our experience with their management. A mailed questionnaire including lesion and chronic pain data was sent to all of the 380 SCI patients who live in the region of Valencia. We received 202 answers, with 145 questionnaires being accurately answered and these were analysed for this study. The results show that chronic pain (that is, lasting more than 6 months) is very common (65.5%). The most frequent type was deafferentation pain (phantom pain), described as burning or a painful numbness. Since 1988 we have been treating a sample of 33 patients suffering from resistant pain according to the following therapies: 1 amitriptyline + clonazepam+NSAID (nonsteroidal antiinflammatory drugs); 2 amitriptyline + clonazepam + 5-OH-tryptophane + TENS (transcutaneous electrical nerve stimulation); 3 amitriptyline + clonazepam + SCS (spinal cord stimulation); 4 morphine, by continuous intrathecal infusion. After almost 4 years using these therapies we can affirm that the results regarding analgesia reached 80% in all cases, and that morphine used by intrathecal route is very safe and useful in selected patients.

Adolescent↗
PubMed1993

Mitosis and protein synthesis 4 turnover of short- and long-lived proteins during the cell cycle of synchronized L-132 and HeLa S-3 cells.

Turnover rates of short- and long-lived proteins throughout the cell cycle were measured in two established mammalian cell lines, L-132 and HeLa S-3, using both selection and induction synchronization. Short-lived, newly synthesized proteins turned over at the same rate during all stages of the cell cycle when this rate was expressed as a percentage of total labelling of proteins present at the start of the chase. Since fewer proteins were made in M-phase, the absolute turnover rate was probably reduced to a small degree during division itself, indicating a close co-ordination between synthesis and degradation. In contrast, long-lived proteins showed a considerable reduction in their rate of turnover specifically during M-phase. One possible explanation for the reduced degradation of long-lived protein is the suppression of lysosomal activity during division, with no apparent effect on the turnover of short-lived proteins which is due to a non-lysosomal system.

Cell Cycle↗
PubMed1992

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.

Adenosine Triphosphate↗
PubMed1991

Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain.

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

Vanadate inhibits degradation of short-lived, but not of long-lived, proteins in L-132 human cells.

Vanadate, at concentrations higher than 0.04 mM, inhibits the intracellular degradation of short-lived proteins in exponentially growing L-132 human cells. The inhibition is not due to a decrease in viability or in the ATP contents of the cells. Since vanadate decreases proteolysis in cell extracts, the inhibition appears to affect the proteinases which degrade these proteins. Under optimal nutritional conditions, the degradation of long-lived proteins is accelerated by vanadate, thus providing additional evidence that in exponentially growing cultured cells degradation of short- and long-lived proteins occurs by different processes. Vanadate also efficiently inhibits the lysosomal degradation of endocytosed proteins and of long-lived proteins under step-down conditions. However, this effect seems to be unrelated to the observed inhibition of degradation of short-lived proteins, because chloroquine and leupeptin, which inhibit degradation of proteins by lysosomes, do not modify the degradation of these proteins. Our results provide for the first time a probe which, owing to its opposite effects on the degradation of short- and long-lived proteins, could be useful to clarify the mechanisms involved in protein degradation in cultured cells.

Cell Division↗
PubMed1989

Effects of centrifugation on the degradation of short-lived proteins in exponentially growing cultured cells.

The degradation mechanisms of short-lived proteins in cultured cells are unknown, probably due to the lack of procedures which specifically affect the degradation of these proteins. We found that centrifugation of cultured cells, growing either in monolayer or in suspension, between 5000 and 25,000g for 30 min, inhibits (more than 50%) the degradation of short-lived proteins but not of long-lived proteins. Protein synthesis or cell viability is not affected. Centrifugation also disorganizes the Golgi apparatus, as checked by routine electron microscopy, and inhibits the degradation of endocytosed proteins (a lysosomal process which is controlled by the Golgi apparatus). Using different centrifugation speeds, a good correlation was found between alteration of the Golgi apparatus and inhibition of protein degradation.

Animals↗
PubMed1988

Modulation of the hydrophobicity of glutamine synthetase by mixed-function oxidation.

Oxidative modification of Escherichia coli glutamine synthetase renders the enzyme susceptible to proteolytic degradation by a specific protease purified from the bacterium; native enzyme is not a substrate for the protease. A model oxidizing system consisting of ascorbate, iron, and oxygen was used to generate a series of glutamine synthetases of increasing oxidative modification. We assessed the effect of oxidative modification on the surface hydrophobicity of the glutamine synthetases, utilizing hydrophobic chromatography on a phenyl matrix. Initial exposure to the oxidizing system caused inactivation of the enzyme and generated a protein that was more hydrophilic than the native form; it was not a substrate for the protease. Continued exposure to the oxidizing system yielded a protein with additional oxidative modification. This form was distinctly more hydrophobic than the native form and it was very susceptible to proteolytic attack by the purified protease. Thus, oxidative modification modulates the surface hydrophobicity of glutamine synthetase, and this modulation can control susceptibility to proteolysis.

Ascorbic Acid↗
PubMed1985

Endocytosis of cationized ferritin in human peripheral blood by resting T-lymphocytes.

We have examined the binding and internalization of cationized ferritin in T-lymphocytes of human peripheral blood, as a model for resting cells. After 30 min of incubation only 8% of endocytotic vesicles contain cationized ferritin. T-cells internalize the equivalent of their entire surface area in approximately 54 h, a longer time than is required by non-resting cells such as PHA-stimulated human lymphocytes. These tracer experiments suggest that the endocytosis of cationized ferritin by T-lymphocytes follows a lysosome pathway similar to that described for other cell types.

Biological Transport, Active↗
PubMed1985

Distribution of concanavalin-A receptor sites on the surface of human resting T lymphocytes. A stereological study using concanavalin-A/colloidal-gold-labelled horseradish peroxidase.

Stereologic techniques were used to analyse the density and distribution of Concanavalin-A (Con-A) receptor sites on the surface of isolated resting human peripheral-blood T lymphocytes using Con-A/colloidal-gold-labelled horseradish peroxidase. The T-lymphocyte surface appeared to be composed of microvilli, smooth areas and uncoated pits. Coated pits and coated vesicles, identified by the preferential staining of clathrin-containing membranes (tannic-acid/saponin fixation), were scarce. Quantitative analysis of the gold labelling on T lymphocytes after glutaraldehyde fixation indicated the presence of 2.13 +/- 0.46 gold particles per micron of cell surface and that these particles were preferentially located on uncoated pits. These results suggest the existence of cell-surface domains for these receptor sites in human resting T lymphocytes.

Coated Pits, Cell-Membrane↗
PubMed1985

Induction of self-tolerance and enhanced stress protein synthesis in L-132 cells by cadmium chloride and by hyperthermia.

The effect of heat shock or cadmium treatment on protein synthesis and cell survival in L-132 cells has been examined. After cadmium treatment, the synthesis of a polypeptide of Mr 68000 (P68) was greatly enhanced over that of untreated cells. Besides P68 the synthesis of another polypeptide of Mr 89000 (P89) was also enhanced in heat-shocked cells. Both heat shock and cadmium treatment induced self-tolerance. The kinetics of the synthesis of induced polypeptides correlated well with the development of self-tolerance. The patterns of peptide maps obtained after partial proteolytic digestion from P68 induced in cadmium-treated and heat-shocked cells were virtually identical. However, neither heat-shocked cells did not confer cadmium tolerance nor did cadmium-treated cells induce thermotolerance.

Actins↗
PubMed1985

The effects of zinc chloride on the RNP structures in HEp-2 cells: accumulation of perichromatin granules.

The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.

Carcinoma, Squamous Cell↗
PubMed1984

Methylation of nucleolar RNA in HeLa cells studied by autoradiography.

Methylation of nucleolar RNA was studied by autoradiography in HeLa cells using L-[methyl-3H]methionine and S-adenosyl-L-[methyl-3H]methionine as radioactive precursors. Pulse-labeling experiments show that nucleolar RNA methylation occurs on the newly synthesized RNA at the nucleolar fibrillar RNP component and mostly on the fibrillar ring of fibrillar centers, where pre-rRNA is being synthesized. Pulse-chase experiments show a shift of silver grains from the nucleolar fibrillar RNP component to the nucleolar granular component first and then to the cytoplasm. Labeling of nucleolar RNA via specific methylation permits the study of intranucleolar processing of pre-rRNA and confirms the sequence of labeling of the two nucleolar RNP components observed with radioactive uridine.

Autoradiography↗
PubMed1984

Increased leucocyte histamine release by Entamoeba histolytica antigen in patients with amoebic abscess of the liver.

Leucocytes (basophils) from non-atopic adult subjects living in an area highly endemic with Entamoeba histolytica release histamine in a dose-dependent fashion upon in vitro exposure to an antigen of axenically grown E. histolytica (histolyticin). Leucocytes of patients with acute amoebic liver abscess were significantly more sensitive to this antigen than leucocytes of control subjects, including patients that had recovered from amoebic liver abscess. By comparison Concanavalin-A induced histamine release found in patients with amoebic liver abscess and healthy controls suggest an immunological mechanism for histolyticin induced in vitro histamine release. This is also suggested by the inability of histolyticin to release histamine from leucocytes of healthy newborn infants and the significant fall in sensitivity to histolyticin following incubation of leucocytes in acid pH. Histamine and other mediators may contribute locally to the early intense inflammatory reaction observed in tissue invasion by E. histolytica.

Adolescent↗
PubMed1983

Cytosine arabinoside induced liver damage: histopathologic demonstration.

Two patients with acute leukemia developed abnormal liver function tests after the administration of cytosine arabinoside. Other possible causes for such abnormalities were not likely. In both patients a close chronologic relationship between the administration of the drug and the appearance of the laboratory abnormalities was noted. The liver damage was also documented by biopsy. To our knowledge this is the first time that the hepatotoxic effects of cytosine arabinoside are histologically demonstrated.

Adult↗