Biomedical subjects

J Avila

Publications and source records attributed to J Avila.

At least 145 records · Page 8Linked to original sources

PubMed1994

Analysis of microtubule-associated protein tau glycation in paired helical filaments.

Alzheimer's disease is typified by the characteristic histopathological lesions of neurofibrillar plaques and tangles. The latter are composed of paired helical filaments (PHFs), the major components of which are modified forms of the microtubule-associated protein tau. The exact nature of these modifications remains unknown, although the presence of hyperphosphorylated tau in PHFs argues strongly that phosphorylation is one of the modifications that result in the polymerization of tau into PHFs. However, hyperphosphorylation alone is insufficient to explain the formation of PHFs. In an attempt to characterize other post-translational modifications of PHF-tau, we have analyzed its glycation. A fraction of PHF-tau seems to be glycated in vivo, whereas soluble tau from either Alzheimer's disease or non-demented human brain is not glycated at all. Purified tau from bovine brain can be efficiently glycated in vitro. Tau glycation is accompanied by a decrease in the tau binding to tubulin. These results support the view that glycation may be one of the modifications hampering the binding of tau to tubulin in Alzheimer's disease, thus facilitating tau aggregation into PHFs.

Aged↗

PubMed1994

Binding of heat-shock protein 70 (hsp70) to tubulin.

Binding of heat-shock protein (hsp70) to polymerized tubulin has been investigated by in vitro experiments. The tubulin region involved in binding to hsp70 corresponds to the carboxy-terminal residues 431-444, also involved in the association with other microtubule-associated proteins (MAPs). Additionally, the putative tubulin binding motif in the hsp70 protein contains a sequence related to the motif described for MAP1B protein.

Amino Acid Sequence↗

PubMed1994

Carboxyl terminal sequences of beta-tubulin involved in the interaction of HMW-MAPs. Studies using site-specific antibodies.

After the finding of the involvement of the C-terminal moieties of tubulin subunits in the interaction of MAPs, different studies have focused on the substructure of the binding domains for the different MAPs. Current biochemical evidence point to the role of a low-homology sequence between alpha and beta-subunits within the conserved region of the C-terminal domain of tubulin, in the binding of MAP-2 and tau. Another line of studies indicates that a site for interaction of the high molecular weight MAPs is located in the variable region defined by the glutamic-rich C-terminus of beta-tubulin. Here, we report the usefulness of idiotypic site-directed antibodies, produced by immunization with peptides from different beta-tubulin isoforms, to study both MAP-1 and MAP-2 binding sites on tubulin. On the basis of these results with site-specific antibodies along with previous structural information (Cross et al., 1991, Biochemistry 30: 4362-4366), we propose the role of consensus sequences, from the invariant beta-tubulin C-terminal domain in the binding of MAP-2 and from the variable domain in the interactions of MAP-1 and MAP-2.

Amino Acid Sequence↗

PubMed1994

Overexpression of tau protein in COS-1 cells results in the stabilization of centrosome-independent microtubules and extension of cytoplasmic processes.

COS-1 cells were transfected with cDNAs coding for different human tau isoforms. Expressed tau isoforms bind to cellular microtubules in vivo, preferentially at the distal regions of microtubules nucleated by the centrosome, leading to their stabilization. Eventually, tau-coated microtubules without any association with the centrosome were observed. A major difference between tau isoforms containing three tubulin-binding motifs and tau isoforms containing four tubulin-binding motifs is the greater ability of the latter in inducing the formation of long cytoplasmic processes.

Base Sequence↗

PubMed1994

Dephosphorylation of tau protein from Alzheimer's disease patients.

Tau protein-prepared post-mortem from brains of Alzheimer's disease patients was treated with protein phosphatase 1 catalytic subunit, 2A catalytic subunit, and 2B (calcineurin). Dephosphorylation was monitored by immunoblotting with two monoclonal antibodies, TAU-1 and SMI31, which recognize in tau the dephospho- and phospho-states, respectively, of proline-directed protein kinase phosphorylation sites. Out of the three enzymes tested, protein phosphatase 2A was only effective in dephosphorylating tau at these Alzheimer-type epitopes.

Alzheimer Disease↗

PubMed1994

Microtubule-associated protein 1B (MAP1B) is present in glial cells phosphorylated different than in neurones.

A panel of four anti-MAP1B antibodies have been used to study the presence and post-translational modification of MAP1B in primary cultures of glial cells. Two antibodies (150 and 125) recognize phosphorylated epitopes whereas the other two (531 and 842) recognize non-phosphorylated phosphorylatable epitopes on the MAP1B molecule. Immunofluorescence and Western blot analysis with antibodies 531 and 842 revealed the presence of small amounts of MAP1B-like immunoreactivity in type 1 astrocytes and a greater content in more differentiated glial cells found in long-term cultures. By immunofluorescence, these latter cells gave positive immunostaining with antibody 125, which recognizes a phosphorylated epitope phosphorylated by casein kinase II. Antibody 150, which reacts to a phosphorylated epitope on the MAP1B molecule, did not show any detectable immunoreactivity in glial cells cultures, either by immunofluorescence or Western blot. All four antibodies recognized hippocampal neurones in culture, with especially intense immunostaining in cell bodies and axons, and reacted strongly with protein present in hippocampal neurones extracts showing an electrophoretic mobility similar to that of brain MAP1B. In mixed optic nerve glial cell cultures, anti-galactocerebroside (GalC) positive cells gave also positive staining with antibodies 531 and 125. We propose that MAP1B is present in cultures of glial cells in moderate amounts and with a phosphorylation state different than in neurones. Thus, less differentiated glial cells, such as type 1 astrocytes, have a small amount of MAP1B, mainly in a non-phosphorylated form, which is spread diffusely in the cytoplasm and probably does not interact with microtubules. More differentiated glial cells, such as oligodendrocytes, show a greater content in MAP1B which, at least in part, is phosphorylated by a casein kinase II-like activity.

Amino Acid Sequence↗

PubMed1994

Role of phosphorylated MAPlB in neuritogenesis.

The distribution of microtubule-associated protein lB (MAPlB) phosphorylated by either proline-directed protein kinase (PDPK) or casein kinase II (CK II) in neuroblastoma cells and hippocampal neurons has been studied by immunofluorescence using specific antibodies to distinct phosphorylation-sensitive epitopes. A proximo-distal gradient of increasing PDPK-catalyzed phosphorylation of MAPlB is superimposed on a proximo distal gradient of decreasing CK II-catalyzed MAPlB phosphorylation within growing axon-like neurites. Additionally, CK II-phosphorylated MAPlB is present in cell bodies and dendrites where no PDPK-phosphorylated MAPlB is observed. These results suggest distinct roles for both types of modifications of MAPlB in developing neurons.

Animals↗

PubMed1994

Purification of brain microtubule-associated protein MAP1A.

Brain microtubule-associated protein MAP1A has been purified until homogeneity by using a novel procedure involving copolymerization with microtubules, treatment with poly-L-aspartic acid and FPLC. The purified protein retains its capacity to facilitate microtubule assembly.

Animals↗

PubMed1994

Depletion of catalytic and regulatory subunits of protein kinase CK2 by antisense oligonucleotide treatment of neuroblastoma cells.

1. The use of antisense oligonucleotides to inhibit expression of the genes coding for the catalytic (alpha/alpha') and regulatory (beta) subunits of protein kinase casein kinase 2 (CK2) has allowed study of the role of this enzyme in mouse neuroblastoma cells. 2. Selective depletion of catalytic (alpha/alpha') subunits results in the blocking of neuritogenesis. The depletion of catalytic subunits also affects the sorting of the regulatory (beta) subunit of CK2, as the absence of catalytic subunits prevents the translocation of the regulatory subunit to the nuclei. These results emphasize the existence of a control mechanism linking the expression and sorting of CK2 catalytic and regulatory subunits. 3. Selective depletion of the regulatory (beta) subunit of protein kinase CK2 by an specific antisense oligonucleotide causes partial inhibition of neurite extension.

Allosteric Site↗

PubMed1994

Microtubule-associated protein MAP1B showing a fetal phosphorylation pattern is present in sites of neurofibrillary degeneration in brains of Alzheimer's disease patients.

Alzheimer's disease results in the appearance of cytoskeletal disorders yielding pathological structures such a neurofibrillary tangles or dystrophic neurites. It has been previously described that the microtubule-associated protein, tau, modified by phosphorylation in serines adjacent to prolines, is a major component of these structures. Here, we show that another microtubule associated protein, MAP1B, aberrantly phosphorylated by a proline-dependent protein kinase, is a component of these previously mentioned structures. Thus, a possible common phosphorylation of axonal MAPs such as tau or MAP1B may correlate with their association with those aberrant cytoskeletal structures present in AD.

Aged↗

PubMed1994

Expression of the beta-subunit isoforms of the Na,K-ATPase in rat embryo tissues, inner ear and choroid plexus.

We report evidence of the apical localization of the two Na,K-ATPase beta-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na,K-ATPase beta 1 and beta 2 subunits. These polyclonal rabbit antisera were raised against truncated beta-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the beta 1 isoform and five bands in the beta 2 isoform immunoblots. In E15 rat embryos, the beta 1 isoform was detected in brain, heart and kidney and the beta 2 isoform only in brain. While beta-subunit mRNA expression (Watts AG, Sanchez-Watts G, Emanuel JR, Levenson R (1991) Proc Natl Acad Sci USA 88, 7425-7429), immunoblotting and enzymatic activity have been determined (Zlokovic BV< mackic JB, Wang L, McComb JG, McDonough A (1993) J Biol Chem 268, 8019-8025), very little is known about the specific localization of each beta-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the beta 1 and beta 2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus.

Animals↗

PubMed1994

Localization of differentially phosphorylated isoforms of microtubule-associated protein 1B in cultured rat hippocampal neurons.

The development and plasticity of axons and dendrites in mammalian neurons may depend on the presence and phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins. One of these proteins, microtubule-associated protein 1B, is modified by different protein kinases, which give rise to two major types of phosphorylated isoforms. The distribution of these isoforms in cultured hippocampal neurons has been studied using antibodies to specific phosphorylation-sensitive epitopes. Mode I-phosphorylated MAP1B is largely restricted to developing axonal processes, particularly at their distal regions including their growth cones where no mode I-dephosphorylated MAP1B is present. Axonal maturation is accompanied by dephosphorylation of MAP1B at mode I sites. Thus, mode I-phosphorylated MAP1B may be a marker for axonal growth. In contrast, mode II-phosphorylated MAP1B is abundant in the axonal and somatodendritic compartments, and no increased dephosphorylation occurs during maturation. These results are compatible with a role for the mode I phosphorylation of MAP1B (which might be catalysed by proline-directed protein kinases) in supporting a rapid axonal-specific growth mechanism and a more general role for the mode II phosphorylation of MAP1B (which seems to be catalysed by casein kinase II) in controlling axonal and dendritic growth and remodeling.

Amino Acid Sequence↗

PubMed1994

Nitrite causes reversible inactivation of nitrate reductase in the yeast Hansenula anomala.

The addition of nitrite, the product of the reaction catalysed by nitrate reductase, to cell suspensions of the yeast Hansenula anomala caused a reversible inactivation of NADPH-dependent nitrate reductase activity. The haem- and Mo-dependent and Mo-dependent activities of nitrate reductase, determined with the non-physiological electron donors FMNH2 and reduced methyl viologen respectively, were less affected. A similar inactivation was found with the proton ionophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The inactive enzyme was found in the particulate fraction and cosedimented with the mitochondrial fraction. When the NADPH-dependent nitrate reductase activity was restored in vivo the enzyme was found in the soluble fraction. The inactivation of nitrate reductase by nitrite, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone was dependent on the external pH. The treatment of isolated mitochondria at alkaline pH with Triton X-100 solubilized about 30% of the inactive enzyme.

2,4-Dinitrophenol↗

PubMed1994

Phosphorylation of microtubule-associated proteins by protein kinase CK2 in neuritogenesis.

Phosphorylation of microtubule-associated protein MAP1B and the neuronal-specific beta III-tubulin isoform takes place during neurite growth in neuroblastoma cells. Protein kinase CK2 (formerly referred to as casein kinase 2) is possibly involved in beta III-tubulin phosphorylation. As for MAP1B, there are at least two types of phosphorylation; one catalyzed by proline-directed protein kinases and another catalyzed by CK2. Protein kinase CK2 is primarily localized to the nuclei in proliferating neuroblastoma cells, whereas an increased amount of the enzyme is present in the cytoplasm of postmitotic cells bearing neurites. Treatment of neuroblastoma cells with an antisense oligonucleotide which specifically results in CK2 catalytic subunit depletion inhibits neuritogenesis. CK2 depletion is accompanied by dephosphorylation of MAP1B on the corresponding phosphorylatable sites. This dephosphorylation is paralleled by a release of MAP1B from microtubules. These results suggest that MAP1B phosphorylation by CK2 may be required for the assembly of microtubules within neurites. Other neuronal cytoskeletal proteins including MAP1A and tau are also substrates for CK2, indicating a role for the enzyme in the regulation of cytoskeletal functions also in mature neurons.

Amino Acid Sequence↗

PubMed1994

Regulation of microtubule dynamics by microtubule-associated protein expression and phosphorylation during neuronal development.

Neuronal morphogenesis is driven by cytoskeletal changes in which microtubules play a leading role. A very heterogeneous group of microtubule-associated proteins (MAPs) seems to control the dynamics and contribute to the organization of the microtubule cytoskeleton. Of great importance in this regard is the developmental regulation of the expression of certain MAPs in specific neuronal compartments. Furthermore, MAP functionality is also modulated by phosphorylation and dephosphorylation events. A correlation between the expression and/or phosphorylation of distinct MAPs and definite stages of neuronal development may be established. A putative role in synaptic plasticity for MAP modifications similar to those occurring during development can be anticipated. Interestingly, gross alterations in microtubule-associated proteins are found in several neuropathologies including Alzheimer's disease. In this review we focus on recent advances in the understanding of the molecular properties of major neuronal MAPs which may be relevant to these issues.

Animals↗

PubMed1994

A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome.

The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.

Amino Acid Sequence↗

PubMed1993

Differential distribution in white and grey matter of tau phosphoisoforms containing four tubulin-binding motifs.

The microtubule-associated protein tau has been isolated and purified from both white and grey brain matter. Tau isoforms were fractionated, based on their different phosphate contents, by iron-chelating affinity chromatography. Differences were observed in the proportions of phosphorylated isoforms of tau protein (containing four tubulin-binding motifs) present in white and grey matter. In white matter, isoforms containing four tubulin-binding motifs are mainly present in a phosphorylated form. Thus there appears to be a correlation between the modification, by phosphorylation, of some sites in the tau molecule and the subcellular localization (axonal or somatodendritic compartments) of the modified isoforms.

Alkaline Phosphatase↗

PubMed1993

Dephosphorylation of distinct sites on microtubule-associated protein MAP1B by protein phosphatases 1, 2A and 2B.

Rat brain microtubule-associated protein MAP1B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAP1B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline-directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.

Animals↗