Biomedical subjects

J Avila

Publications and source records attributed to J Avila.

At least 163 records · Page 9Linked to original sources

PubMed1993

Role of the carboxy terminal region of beta tubulin on microtubule dynamics through its interaction with the GTP phosphate binding region.

The dynamic instability of microtubules depends on the GTP binding to tubulin, the rate of hydrolysis of GTP bound to tubulin molecules, at the microtubule caps, and on the affinity and exchange rate of tubulin for GTP versus GDP. It has been demonstrated that the binding of microtubule-associated proteins (MAPs) such as Tau or MAP2 notably enhances microtubule stability in vivo. These MAPs bind to the tubulin carboxy terminal domain. Consequently, an attractive hypothesis to explain the modulation of microtubule dynamics by MAPs is that the carboxy terminal domain of tubulin interacts with a region close to the GTP binding site, preventing the binding of GTP or exchange of GDP for GTP. By carrying out a combined analysis of crosslinking and limited proteolysis, an intramolecular interaction between the carboxy terminus and the tubulin region containing the GTP binding site in beta tubulin has been observed. It is proposed that this interaction modifies the binding of GTP to the tubulin beta-subunit and, therefore, affects tubulin assembly dynamics. This suggests a molecular explanation for the effect of MAPs in facilitating tubulin polymerization through the regulation of the interaction of GTP.

Amino Acid Sequence↗

PubMed1993

Rapid dephosphorylation of microtubule-associated protein 2 in the rat brain hippocampus after pentylenetetrazole-induced seizures.

We have studied the effect of Pentylenetetrazole (PTZ)-induced seizures on the state of phosphorylation of microtubule-associated protein 2 (MAP-2) from rat hippocampus. A method for the in vivo 32P-labeling of hippocampal proteins has been established, consisting of intracerebro-ventricular injection of 32PO4 of high specific activity. The results obtained indicate that PTZ induces a rapid and transient dephosphorylation of high-molecular-mass MAP-2, which is prevented when the N-methyl-D-aspartate receptor antagonist MK-801 is previously administered. Phosphopeptide mapping of 32P-labeled MAP-2 obtained from hippocampi of PTZ-treated rats reveals a pattern of phosphorylation distinct from that obtained from control saline-treated rats or MK-801 plus PTZ treated rats. We discuss the possible implications of N-methyl-D-aspartate-receptor activation and MAP-2 dephosphorylation on the plastic changes induced in rat brain hippocampus after induced epileptiform activity.

Animals↗

PubMed1993

High external potassium induces an increase in the phosphorylation of the cytoskeletal protein MAP2 in rat hippocampal slices.

Depolarization induced in rat hippocampal slices by a high concentration of extracellular K+ leads to an increase in the phosphorylation of microtubule-associated protein MAP2. The comparison of the major phosphopeptides derived from in situ and in vitro phosphorylated MAP2 suggests the implication of calcium-dependent protein kinases, including calcium/calmodulin-dependent protein kinase type II and protein kinase C, in the up-phosphorylation of MAP2. In particular, a peptide containing the tubulin-binding domain of the MAP2 molecule may be phosphorylated by protein kinase C. As the association of MAP2 with the cytoskeleton may be regulated by phosphorylation, we suggest that changes in the phosphorylation level of MAP2 might be involved in synaptic remodelling in hippocampal neurons.

Animals↗

PubMed1993

Differences in microtubule binding and self-association abilities of bovine brain tau isoforms.

We have fractionated tau isoforms by elution at increasing pH values using iron-chelated affinity chromatography, which discriminates between isoforms phosphorylated to different extents. Microtubule-associated tau elutes from the column at a pH gradient narrower than that of total brain tau. Neither under-phosphorylated nor highly phosphorylated isoforms are found in the microtubule-associated tau protein preparation. This indicates that phosphorylation at certain sites is needed for tau binding to microtubules, whereas phosphorylation at some other sites may prevent the association. The self-association ability of the different tau isoforms has also been analyzed. Tau isoforms containing three tubulin binding motifs form covalently bound dimers more efficiently than tau isoforms containing four motifs. This dimer-forming ability is notably diminished in the presence of a reducing agent, as determined by SDS-polyacrylamide gel electrophoresis, thus suggesting the involvement of cysteine residues. Additionally, tau forms larger aggregates, as detected by gel permeation chromatography, which are solubilized by SDS and cannot, therefore, be observed by SDS-polyacrylamide gel electrophoresis. These tau aggregates are observed even in the presence of reducing agents. These results support the idea that other regions in the tau molecule, besides the Cys-containing tubulin binding region, also contribute to tau self-association. Tau dimerization and aggregation may be prior steps to the formation of paired helical filaments.

Animals↗

PubMed1993

Depletion of casein kinase II by antisense oligonucleotide prevents neuritogenesis in neuroblastoma cells.

Casein kinase II is a multifunctional protein kinase which has been implicated in the regulation of cell growth and differentiation. This enzyme is much more abundant in neurons than in any other cell type. The treatment of neuroblastoma cells with an antisense oligodeoxyribonucleotide which specifically results in the depletion of casein kinase II catalytic subunits blocks neuritogenesis. Accordingly, this enzyme may perform an essential role during neurite growth in developing neurons. Casein kinase II depletion induced by antisense oligodeoxyribonucleotide is accompanied by a site-specific dephosphorylation of microtubule-associated protein MAP1B (also referred to as MAP5, MAP1.X or MAP1.2), which is paralleled by a release of MAP1B from microtubules. We therefore propose that phosphorylation by casein kinase II may be required for the proper MAP1B functioning in the promotion of the assembly of microtubules which constitute the cytoskeletal scaffolding of growing axon-like neurites.

Animals↗

PubMed1993

Effect of superoxide dismutase, allopurinol and glucocorticoids on liver and lung metallothionein induction by endotoxin in the rat.

Liver and lung metallothionein (MT) levels were increased by endotoxin. The administration of superoxide dismutase (SOD) or allopurinol (ALLO) before (30-60 min) or after (24-32 h) the endotoxin treatment either increased or did not affect the effect of endotoxin on MT levels, depending on the particular treatment and tissue. SOD and ALLO also increased liver and lung MT levels in control rats. In contrast, liver MT levels tended to be decreased by the glucocorticoid prednisolone (PRED) when administered before the endotoxin and were significantly decreased when it was administered after endotoxin. The effect of PRED on lung MT levels was completely different, since it decreased the effect of endotoxin when injected before the lipopolysaccharide, but increased it when injected after the endotoxin. Liver lipid peroxidation, as measured by thiobarbituric acid reactants (TBARs), increased after endotoxin in the liver but not in the lung, an effect even potentiated in some cases by the antioxidants studied. As expected, tissue MT and TBARs could not be correlated.

Allopurinol↗

PubMed1993

N-methyl-D-aspartate stimulates the dephosphorylation of the microtubule-associated protein 2 and potentiates excitatory synaptic pathways in the rat hippocampus.

We have studied the effect of brief (50-150 s) applications of N-methyl-D-aspartate (10-100 microM) on the phosphorylated state of the microtubule-associated protein 2 in slices of rat hippocampus. Following a similar experimental protocol we also studied the pattern of excitatory postsynaptic potentials intracellularly recorded in CA1 pyramidal cells elicited by stimulation of the Schaffer collateral-commissural pathway. N-Methyl-D-aspartate treatment produced a marked and specific dephosphorylation of the cytoskeletal microtubule-associated protein 2, which was not due to enhanced proteolytic activity. Dephosphorylation of the microtubule-associated protein 2 affects mainly the tubulin-binding domain of the molecule and seems to be a consequence of the activation of the Ca2+/calmodulin-dependent phosphatase calcineurin, as it is partially inhibited by calmidazolium but not by okadaic acid. A few minutes after N-methyl-D-aspartate treatment we observed a 23 +/- 17% increase in the amplitude of the monosynaptic excitatory postsynaptic potential recorded in the cells and the appearance of a large polysynaptic excitatory postsynaptic potential. Both effects lasted for several tens of minutes. The late polysynaptic potential was not observed when the CA3 and CA1 subfields were surgically separated. Our results indicate that the N-methyl-D-aspartate receptor activation leads to the dephosphorylation of the microtubule-associated protein 2 via a Ca2+/calmodulin phosphatase, probably calcineurine. This may, in turn, participate in the potentiation of synaptic efficacy.

Animals↗

PubMed1993

Petunia hybrida genes related to the maize regulatory C1 gene and to animal myb proto-oncogenes.

The maize C1 gene is a regulatory gene of the anthocyanin pathway. It is a part of a multigene family whose predicted protein products contain a region with homology to the DNA-binding domain of myb proto-oncogene products. Using as a probe an oligonucleotide mixture encoding a highly conserved region in plant MYB proteins, cDNAs encoding three related polypeptides expressed in the flower of Petunia hybrida have been isolated. Predicted proteins are 280-517 amino acids long and show sequence similarities among themselves as well as with other MYB proteins (45-80% amino acid identity) in the MYB DNA-binding domain. Outside this domain no significant sequence similarity was found. However, in all cases regions with structural characteristics of activator domains were found. Analysis with Southern blots allowed an estimation of at least 20-30 myb genes in P. hybrida. Expression patterns were specific for each gene as shown by Northern blotting analysis. Subcellular location studies of the largest MYB product, by both immunoblotting of the protein in subcellular fractions and by 'in situ' immunolocation experiments, showed that it is a nuclear protein. The role of MYB proteins as transcriptional activators regulating the synthesis of different anthocyanin(flavonoid)-related compounds is discussed.

Amino Acid Sequence↗

PubMed1993

MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture.

Cultures of hippocampal neurones have been used to study a possible correlation between the pattern of dendritic growth and post-translational modification of Microtubule-Associated Protein 2 (MAP2). During the first three days in vitro, a small increase in the total amount of MAP2 is observed, whereas the level of phosphorylation increases exponentially, being particularly dramatic after three days in vitro. Analysis of dendrite morphology by MAP2 immunofluorescence, revealed a parallel exponential growth in dendrite arborization with maximum rates at the third day of culture. We propose a correlation between dendrite arborization and phosphorylation of MAP2, that could be mediated by the establishment of cell contacts.

Animals↗

PubMed1993

Heterogeneity in the phosphorylation of microtubule-associated protein MAP1B during rat brain development.

The patterns of isoforms and of immunoreactivity of the microtubule-associated protein MAP1B toward a panel of antibodies to phosphorylation-sensitive epitopes are different in distinct rat brain regions and change during development. This suggests the occurrence of a considerable degree of heterogeneity in the phosphorylation state of rat brain MAP1B. It appears that MAP1B can be phosphorylated at multiple sites that may be conventionally classified into at least two modes of phosphorylation. Mode I of phosphorylation induces significant upward shifts in the electrophoretic mobility of the protein, giving rise to "high" MAP1B isoforms, whereas the mode II of MAP1B phosphorylation does not greatly affect the electrophoretic mobility of the protein. These MAP1B phosphorylation modes are differentially regulated throughout development and show some regional specificity. Cytosolic MAP1B is highly phosphorylated both at mode I and mode II sites in the developing rat brain, as well as in the adult olfactory bulb, where axonal growth takes place. In most adult rat brain regions, cytosolic MAP1B is highly phosphorylated at mode II sites but largely dephosphorylated at certain mode I sites. However, MAP1B present in the particulate fraction of most rat brain region homogenates may be partially dephosphorylated at certain mode II sites, although it contains some phosphorylated mode I sites. These data are compatible with the view that different protein kinases, possibly including casein kinase II and proline-directed protein kinases, might regulate the state of phosphorylation of MAP1B in distinct localizations along the development of different neuronal populations in the brain.

Aging↗

PubMed1993

Incorporation of tubulin subunits into dimers requires GTP hydrolysis.

A toroid multisubunit complex of 800-900 kDa has been implicated in assisting protein folding of at least two cytoplasmic proteins, actin and tubulin. This process is dependent on the presence of magnesium ions and ATP hydrolysis. In vitro translation of cDNAs encoding different alpha- and beta-tubulin isotypes also gives rise to the formation of complexes of about 300 kDa. These complexes have been functionally implicated in the incorporation of tubulin monomers within the tubulin heterodimer. This work shows that, in addition to ATP hydrolysis, the incorporation of newly synthesized tubulin subunits into functional heterodimers requires GTP hydrolysis in the presence of magnesium ions. A two-step process is suggested, a first ATP-dependent step in which the 900 kDa complexes are implicated in a similar way to the step taking place in actin folding, and a second GTP-dependent step in which the 300 kDa complexes are involved in the assembly of the heterodimer.

Adenosine Triphosphate↗

PubMed1993

[Infected pancreatic necrosis. Reexaminations planned with locoregional anesthesia].

Eleven patients with infected pancreatic necrosis are presented. They were treated with necrosectomy and lavage with programmed reexploration and peridural anesthesia. Nine of these patients, had biliary stone disease as associated pathology and two were chronic alcoholics. We used the contrast computerized tomography and CT-guided percutaneous aspiration to obtain fluid for Gram stains and culture, as a diagnostic method. The patients were reoperated 3 to 5 times each 48-72 hs., with a mortality of 18.2%.

Aged↗

PubMed1993

Unreliable supply of potable water and elevated Aedes aegypti larval indices: a causal relationship?

We investigated the hypothesis that a deficient supply of piped water was causing a high prevalence of water storage containers, which in turn, become important aquatic habitats of Aedes aegypti in a small town in Venezuela. The House (71.2%) and Breteau indices (229) were considerably elevated. Prevalent positive containers were: metal drums, small disposable containers (bottles, tins, etc.), tires, house plants (flowers in vases and plants in pots with earth) and tanks. Most people reported frequent interruptions in the supply of piped water and considered it to be unreliable. The frequency of interruptions in the supply of water was positively correlated with the House and Container indices, and with the number of positive containers, water-storage devices and positive water-storage devices per house. Even people who considered that they had an adequate supply of water kept numerous water storage containers. Most people (60%) said they would not stop storing water even in the event of the establishment of a reliable supply of piped water.

Aedes↗

PubMed1992

Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease.

Brain tau protein is phosphorylated in vitro by cdc2 and MAP2 kinases, obtained through immunoaffinity purification from rat brain extracts. The phosphorylation sites are located on the tau molecule both upstream and downstream of the tubulin-binding motifs. A synthetic peptide comprising residues 194-213 of the tau sequence, which contains the epitope recognized by the monoclonal antibody tau-1, is also efficiently phosphorylated in vitro by cdc2 and MAP2 kinases. Phosphorylation of this peptide markedly reduces its interaction with the antibody tau-1, as it has been described for tau protein in Alzheimer's disease. Both cdc2 and MAP2 kinases are present in brain extracts obtained from Alzheimer's disease patients. Interestingly, the level of cdc2 kinase may be increased in patient brains as compared with non-demented controls. These results suggest a role for cdc2 and MAP2 kinases in phosphorylating tau protein at the tau-1 epitope in Alzheimer's disease.

Alzheimer Disease↗

PubMed1992

Microtubule-associated protein tau is phosphorylated by protein kinase C on its tubulin binding domain.

We have analyzed the in vitro phosphorylation of tau protein by Ca2+/calmodulin-dependent protein kinase, casein kinase II, and proline-directed serine/threonine protein kinase. These kinases phosphorylate tau protein in sites localized in different regions of the molecule, as determined by peptide mapping analyses. Focusing on the phosphorylation of tau by protein kinase C, it was calculated as an incorporation of 4 mol of phosphate/mol of tau. Limited proteolysis assays suggest that the phosphorylation sites could be located within the tubulin-binding domain. Direct phosphorylation of synthetic peptides corresponding to the cysteine-containing tubulin-binding region present in both fetal and adult tau isoforms demonstrates that serine 313 is modified by protein kinase C. Phosphorylation of the synthetic peptide by protein kinase C diminishes its binding to tubulin, as compared with the unphosphorylated peptide.

Adenosine Triphosphate↗

PubMed1992

Differentiation of neuroblastoma cells correlates with an altered splicing pattern of tau RNA.

Morphological differentiation of N2A neuroblastoma cells is associated with an altered splicing of the gene of the microtubule-associated protein, tau. Two populations of RNA (coding for tau proteins containing three or four tubulin-binding motifs) are present in a similar proportion in undifferentiated neuroblastoma cells while in differentiated cells the proportion is changed in favour of that population coding for tau protein containing four tubulin-binding motifs. An increase in a high molecular weight tau isoforms correlates with the increase in the RNA population coding for four tubulin-binding motifs. A possible consequence of expressing a higher proportion of the tau protein containing four tubulin-binding motifs could be an increase in microtubule stability of differentiated neuroblastoma cells.

Animals↗

PubMed1992

Decreased tubulin synthesis in synoviocytes from adjuvant-induced arthritic rats.

The first microscopical alterations along adjuvant arthritis induction in rats seem to appear in the synovium. We have studied the protein synthesis pattern of the cells constitutively present in synovial membrane (synoviocytes) and have found an impairment of synthesis of some proteins when synoviocytes are derived from adjuvant arthritic rats. One of these polypeptides was identified as beta tubulin by two-dimensional gel electrophoresis, a membrane transfer assay using a specific monoclonal antibody and peptide mapping. We postulate that a repressed synthesis of tubulin may be an initial step in the triggering of the disease, since the effect was evident at pre-arthritic stages, when infiltration by inflammatory cells had not yet occurred.

Animals↗

PubMed1992

Microtubule functions.

Microtubules, with intermediate filaments and microfilaments, are the components of the cell skeleton which determinates the shape of a cell. Microtubules are involved in different functions including the assembly of mitotic spindle, in dividing cells, or axon extension, in neurons. In the first case, microtubules are highly dynamic, while in the second case microtubules are quite stable, suggesting that microtubule with different physical properties (stability) are involved in different functions. Thus, to understand the mechanisms of microtubule functions it is very important to understand microtubule dynamics. Historically, tubulin, the main component of microtubules, was first characterized as the major component of the mitotic spindle that binds to colchicine. Afterwards, it was found that tubulin is particularly more abundant in brain than in other tissues. Therefore, the roles of microtubules in mitosis, and in neurons, have been more extensively analyzed and, in this review, these roles will be discussed.

Animals↗