Biomedical subjects

J Avila

Publications and source records attributed to J Avila.

At least 55 records · Page 3Linked to original sources

PubMed2000

Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

3T3 Cells↗

PubMed2000

Cloning, sequencing, and expression of H.a.YNR1 and H.a.YNI1, encoding nitrate and nitrite reductases in the yeast Hansenula anomala.

A single Hansenula anomala genomic DNA fragment containing the genes H.a.YNR1 (yeast nitrate reductase) and H.a.YNI1 (yeast nitrite reductase) encoding nitrate and nitrite reductase, respectively, was isolated from a lambda EMBL3 genomic DNA library. As probe, a 3.2 kb DNA fragment isolated from a lambda gt11 H. anomala genomic DNA library screened with antiserum anti-NR from H. anomala was used. H. a.YNR1 and H.a.YNI1 genes are separated by 473 bp and encode putative proteins of 870 and 1077 amino acids, respectively, with great similarity to nitrate and nitrite reductases from other organisms. Northern blot analysis revealed that both genes are highly expressed in nitrate, very low in nitrate plus ammonium, and no expression was detected in ammonium or nitrogen-free media. Levels of nitrate reductase and nitrite reductase were very low or undetectable by Western blot analysis in nitrogen-free and ammonium media, whereas both proteins were present in nitrate and ammonium plus nitrate media. The nucleotide sequence Accession No. is AF123281.

Amino Acid Sequence↗

PubMed2000

A study of the response of Y3Al5O12:Ce phosphor powder screens in the vacuum ultraviolet and soft X-ray regions using synchrotron radiation.

Phosphor screens find application in many fields because of their ability to convert incident radiation to wavelengths that are readily measured by modern detectors. While the response of such screens in the X-ray region has been widely studied, much work still remains to be done regarding their response in the vacuum ultraviolet and soft X-ray regions, where the response is predicted to be non-linear owing to the presence of elemental absorption edges. Here, an experiment using synchrotron radiation to determine the response of thin Y(3)Al(5)O(12):Ce (1-21 mg cm(-2)) and Y(2)O(3):Eu (2.64 mg cm(-2)) powder phosphor screens in the spectral range 20-900 A (13.8-620 eV) is reported. Also, a custom-built camera is described which permits simultaneous collection of the forward- and backward-emitted light and that enables measurements to be made at various positions across the screens and at several screen/incident beam angles. Finally, features in the response spectra are identified, and efficiencies across the spectral range indicated for different screen thicknesses and operating modes are plotted, before a curve of the intrinsic radiant efficiency of Y(3)Al(5)O(12):Ce is produced. The results are discussed in the context of other measurements.

Journal Article↗

PubMed2000

Tau aggregation into fibrillar polymers: taupathies.

Different neurological disorders, known as taupathies have been recently described. In these disorders it has been suggested that modifications in the microtubule-associated protein tau could cause neural degeneration in specific regions. Although these regions are different in the different taupathies, some common features appear to occur in all of them: abnormal hyperphosphorylation of tau and aberrant tau aggregation. These two features are commented upon in this review.

Alzheimer Disease↗

PubMed2000

The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers.

Spisulosine is a novel antiproliferative (antitumoral) compound of marine origin. In this work the molecular target for this toxic agent has been analyzed. In the presence of spisulosine, cultured cells change their morphology, first acquiring a fusiform morphology, and later becoming rounded without focal adhesions. Analysis of the cytoskeleton of treated cells indicate the absence of actin stress fibers.

Actins↗

PubMed2000

Regulation of phosphorylation of neuronal microtubule-associated proteins MAP1b and MAP2 by protein phosphatase-2A and -2B in rat brain.

The function of the neuronal high molecular weight microtubule-associated proteins (MAPs) MAP1b and MAP2 is regulated by the degree of their phosphorylation, which in turn is controlled by the activities of protein kinases and protein phosphatases (PP). To investigate the role of PP in the regulation of the phosphorylation of MAP1b and MAP2, we used okadaic acid and cyclosporin A to selectively inhibit PP2A and PP2B activities, respectively, in metabolically competent rat brain slices. The alteration of the phosphorylation levels of MAP1b and MAP2 was examined by Western blots using several phosphorylation-dependent antibodies to these proteins. The inhibition of PP2A, and to a lesser extent of PP2B, was found to induce an increased phosphorylation of MAP1b and inhibit its microtubule binding activity. Immunocytochemically, a marked increase in neuronal staining in inhibitor-treated tissue was observed with antibodies to the phosphorylated MAP1b. The inhibition of PP2A but not of PP2B also induced phosphorylation of MAP2 at multiple sites and impaired its microtubule binding activity. These results suggest that PP2A might be the major PP that participates in regulation of the phosphorylation of MAP1b and MAP2 and their biological activities.

Animals↗

PubMed2000

A polymorphism in the tau gene associated with risk for Alzheimer's disease.

Searching for tau genetic variations which could be associated with risk for Alzheimer's disease (AD), we have performed a mutational analysis of a region containing the whole exon 11 of the tau gene, which encodes a microtubule binding region critical for tau self-assembly, and we have found a biallelic polymorphism at position +34 of intron 11 (IVS11 + 34G/A). We have analyzed the allelic frequencies of this polymorphism in a case-control sample (167 clinically diagnosed AD and 194 controls) and found that the presence of any G allele (genotypes AG + GG) is associated with a five-fold AD risk in individuals carrying the apolipoprotein E4 allele, strongly suggesting that the combined effect of tau and apoE is relevant in relation with AD pathogenesis.

Age of Onset↗

PubMed2000

Perinatal lethality of microtubule-associated protein 1B-deficient mice expressing alternative isoforms of the protein at low levels.

Microtubule-associated protein 1B (MAP1B) has been implicated in axogenesis in cultured cells. To gain insight into the functions that MAP1B plays in vivo, we analyzed a strain of Map1B mutant mice generated by a gene trapping approach. Homozygous mice die on the first day after birth, probably due to a severe abnormal development of the nervous system. They present alterations in the structure of several brain regions. The normal Map1B gene yields different protein isoforms from alternatively spliced transcripts. The smaller isoforms were present in wild type, hetero-, and homozygous mice, but their expression was higher in the mutants than in the wild-type. Moreover, trace amounts of MAP1B protein were also observed in Map1B homozygous mutants, indicating an alternative splicing around the gene trap insertion. Thus, the Map1B gene trapped mutation reported in this work did not generated a null mutant, but a mouse with a drastic deficiency in MAP1B expression. Analyses of these mice indicate the presence of several neural defects and suggest the participation of MAP1B in neuronal migration.

Alternative Splicing↗

PubMed2000

Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function.

Neurons, the basic information processing units of the nervous system, are characterized by a complex polar morphology which is essential for their function. To attain their precise morphology, neurons extend cytoplasmatic processes (axons and dendrites) and establish synaptic connections in a highly regulated way. Additionally, neurons are also subjected to small plastic changes at the adult stage which serve to regulate synaptic transmission. Every step of neuronal development is genetically controlled by endogenous determinants, as well as by environmental signals including intercellular contacts, extracellular matrix and diffusible signals. Cytoskeletal components are among the main protein targets modified in response to most of those extracellular signals which ultimately determine neuronal morphology. One of the major mechanisms controlling the neuronal cytoskeleton is the modification of the phosphorylation state of cytoskeletal proteins via changes in the relative activities of protein kinases and phosphatases within neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins is an abundant group of cytoskeletal components which are predominantly expressed in neurons and serve as substrates for most of protein kinases and phosphatases present in neurons. MAP2 phosphorylation seems to control its association with the cytoskeleton and it is developmentally regulated. Moreover, MAP2 may perform many functions including the nucleation and stabilization of microtubules (and maybe microfilaments), the regulation of organelle transport within axons and dendrites, as well as the anchorage of regulatory proteins such as protein kinases which may be important for signal transduction. These putative functions of MAP2 have also been proposed to play important roles in the outgrowth of neuronal processes, synaptic plasticity and neuronal cell death. Thus, MAP2 constitutes an interesting case to understand the regulation of neuronal function by the alteration of the phosphorylation state of cytoskeletal proteins in response to different extracellular signals. Here we will review the current knowledge about the regulation of MAP2 function through phosphorylation/dephosphorylation and its relevance in the broader context of neuronal functions.

Animals↗

PubMed2000

Microtubule-associated protein-2 located in growth regions of rat hippocampal neurons is highly phosphorylated at its proline-rich region.

Neuronal morphogenesis is regulated, among other factors, by microtubule-associated proteins (MAPs). A family of these proteins, MAP2, which is very abundant in the mammalian nervous system, has been associated with the formation of neurites at early developmental stages and with the dendritic scaffold upon maturation. The function of MAP2 is regulated by its phosphorylation state. One of the phosphorylation sites that has been described is located in the proline-rich region of the protein. It comprises of the residues 1616-1626 and is specifically recognized by the antibody 305. However, little is known about the functional consequences of its modification in vivo. To gain insight into this, we have analysed the expression levels and intracellular distribution of MAP2 phosphorylated at this site (MAP2-P), in primary cultures of rat hippocampal neurons at different developmental stages. Western blot analysis of hippocampal neuron protein extracts revealed that the ratio of MAP2-P:MAP2 was 4:1 at early developmental stages and became 1:4 at later developmental stages, suggesting a role of such phosphorylated forms of the protein in neuritogenesis. Consistent with this view, immunofluorescence microscopy analysis showed that the ratio MAP2-P:MAP2 was 2 in the neurite growth cones, sites where net elongation takes place. A higher presence of phosphorylated MAP2 was observed in growth regions with higher levels of microfilaments, which may be related with the growth region stability. Indeed, when growth-cone collapse was induced in hippocampal neurons after cytochalasin D treatment, which depolymerizes microfilaments, the ratio MAP2-P:MAP2 in these growing regions decreased down to 1. Finally, acceleration of neuronal maturation induced by the activation of glutamate-receptors triggered a dramatic decrease in the phosphorylation of MAP2 at the site recognized by antibody 305. From these results we suggest that the phosphorylation of MAP2 at its proline-rich region is an important event during neuritogenesis.

Animals↗

PubMed2000

Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia.

Axonal regeneration in the lesioned mammalian central nervous system is abortive, and this causes permanent disabilities in individuals with spinal cord injuries. In adult rats, olfactory ensheathing glia (OEG) transplants successfully led to functional and structural recovery after complete spinal cord transection. From 3 to 7 months post surgery, all OEG-transplanted animals recovered locomotor functions and sensorimotor reflexes. They presented voluntary hindlimb movements, they supported their body weight, and their hindlimbs responded to light skin contact and proprioceptive stimuli. In addition, relevant motor axons (corticospinal, raphespinal, and coeruleospinal) regenerated for long distances within caudal cord stumps. Therefore, OEG transplantation provides a useful repair strategy in adult mammals with traumatic spinal cord injuries. Our results with these cells could lead to new therapies for the treatment of spinal cord lesions in humans.

Animals↗

PubMed2000

Na+, K+-ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interactions.

Na+, K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where it serves as the principal regulator of intracellular ion homeostasis. Na+, K+-ATPase is responsible for generating and maintaining transmembrane ionic gradients that are of vital importance for cellular function and subservient activities such as volume regulation, pH maintenance, and generation of action potentials and secondary active transport. The diversity of Na+, K+-ATPase subunit isoforms and their complex spatial and temporal patterns of cellular expression suggest that Na+, K+-ATPase isozymes perform specialized physiological functions. Recent studies have shown that the alpha subunit isoforms possess considerably different kinetic properties and modes of regulation and the beta subunit isoforms modulate the activity, expression and plasma membrane targeting of Na+, K+-ATPase isozymes. This review focuses on recent developments in Na+, K+-ATPase research, and in particular reports of expression of isoforms in various tissues and experiments aimed at elucidating the intrinsic structural features of isoforms important for Na+, K+-ATPase function.

Animals↗

PubMed2000

Tau dephosphorylation at tau-1 site correlates with its association to cell membrane.

It has been considered that tau protein is mainly a cytoplasmic protein since it is a microtubule associated protein. However, it has also been suggested that tau could be located in the cell nucleus and membrane. In our work, the cellular distribution of tau has been studied by immunofluorescence and western blot analysis, after subcellular fractionation in neuroblastoma cells and in tau-transfected non neural cells using, mainly, two types of tau antibodies; antibody 7.51 (that recognizes tau independent of its phosphorylation level); and antibody Tau-1 (that recognizes tau only in its dephosphorylated form). Also, tau was expressed in COS-1 cells to test for the features involved in the sorting of tau to different cell localizations. Our results show that tau associated to cell membrane has a lower phosphorylation level in its proline-rich region. Additionally, in differentiated neuroblastoma cells, tau phosphorylation, at that region, decreases and the amount of tau associated to cell membrane increases.

Animals↗

PubMed2000

The FTDP-17-linked mutation R406W abolishes the interaction of phosphorylated tau with microtubules.

The recent finding that several point mutations in the gene encoding for the microtubule-binding protein tau correlate with neurological disorders has heightened interest in the mechanisms of destabilization of this protein. In this study the functional consequences of the tau mutation R406W on the interaction of the protein with microtubules have been analyzed. Mutated tau is less phosphorylated than its normal counterpart at serines 396 and 404. Furthermore, the phosphorylated mutant protein is unable to bind to microtubules, and, as a consequence, microtubules assembled after transient nocodazole treatment in the presence of this tau variant contain only unmodified tau and appear to form more and longer bundles than those assembled in the presence of wild-type tau. We propose that phosphorylated tau, unbound to microtubules, could accumulate in the cytoplasm.

Amino Acid Sequence↗

PubMed2000

GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling.

A major determinant of neuronal morphology is the cytoskeleton. And one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of protein kinases and phosphatases in neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins are abundant cytoskeletal components predominantly expressed in neurons and have been found to be substrates for most of protein kinases and phosphatases present in neurons, including glycogen-synthase kinase 3 (GSK3). It has been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could control neuronal development. We have previously shown that MAP2 is phosphorylated in vitro and in situ by GSK3 at Thr1620 and Thr1623, located in the proline-rich region of MAP2 and recognized by antibody 305. However, the function of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-1 cells have been co-transfected with cDNAs encoding MAP2C and either wild type or mutated GSK3beta to analyze possible effects on microtubule stability and on the association of MAP2 with microtubules. We have found that GSK3beta phosphorylates MAP2C in co-transfected cells. Moreover, this phosphorylation is inhibited by the specific GSK3 inhibitor lithium chloride. Additionally, the formation of microtubule bundles, which is observed after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3beta wild type. Microtubule bundles were not observed in cells expressing MAP2C phosphorylated at the site recognized by antibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3beta wild type transfected cells with lithium chloride. Highly phosphorylated MAP2C species, which were phosphorylated at the site recognized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3beta wild type. Interestingly, these MAP2C species were enriched in cytoskeleton-unbound protein preparations. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulate microtubule stability.

Animals↗

PubMed2000

Molecular genetic approaches to microtubule-associated protein function.

Protein function in vivo can be studied by deleting (knock-out) the gene that encodes it, and search for the consequences. This procedure involves different technologies, including recombinant DNA procedures, cell biology methods and histological and immunocytochemical analysis. In this work we have reviewed these procedures when they have been applied to ascertain the function of several microtubule-associated proteins. These proteins have been previously involved, through in vitro experiments, in having a role in the microtubule stabilization. Here, we will summarize the generation and characterization of different microtubule-associated protein knock-out mice. Special attention will be paid to MAP1B knock-out mice. Amongst the different MAPs knock-out mice these show the strongest phenotype, the most likely for being MAP1B, the MAP that is expressed earliest in neurogenesis. Molecular genetics could be considered as a valid and useful procedure to truly establish the in vivo functions of a protein, although it is necessary to be aware of possible artifacts such as the generation of some kinds of RNA alternative splicing. To avoid this the best strategy to be used must consider the deletion of the exon that contains the functional domains of the protein.

Animals↗

PubMed2000

Initial studies with high resolution TEM and electron energy loss spectroscopy studies of ferritin cores extracted from brains of patients with progressive supranuclear palsy and Alzheimer disease.

Studies of crystallographic structure and composition of core nanocrystals of ferritin bound to aberrant tau filaments extracted from progressive supranuclear Palsy (PSP) and Alzheimer disease (AD) brain tissues were performed using high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). The results were compared with those obtained from synthetic Fe3O4 crystal (magnetite) and horse spleen ferritin cores. Core dimensions of ferritin molecules from PSP and AD were similar to those found in normal brain. Ferritin cores nanocrystals in AD seems to have less ordered structure than in PSP. Some nanocrystals did not have the hexagonal ferrihydrite structure generally found in healthy ferritin but rather a cubic structure similar to magnetite, a crystalline form in which both Fe2+ and Fe3+ are present. The presence of ferrous ion, Fe2+, may indicate some dysfunction in these pathological ferritins that might contribute to production of free radicals via the Fenton reaction involved in neurodegeneration.

Alzheimer Disease↗

PubMed1999

The neurite retraction induced by lysophosphatidic acid increases Alzheimer's disease-like Tau phosphorylation.

The bioactive phospholipid lysophosphatidic acid (LPA) causes growth cone collapse and neurite retraction in neuronal cells. These changes are brought about by the action of a cell surface receptor coupled to specific G proteins that control morphology and motility through the action of a group of small GTPases, the Rho family of proteins. Many studies have focused on actin reorganization modulated by Rho-GTPases, but almost no information has been obtained concerning microtubular network reorganization after LPA-induced neurite retraction. In the present study, we demonstrate an increase in site-specific Alzheimer's disease-like Tau phosphorylation during LPA-induced neurite retraction in differentiated SY-SH5Y human neuroblastoma cells. The phosphorylation state of Tau was inferred from its immunoreactivity with antibodies that recognize phosphorylation-sensitive epitopes. The effects of specific kinase inhibitors indicate that this phosphorylation is mediated by glycogen synthase kinase-3 (GSK-3). In support of this idea, we observed an increase of GSK-3 activity upon growth cone collapse. Our results are consistent with the hypothesis that activation of GSK-3 occurs in the Rho pathway and may represent an important link between microtubules and microfilaments dynamics during neuritogenesis and in pathological situations such as Alzheimer's disease.

Alzheimer Disease↗