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J Avila

Publications and source records attributed to J Avila.

At least 109 records · Page 6Linked to original sources
PubMed1997

Lithium inhibits Alzheimer's disease-like tau protein phosphorylation in neurons.

In Alzheimer's disease, tau protein becomes hyperphosporylated, which can contribute to neuronal degeneration. However, the implicated protein kinases are still unknown. Now we report that lithium (an inhibitor of glycogen synthase kinase-3) causes tau dephosphorylation at the sites recognized by antibodies Tau-1 and PHF-1 both in cultured neurons and in vivo in rat brain. This is consistent with a major role for glycogen synthase kinase-3 in modifying proline-directed sites on tau protein within living neurons under physiological conditions. Lithium also blocks the Alzheimer's disease-like proline-directed hyperphosphorylation of tau protein which is observed in neurons treated with a phosphatase inhibitor. These data raise the possibility of using lithium to prevent tau hyperphosphorylation in Alzheimer's disease.

Alzheimer Disease↗
PubMed1997

The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate.

DNA sequencing in the phage lambda JA13 isolated from a lambda EMBL3 Hansenula polymorpha genomic DNA library containing the nitrate reductase-(YNR1) and nitrite reductase-(YNI1) encoding genes revealed an open reading frame (YNT1) of 1524 nucleotides encoding a putative protein of 508 amino acids with great similarity to the nitrate transporters from Aspergillus nidulans and Chlamydomonas reinhardtii. Disruption of the chromosomal YNT1 copy resulted in incapacity to grow in nitrate and a significant reduction in rate of nitrate uptake. The disrupted strain is still sensitive to chlorate, and, in the presence of 0.1 mM nitrate, the expression of YNR1 and YNI1 and the activity of nitrate reductase and nitrite reductase are significantly reduced compared with the wild-type. Northern-blot analysis showed that YNT1 is expressed when the yeast is grown in nitrate and nitrite but not in ammonium solution.

Amino Acid Sequence↗
PubMed1997

The zeta isozyme of protein kinase C binds to tubulin through the pseudosubstrate domain.

It has been suggested that the protein kinase C zeta (zeta PKC) isoform is involved in mitogenic signaling in Xenopus oocytes and mammalian cells. Thus, the characterization of potential regulatory molecules that bind to zeta PKC is of great interest. We report here the identification by affinity chromatography of tubulin as a zeta PKC-binding protein. Further immunofluorescence and microtubule copolymerization studies are consistent with this interaction. It is suggested that tubulin binds to zeta PKC through its pseudosubstrate domain. Furthermore, results demonstrate that treatment of cells with nocodazole, which disrupts microtubule structures, severely impairs the activity of native zeta PKC, stressing the potential functional relevance of zeta PKC binding to tubulin.

3T3 Cells↗
PubMed1997

Differential expression of microtubule-associated protein 1B phosphorylated isoforms in the adult rat nervous system.

Phosphorylated microtubule-associated protein 1B isoforms are thought to be involved in the plastic events taking place in neurons during development. However, little is known about their expression and possible role in the mature nervous system. To gain insight into the mechanisms underlying neuronal plasticity in the adult, we studied the pattern of expression of three microtubule-associated protein 1B isoforms in the entire adult rat nervous system. Accordingly, we performed western blots and immunohistochemistries using the antibodies 125, 150 and 531, which specifically recognize phosphorylated and unphosphorylated microtubule-associated protein 1B epitopes. Two electrophoretically distinct microtubule-associated protein 1B isoforms, slow-migrating and fast-migrating, were detected with the antibodies. The pattern of expression of these isoforms in the adult rat nervous system was region specific. Phosphorylated slow-migrating microtubule-associated protein 1B was expressed at all cellular compartments of primary sensory neurons in the central and peripheral nervous systems. In addition to primary sensory axons, slow-migrating microtubule-associated protein 1B was encountered at some other axons within the central nervous system. We discuss the correlation between slow-migrating microtubule-associated protein 1B axonal content and the regenerative potential of neurons. Phosphorylated fast-migrating microtubule-associated protein 1B was exclusively found in central nervous system dendrites where synaptic plasticity with morphological changes occurs in the adult. Unphosphorylated fast-migrating microtubule-associated protein 1B was the only isoform present in the bodies and dendrites of all motor neurons, and in peripheral and central nervous system glial cells of myelinated tracts with slow-migrating microtubule-associated protein 1B-containing axons. In summary, this report describes the pattern of expression of microtubule-associated protein 1B isoforms in the entire adult rat nervous system. In addition, it provides some information about the possible functional implications of phosphorylated microtubule-associated protein 1B isoforms in the adult.

Animals↗
PubMed1997

Analysis of the expression, distribution and function of cyclin dependent kinase 5 (cdk5) in developing cerebellar macroneurons.

Cultures of cerebellar macroneurons were used to study the expression, activity, subcellular localization, and function of cdk5 during neuronal morphogenesis. The results obtained indicate that in non-polarized neurons cdk5 is restricted to the cell body but as soon as polarity is established it becomes highly concentrated at the distal tip of growing axons where it associates with microtubules and the subcortical cytoskeleton. In addition, we show that laminin, an extracellular matrix molecule capable of stimulating axonal extension and promoting MAP1b phosphorylation (DiTella et al., 1996), accelerates the redistribution of cdk5 to the axonal tip and dramatically increases its activity. Finally, our results indicate that cdk5 suppression by antisense oligonucleotide treatment selectively reduces axonal elongation and decreases the phosphorylation status of MAP1b, as well as its binding to microtubules. Taken collectively, our observations suggest that cdk5 may serve as an important regulatory linker between environmental signals (e.g. laminin) and constituents of the intracellular machinery (e.g. MAP1b) involved in axonal formation.

Animals↗
PubMed1997

Role of glycosaminoglycans in determining the helicity of paired helical filaments.

It is known from previous work that tau is the main component of paired helical filaments (PHFs) and that it can assemble in vitro into polymers resembling PHFs when high concentrations of protein are used. In the search for molecules that can facilitate tau polymerization, a component of neurofibrillary tangles, heparan sulfate (or its more sulfated form, heparin), and other glycosaminoglycans have been tested. Glycosaminoglycans, in the sulfated but not in the unsulfated form, facilitate not only tau assembly but also the formation of polymers resembling PHFs. Conversely, PHFs were found to contain heparan sulfate and chondroitin sulfate. Heparinase or chondroitinase treatment of PHFs results in the formation of straight structures. All of these results suggest a role for sulfated glycosaminoglycans in determining the helicity of PHFs.

Binding Sites↗
PubMed1996

Glycogen synthase kinase 3 phosphorylation of different residues in the presence of different factors: analysis on tau protein.

Several peptides derived from microtubule-associated tau protein, have been tested as substrates for glycogen synthase kinase 3 (GSK 3). In the absence of cofactors, GSK 3 can modify serines or threonines followed by prolines. In other cases, a phosphorylation in position +4 is required for the phosphorylation of threonine/serine residues. A third type of substrate can be modified by GSK 3 in the presence of heparin. The comparison of GSK 3 with other kinases suggests some similar features of this kinase with proline-directed protein kinases, such as cdc-2 or mitogen-activated protein kinase (MAP Kinases,) and also with casein kinase 2 (CK 2). Thus, all these kinases are specifically inhibited by 5,6-Dichloro-1-(beta-D-ribofuranosyl)-benzimidazole (DRB). However, heparin is an inhibitor of CK 2 whereas it activates the modification of certain substrates by GSK 3. A possible explanation for the obtained results is that the consensus sequence for GSK 3 phosphorylation is a serine/threonine adjacent to a proline or other beta-turn former residue and that such recognition could be favoured by the presence of adjacent negative charges or the addition of polyanions.

Amino Acid Sequence↗
PubMed1996

The beta-tubulin monomer release factor (p14) has homology with a region of the DnaJ protein.

p14 is a molecular chaperone involved in beta-tubulin folding which catalyzes the release of beta-tubulin monomers from intermediate complexes. Here we demonstrate that active p14 protein which we have purified from an overproducing Escherichia coli strain can also release beta-tubulin monomers from tubulin dimers in the presence of an additional cofactor (Z). Analysis of p14 secondary structure suggests that this protein may belong to a family of conserved proteins which share structural similarities with the J-domain of DnaJ. We have constructed deletions and site-directed mutations in the p14 gene. A single D to E mutation in the region shown in DnaJ to be an essential loop for its function affected the monomer-release activity of p14. These results support the hypothesis that this p14 loop interacts with beta-tubulin in a similar fashion as DnaJ interacts with DnaK and suggest a possible role of p14 in the folding process.

Amino Acid Sequence↗
PubMed1996

Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2.

The C-terminal domain of microtubule-associated protein 2 (MAP2) contains a proline-rich region and the tubulin-binding domain. We have generated antibodies to follow the phosphorylation state of the proline-rich domain. One of these antibodies (no. 305) has been raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the threonine residues. This sequence is present in the proline-rich region of MAP2 and is phosphorylated in vitro by at least three different proline-directed protein kinases: p42mpk, p34cdc2, and GSK3 (glycogen-synthase kinase 3) alpha/beta. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as determined by Staphylococcus aureus V8 protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all three kinases studied with similar efficiency. In high-molecular-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.

Animals↗
PubMed1996

The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated.

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages lambdaNB5 and lambdaJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the delta ynil::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

Amino Acid Sequence↗
PubMed1996

Protein kinases involved in the phosphorylation of human tau protein in transfected COS-1 cells.

Human tau phosphorylation has been studied in transfected COS-1 cells. Treatment with okadaic acid alters the electrophoretic mobility of human tau protein transiently expressed in transfected cells, due to an increase in the level of phosphorylation. Treatment with okadaic acid also results in an increased phosphorylation of Alzheimer's disease-type phosphoepitopes. Tau phosphorylation within COS-1 cells is partially inhibited by in vivo treatment with DRB, a protein kinase inhibitor. Double treatment of transfected cells with okadaic acid and DRB reveals that phosphorylation of tau protein at the AT8 epitope is achieved by a DRB-resistant protein kinase which is different from that responsible for tau phosphorylation at the SMI-31 epitope, which appears to be sensitive to DRB.

Alzheimer Disease↗
PubMed1996

Characterization of microtubule-associated protein MAP1B: phosphorylation state, light chains, and binding to microtubules.

We have recently described a procedure for the purification of microtubule associated protein 1B (MAP1B) from calf brain [Pedrotti, B., & Islam K. (1995) Cell Motil. Cytoskeleton 30, 301-309], and this study further characterizes the purified protein and its interaction with microtubules. We show that purified MAP1B (1) is thermostable; (2) is mainly phosphorylated at the casein kinase II (CKII) sites but only partially phosphorylated at the proline-directed protein kinase (PDPK) sites; (3) both the CKII and PDPK sites can be dephosphorylated by alkaline phosphatase; and (4) dephosphorylation results in an increased mobility on SDS-PAGE gels. The ability of MAP1B to interact with microtubules was also examined and shows that (1) phosphorylated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-HT) MAP1B bind to taxol-stabilized microtubules; (2) 1 mol of 1B-P, 1B-AP, or 1B-HT each binds about 13-14 tubulin dimers; (3) light chain interaction with MAP1B heavy chain is not affected by AP- or heat-treatment; (4) MAP1B can be displaced from taxol-stabilized microtubules by titration with salt; (5) higher salt concentrations are required to displace 1B-AP compared with 1B-P from taxol-stabilized microtubules; and (6) MAP2 is able to displace both 1B-P and 1B-AP from taxol-stabilized microtubules. The role of phosphorylation in regulating MAP1B interaction with microtubules and light chains is discussed.

Alkaline Phosphatase↗
PubMed1996

The antitumoral compound Kahalalide F acts on cell lysosomes.

The target for the antitumoral peptidic drug, Kahalalide F, has been studied in cultured cells. In the presence of the compound, the cells became impressively swollen, showing the formation of large vacuoles. The formation of these vacuoles appears to be the consequence of changes in lysosomal membranes. Thus, lysosomes are a target for Kahalalide F action.

Amino Acid Sequence↗
PubMed1996

The in vitro formation of recombinant tau polymers: effect of phosphorylation and glycation.

Tau Isolated from paired helical filaments, aberrant structures that appear in Alzheimer disease (AD) patients' brains, show at least two posttranslational modifications: phosphorylation (Grundke-Iqbal et al., 1986; Ihara et al., 1986) and glycation (Ledesma et al., 1994; Yan et al., 1994). To test whether these modifications could affect the capacity of tau to self-aggregate, recombinant tau was phosphorylated and glycated, and its capacity to form polymers analyzed. Our results indicate that on phosphorylation and glycation, the capacity of tau to form aggregates increases, and that glycation of tau could stabilize the assembled polymers and could facilitate formation of bundles from these polymers.

Alzheimer Disease↗
PubMed1996

Expression of the beta 1 and beta 2(AMOG) subunits of the Na,K-ATPase in neural tissues: cellular and developmental distribution patterns.

We have used isoform-specific antisera against the Na,K-ATPase beta 1 (SpETb1) and beta 2(AMOG) (SpETb2) subunit isoforms in order to establish their specific cellular and subcellular localization in several developmental stages of the rat central nervous system. Immunocytochemical preparations revealed beta 1 Isoform protein in most neural cells, being predominantly located in the soma of neurons and astrocytes, with no appreciable developmental variations. In the newborn rat, beta 2(AMOG) immunoreactivity was present in cellular processes of astroglia and in the somas of neurons and decreasing in intensity with maturation until adulthood, where no beta 2 isoform was detected in neurons. The differential location of these isoforms, both developmentally and at the cellular level suggest a complex regulation of their genes expression and mechanisms of subcellular distribution, as well as functional differences.

Adenosine Triphosphatases↗
PubMed1996

Characterisation of antimitotic products from marine organisms that disorganise the microtubule network: ecteinascidin 743, isohomohalichondrin-B and LL-15.

The effect of selected marine compounds with anti-tumoral activity on the cell microtubule network was tested by immunofluorescence analyses, or by other in vitro analyses involving competition with colchicine or with GTP for tubulin binding and tubulin polymerisation, studies that were carried out in parallel with other microtubule poisons used as controls. Three compounds were found to disorganise the microtubule network: isohomohalichondrin B, LL-15 and ecsteinascidin 743. The first two compounds prevent microtubule assembly and GTP binding to tubulin. Ecteinascidin 743 disorganises the microtubule network but it does not seem to interact directly with tubulin.

Actin Cytoskeleton↗