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J Avila

Publications and source records attributed to J Avila.

At least 253 records · Page 14Linked to original sources
PubMed1986

The interaction between a Na+-channel toxin and brain microtubule proteins in vitro.

The aim of this work was to characterize the interaction of the Na+-channel toxin, purified from venom of the scorpion Leiurus quinquestriatus, with microtubule proteins in vitro. The toxin enhanced microtubule assembly, causing the formation of microtubule 'bundles'. It interacted with the Na+-channel 270 kDa subunit and was subsequently found to be unrelated to high molecular weight microtubule-associated protein with respect to apparent molecular weight and toxin binding. However, the radiolabelled toxin bound to tubulin, although with a much lower affinity than that published for the reconstituted Na+-channel. This binding appears to occur in the carboxy terminal 4 kDa region of tubulin. These results may reflect a secondary action of the toxin involved in binding to its receptors in the neural plasma membrane.

Animals↗
PubMed1986

Analysis of drug-tubulin interaction by trypsin cleavage: comparison for colchicine, podophyllotoxin, griseofulvin, vinblastine and taxol.

Trypsin preferentially cleaves the alpha subunit of depolymerized tubulin or vinblastine induced aggregates (in which longitudinal interactions between tubulin molecules could take place). No cleavage was found for tubulin polymerized into microtubules (containing lateral and longitudinal tubulin interactions), in the presence of taxol. In the presence of colchicine or podophyllotoxin the alpha subunit was partially protected from proteolytic digestion. Trypsin digestion pattern varied upon the addition of different concentrations of griseofulvin. At the higher concentration used, in which microtubules assembly was inhibited, both tubulin subunits were cleaved.

Alkaloids↗
PubMed1986

Centromere pattern in different mouse seminiferous tubule cells.

Centromere arrangement in different mouse seminiferous tubule cells was analyzed using an anticentromere antiserum from a patient with the CREST syndrome of scleroderma. A peptide of 18 kd was recognized by this serum on immunoblotting of mouse nuclear proteins from seminiferous tubule cells. In the cells studied by immunofluorescence, different patterns of centromere arrangement were observed. A speckled arrangement of centromeres was found in spermatogonia, double spots corresponding to meiotic bivalents were found in pachytene cells, and clusters of a haploid numer of centromeres were found in early and acrosome phase spermatids. In Sertoli cells, only three centromeric spots were detected, corresponding to the nucleolar organizer chromosome pairs. A relationship between the functional stage of the cell and the arrangement of and conformational changes in the centromeres is considered.

Animals↗
PubMed1986

The localization of tau proteins on the microtubule surface.

The localization of porcine brain tau factor on in vitro assembled microtubules has been carried out by immunoelectron microscopy, using affinity-purified antibodies and protein A-gold particles. A parallel experiment was done using antibody against microtubule associated protein 2 (MAP2) and also a double labelling experiment using both antibodies with different sized gold particles. Our results indicate that, within the limits of resolution imposed by immunolabelling, the distribution patterns of tau factor and MAP2 on the microtubule are indistinguishable.

Animals↗
PubMed1985

Localization of the tubulin binding site for tau protein.

Limited proteolysis of tubulin with subtilisin resulted in the removal of the carboxyl-terminal moiety of tubulin subunits. The remaining peptides from both alpha and beta tubulin lacking the carboxyl terminal did not bind to tau factor nor to MAP2 or MAP1. The carboxyl-terminal fragments bind to tau factor and MAP2 and both compete for the same binding sites in the tubulin molecule. Our results suggest that the carboxyl-terminal region of tubulin is a regulatory domain for the assembly of tubulin and the site for interaction with MAPs.

Animals↗
PubMed1985

The interaction between subunits in the tubulin dimer.

Limited proteolysis and chemical cross-linking techniques have been used to study the interaction between alpha- and beta-tubulin subunits. Trypsin digestion of tubulin dimer resulted in the cleavage of the alpha-subunit into two fragments, whereas chymotrypsin cleaved the beta-subunit into two distinct fragments. All of these fragments have been mapped on the tubulin subunits by further proteolysis with formic acid. Cross-linking of trypsin- and chymotrypsin-cleaved subunits has been performed with two different cross-linker agents of different cross-linking distance. The addition of formaldehyde resulted in the cross-linking of the alpha-tubulin N-terminal fragment with beta-tubulin C-terminal domain. The same result was obtained when methyl 4-mercaptobutyrimidate was used.

Animals↗
PubMed1985

Differential association of the different brain microtubule proteins in different in vitro assembly conditions.

Microtubule protein was assembled in vitro for different time periods and at different protein concentrations near and far from the critical concentration needed for assembly. When the isolated polymers were characterized by electrophoresis, a higher association of high-molecular-weight microtubule-associated proteins, particularly of the larger microtubule-associated protein, was found in the polymers assembled close to the critical concentration, while tau factor polypeptides are predominantly present in those polymers assembled at higher protein concentration. Also, a higher proportion of high-molecular-weight microtubule associated proteins MAP1 and MAP2 was observed in microtubules assembled for short time periods, compared with those obtained after the monomer-polymer equilibrium was reached. When the assembled protein was further characterized by isoelectric focusing, no differences were found in the proportion of the different isotubulins present in the polymers assembled under different conditions tested.

Animals↗
PubMed1985

Quantitative determination of tubulin and characterization of tubulin forms during development in Drosophila melanogaster.

The proportion of tubulin and its isoform pattern have been analyzed at different stages in the development of Drosophila melanogaster. Tubulin proportion varied during development, the highest proportion being found at embryogenesis where two alpha- and beta- (one of them transitory) tubulin subunits were found. During the larval stage, the proportion of total tubulin decreased but new alpha-isotubulins were identified. These alpha-isotubulins were also present at the adult stage and all of them could be incorporated into microtubules assembled in vitro.

Animals↗
PubMed1985

A cell division mutant of Drosophila with a functionally abnormal spindle.

Normal distribution of chromosomes to daughter cells is insured by the proper functioning of the spindle. Homozygosity for a semi-lethal mutation of Drosophila melanogaster (abnormal spindle) altering this structure has the following effects: the mitotic cycle is arrested in metaphase, leading to a high frequency of polyploid cells; sex chromosome disjunction during male meiosis is severely affected, as revealed by the resulting exceptional (diplo and nullo) gametes (microscopic examination of spermiogenesis confirms this aberrant segregation); meiotic spindles of living cells are morphologically abnormal; and tubulins extracted from mutant larvae are normal in amount, electrophoretic mobility, and ability to form microtubules in vitro. The results suggest that the mutant phenotype is due to an altered structural component of the spindle other than tubulins.

Animals↗
PubMed1985

Homogeneity of lung tubulin isoforms during lung maturation.

The rat and rabbit lung isoform pattern before (fetal) and after (adult) lung maturation has been analyzed by isoelectric focusing. This pattern has been compared to that of brain tubulin at the same developmental stages. No changes in the pattern of fetal and adult lung tubulin were found. However an increase in brain tubulin heterogeneity was detected from fetal to adult stage.

Aging↗
PubMed1985

Localization and characterization of tubulin-like proteins associated with brain mitochondria: the presence of a membrane-specific isoform.

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.

Animals↗
PubMed1985

Effects of high doses of iodide on thyroid secretion: evidence for the presence of iodinated membrane tubulin.

The transient inhibitory effect on thyroid secretion produced by high doses of iodide was investigated with respect to changes in the level of in vivo iodination of membrane tubulin. Iodinated tubulin, characterized by gel electrophoresis, immunoprecipitation, and peptide mapping, was shown to be associated with a thyroid membrane fraction, and totally absent from cytoplasmic proteins. The administration of an acute dose of 5 mg KI, although it inhibited thyroid secretion (as shown by an increase in TSH), did not have a significant effect on the level of iodination of membrane tubulin. Thus, the observed inhibition of thyroid secretion is probably unrelated to the level of iodination of tubulin. Although its function is not known at present, iodinated tubulin is probably involved in membrane-related phenomena.

Animals↗
PubMed1985

Antibodies to vimentin intermediate filaments in sera from patients with SLE and RA: quantitation by solid phase radioimmunoassay.

A solid phase radioimmunoassay was used to quantify levels of antibodies to vimentin intermediate filaments (VIF) of the cytoskeleton in sera from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Significantly higher levels of these antibodies were found in 52% of the SLE sera studied, compared to 6% in RA sera and 10% of sera from healthy controls. These antibodies include IgG and IgM classes of immunoglobulins. Our results show that high levels of anti-VIF antibodies are more prevalent in SLE sera, whereas no significant differences are found between sera from patients with RA and healthy controls.

Adult↗
PubMed1985

Human vimentin autoantibodies preferentially interact with a peptide of 30kD mol. wt, located close to the amino-terminal of the molecule.

Patients of some rheumatic autoimmune diseases like systemic lupus erythematosus have in their sera autoantibodies to vimentin, the main protein of intermediate sized filaments of the cytoskeleton of connective tissue cells. Immunoprecipitation and Western blotting techniques have been used to detect the interaction between human anti-vimentin autoantibodies and the two peptides that resulted from the cleavage of vimentin by N-chlorosuccinimide. Both methods and also immunoabsorption of sera with the isolated peptides suggest that the interaction of vimentin and immunoglobulins is mainly carried out through a peptide of 30kD mol. wt located close to the amino-terminal part of the vimentin molecule. The same results were obtained when a serum from a rabbit immunized with human vimentin was used.

Animals↗
PubMed1984

Controlled proteolysis of tubulin by subtilisin: localization of the site for MAP2 interaction.

The treatment of tubulin with subtilisin resulted in a significant decrease in the ability of tubulin to assemble. The addition of taxol reduced the effect of subtilisin on the assembly of digested protein. Limited proteolysis of tubulin by subtilisin affected simultaneously both alpha- and beta-subunits, and it resulted in the appearance of two major cleavage fragments (32 and 20 kilodaltons) or an alternative pattern yielding two fragments (48 and 4 kilodaltons). The smallest peptide (4 kilodaltons) and also the 20-kilodalton fragment are localized in the C-terminal region of the tubulin alpha-subunit. Digested tubulin can assemble into sheet-shaped polymers, which cannot incorporate MAP2. On the other hand, the isolated C-terminal fragments can bind to MAP2. These results suggest that the carboxyl-terminal domain of the tubulin molecule is the site for the MAP2 interaction.

Animals↗
PubMed1984

Limited proteolysis of tubulin and the localization of the binding site for colchicine.

Limited proteolysis of porcine brain tubulin with trypsin resulted in a gradual loss of its colchicine binding activity as well as its ability of assembly into microtubules. The analysis of the tryptic degradation products showed a preferential proteolysis of alpha-tubulin subunit. This enzymatic proteolysis cleaved tubulin in one major site producing fragments of 36,000 and 16,000 daltons, the smaller polypeptides containing the carboxyl-terminal residue as shown by 14C tyrosination . However, proteolysis after incubation with 1 X 10(-3) M colchicine resulted in formation of the indicated fragments plus a 41,000-dalton fragment and smaller size peptides indicating the drug induces a second cleavage site closer to the carboxyl-terminal alpha-tubulin. Preincubation of tubulin with [3H]colchicine followed by proteolysis and separation of the fragments by Sephadex G-75 chromatography showed radioactive colchicine associated with the 16,000-dalton fragment and to the smaller size peptides resulting from digestion in the presence of the drug. The data indicate a localization of the colchicine-binding site in the 16,000-dalton segment containing the COOH-terminal region of alpha-tubulin subunit.

Animals↗
PubMed1984

Quantitative determination, isolation and characterization of pig lung tubulin.

Tubulin from pig lung was quantitatively determined, isolated and characterized. It accounted for about 0.3-0.4% of the total soluble protein of pig lung, as measured by colchicine binding or radioimmunoassay. Purified tubulin was obtained by several cycles of polymerization and depolymerization in the presence of dimethyl sulphoxide and 2H2O as stabilizing agents. The proteolytic cleavage patterns of the lung tubulin subunits closely resembled those of other mammalian cytoplasmic tubulin subunits, such as those of brain and kidney. However, the pattern of lung isotubulins on isoelectric focusing differed substantially from that of brain isotubulins . These differences did not appear to be the result of major lung tubulin post-translational modifications, since approximately the same pattern of isotubulins was found for the tubulin synthesized by lung poly(A)-containing mRNA in a reticulocyte system in vitro.

Animals↗