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1.
J Cell Biol ; 108(3): 965-71, 1989 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2921287

RESUMO

The axonal transport of the diverse isotubulins in the motor axons of the rat sciatic nerve was studied by two-dimensional polyacrylamide gel electrophoresis after intraspinal injection of [35S]methionine. 3 wk after injection, the nerve segments carrying the labeled axonal proteins of the slow components a (SCa) and b (SCb) of axonal transport were homogenized in a cytoskeleton-stabilizing buffer and two distinct fractions, cytoskeletal (pellet, insoluble) and soluble (supernatant), were obtained by centrifugation. About two-thirds of the transported-labeled tubulin moved with SCa, the remainder with SCb. In both waves, tubulin was found to be associated mainly with the cytoskeletal fraction. The same isoforms of tubulin were transported with SCa and SCb; however, the level of a neuron-specific beta-tubulin subcomponent, termed beta', composed of two related isotubulins beta'1 and beta'2, was significantly greater in SCb than in SCa, relative to the other tubulin isoforms. In addition, certain specific isotubulins were unequally distributed between the cytoskeletal and the soluble fractions. In SCa as well as in SCb, alpha''-isotubulins were completely soluble in the motor axons. By contrast, alpha''' and beta'2-isotubulins, both posttranslationally modified isoforms, were always recovered in the cytoskeletal fraction and thus may represent isotubulins restricted to microtubule polymers. The different distribution of isotubulins suggests that a recruitment of tubulin isoforms, including specific posttranslational modifications of defined isoforms (such as, at least, phosphorylation of beta' and acetylation of alpha'), might be involved in the assembly of distinct subsets of axonal microtubules displaying differential properties of stability, velocity and perhaps of function.


Assuntos
Axônios/metabolismo , Neurônios Motores/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Transporte Biológico , Citoesqueleto/metabolismo , Feminino , Microtúbulos/metabolismo , Ratos , Ratos Endogâmicos , Nervo Isquiático
2.
Eur J Cell Biol ; 59(2): 425-32, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1493808

RESUMO

A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.


Assuntos
Anticorpos Monoclonais , Glutamatos/metabolismo , Tubulina (Proteína)/análise , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Astrócitos/química , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Pulmão/química , Masculino , Camundongos , Dados de Sequência Molecular , Neurônios/química , Baço/química , Testículo/química
5.
C R Seances Acad Sci D ; 289(10): 737-40, 1979 Oct 29.
Artigo em Francês | MEDLINE | ID: mdl-93521

RESUMO

Adult mouse liver cells obtained by enzymatic dispersion were maintained in primary cultures for up to 4 weeks. They retained some of the typical morphological and ultrastructural characteristics of hepatocytes. After 2 days of culture, structures similar to bile canaliculi were found and after 6 days clusters of small proliferating cells were noticed in the gaps of the monolayer formed by large well-spread hepatocytes. Almost all 3-day cultures began to secrete alphafetoprotein for about 2 weeks, whereas albumin was secreted throughout the period of culture.


Assuntos
Albuminas/metabolismo , Fígado/metabolismo , alfa-Fetoproteínas/metabolismo , Animais , Células Cultivadas , Fígado/citologia , Fígado/ultraestrutura , Masculino , Métodos , Camundongos , Camundongos Endogâmicos
6.
Biol Cell ; 63(3): 319-26, 1988.
Artigo em Inglês | MEDLINE | ID: mdl-2465048

RESUMO

The production and identification of a monoclonal antibody, 111 B52 C2, raised against fragments obtained after limited proteolysis of purified tubulin is described. The recognized epitope is located on the aminoterminal domain of the alpha-tubulin subunit and differs from the antigenic sites reacting with the presently existing panel of available monoclonal antibodies. This monoclonal antibody thus constitutes a potentially useful tool to explore interactions between tubulin and other specific ligands.


Assuntos
Anticorpos Monoclonais , Epitopos/análise , Tubulina (Proteína)/análise , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Immunoblotting , Camundongos , Dodecilsulfato de Sódio , Tubulina (Proteína)/imunologia
7.
J Neurochem ; 46(3): 708-16, 1986 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2419495

RESUMO

After transection of the mouse sciatic nerve, the sequence of events occurring in the distal degenerating segment was followed by the biochemical changes related to the cytoskeletal components and to the myelin protein markers. The components of the intermediate filaments and of the microtubules undergo early changes. Within 3 days, the neurofilament triplet and the peripherin disappear whereas many peptides bearing the antigenic determinant common to all classes of intermediate filaments accumulate. Several of them persist after 1 month. The tubulin pattern changes from a high level of microheterogeneity--reflecting mostly the axonal contribution--to a lower level displayed by the predominant Schwann cells. A decrease in the amount of the myelin markers is also observed. However, a month after transection, immunoreactive basic protein is still present in the degenerated segment homogenate.


Assuntos
Citoesqueleto/metabolismo , Glicoproteínas de Membrana , Proteínas da Mielina/metabolismo , Degeneração Neural , Proteínas do Tecido Nervoso , Nervo Isquiático/fisiologia , Degeneração Walleriana , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas de Filamentos Intermediários/metabolismo , Ponto Isoelétrico , Camundongos , Camundongos Endogâmicos CBA , Peso Molecular , Proteína Básica da Mielina/metabolismo , Proteínas de Neurofilamentos , Periferinas , Nervo Isquiático/ultraestrutura , Tubulina (Proteína)/metabolismo , Vimentina/metabolismo
8.
Dev Biol ; 123(2): 549-58, 1987 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3653524

RESUMO

Mouse neuroblastoma and teratocarcinoma constitute adequate cellular systems to study the expression of tubulin isoforms during early as well as later steps of neuronal differentiation. Tubulin heterogeneity is extensively analyzed using both isoelectric focusing and two-dimensional electrophoresis. Multipotential embryonal carcinoma cells express mainly one alpha-tubulin isoform (alpha 1) and three beta-tubulin isoforms: a major one (beta 3) and two minor ones (beta 4 and beta 5). Early events of neuronal differentiation are shown to induce the expression of an additional beta-tubulin isoform, beta'1, which is encoded by a specific mRNA. Neurite extension further increases tubulin heterogeneity and leads to the appearance of post-translationally modified isoforms: beta'2 in neuroblastoma and alpha 2 in teratocarcinoma cells. beta' 2 is shown to derive from the above mentioned beta'1 by phosphorylation, while alpha 2 is probably an acetylated form of the common alpha 1-tubulin. These results show that specific changes in tubulin heterogeneity are induced at different steps of neuronal differentiation and are controlled both at the transcriptional (or post-transcriptional) and post-translational levels.


Assuntos
Regulação da Expressão Gênica , Neuroblastoma/patologia , Neurônios/citologia , Teratoma/patologia , Tubulina (Proteína)/genética , Animais , Diferenciação Celular , Linhagem Celular , Células Clonais , Camundongos , Fosforilação , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Tubulina (Proteína)/biossíntese
9.
Biol Cell ; 92(6): 397-407, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11132701

RESUMO

Three distinct mRNAs have been shown to be produced by alternative splicing from the unique mouse peripherin gene. They generate three translation products, one major form, Pe-58, and two minor forms, Pe-56 which possess a shorter C-terminal sequence, and Pe-61 in which an additional sequence has been inserted in the central rod domain (Landon et al., 1989, EMBO J. 8, 1719-1726). In this study, the simultaneous occurrence of multiple transcripts in murine nervous tissues and neuroblastoma cell lines was shown by PCR amplification of fragments overlapping the sites of alternative splicing. Recombinant peripherin isoforms were purified from E. coli expressing full-length cDNAs. Rabbit antisera were raised against synthetic peptides mimicking parts of the two C-terminal sequences and of the inserted sequence of Pe-61 and were immunoadsorbed until they became monoreactive. By western blot analysis, the peripherin isoforms were localised in neuroblastoma NB2a cell lysates and detergent insoluble fractions separated by two-dimensional electrophoresis. In addition, each isoform was resolved into several charge variants. At the cellular level, each antibody decorated the filament array of the NB2a cells, suggesting the participation of the minor peripherin isoforms in the intermediate filament network.


Assuntos
Proteínas de Filamentos Intermediários , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Animais , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Soros Imunes/imunologia , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/imunologia , Camundongos , Tecido Nervoso/química , Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Neuroblastoma/química , Periferinas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Distribuição Tecidual , Células Tumorais Cultivadas/química
10.
J Biomed Mater Res ; 29(3): 315-23, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7615583

RESUMO

The adsorption of plasma proteins onto biomedical polymers is an important factor in the biocompatibility of biomaterials. We identified the plasma proteins adsorbed onto four polymeric fibers used for synthetic ligament replacement: polyarylamide, polylactic acid, polyester, and polypropylene. The adsorbed proteins were eluted and analyzed by one-dimensional and two-dimensional gel electrophoresis. Fibrinogen, albumin, IgG, high molecular weight kininogen (HMWK), and lipoproteins ApoA-1 and ApoE were the major proteins adsorbed onto polyarylamide. The three others biomaterials bound albumin, fibrinogen, ApoA-1, and ApoE; however, the proportions of proteins bound to each polymer differed. There was an inverse relationship between ApoA-1 and fibrinogen binding for all four biomaterials; polyarylamide bound a high percentage of fibrinogen, but little ApoA-1; polylactic acid, polyester, and polypropylene bound a high percentage of ApoA-1, but little fibrinogen. These results support suggestions that low fibrinogen adsorption might be due to the preferential adsorption of Apo-1. High fibrinogen binding to polyarylamide ligaments may favor fibroblast adherence, growth, and tissue repair.


Assuntos
Proteínas Sanguíneas/química , Ligamentos , Teste de Materiais , Próteses e Implantes , Adsorção , Western Blotting , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Estudos de Avaliação como Assunto , Polímeros , Ligação Proteica
11.
Dev Neurosci ; 6(6): 335-44, 1983.
Artigo em Inglês | MEDLINE | ID: mdl-6399022

RESUMO

Peripherin, a Triton-insoluble protein, whose distribution was found to be restricted to neurons in the rodent and human peripheral nervous system, was characterized by its electrophoretic features (isoelectric point: 5.6; molecular weight: 56,000 daltons) and by its peptidic map after limited proteolysis. Comparative peptide analysis of the 70,000-dalton subunit of neurofilaments (70K NFP), vimentin and peripherin, was performed by two different methods; limited proteolysis with Staphylococcus aureus V8 protease yields a different peptidic map for each protein; treatment with N-chlorosuccinimide, which cleaves preferentially at tryptophan residues, yields only two peptides from each protein: the size of the two fragments indicates that these proteins possess a single tryptophan residue located in the central part of the molecule. A rabbit antiserum raised against mouse peripherin decorated an intracellular filamentous network in mouse neuroblastoma NIE 115 cell line. The IgG fraction of the antiserum recognizes peripherin and the smallest subunit of the neurofilament triplet (70K NFP)--but not vimentin--whereas a monoclonal anti-70K NFP recognizes only the 70K NFP. Moreover, peripherin displays the common antigenic determinant shared by all intermediate filament proteins. Hence, we propose that peripherin represents a new member of the intermediate filament protein family, and might belong to the neurofilament class.


Assuntos
Proteínas de Filamentos Intermediários/isolamento & purificação , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Nervos Periféricos/análise , Animais , Química Encefálica , Linhagem Celular , Células Cultivadas , Técnicas de Cultura , Eletroforese , Imunofluorescência , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Neoplasias/análise , Nervos Periféricos/metabolismo , Periferinas , Codorniz , Coelhos , Ratos , Especificidade da Espécie
12.
J Neurochem ; 50(1): 122-30, 1988 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2826682

RESUMO

A disorder of CNS myelination was found in paralytic tremor ("pt") rabbits. The condition is inherited in a sex-linked recessive mode. Ultrastructurally, an obvious myelin deficiency with aberration of myelin sheath formation is observed. The yield of myelin isolation was reduced to 20-30% of control. Myelin isolated from 4-week-old "pt" rabbits contained reduced amounts of galactosphingolipids and of several myelin protein markers. Moreover, myelin basic protein, analyzed by two-dimensional gel electrophoresis, showed a deficit in its more basic components. All these facts suggest a delay in myelin maturation. Ganglioside content was increased as well as Na+,K+-ATPase specific activity. 2',3'-Cyclic nucleotide phosphodiesterase (CNPase) specific activity was the same in "pt" as in control myelin but differed by having greater sensitivity to detergent activation.


Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Cerebrosídeos/análise , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Ácidos Graxos/análise , Galactolipídeos , Gangliosídeos/análise , Glicolipídeos/análise , Hidroxilação , Microscopia Eletrônica , Proteínas da Mielina/análise , Bainha de Mielina/análise , Bainha de Mielina/patologia , Nervo Óptico/patologia , Coelhos
13.
J Neurosci ; 8(7): 2227-33, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3249221

RESUMO

The proteins carried by the slow axonal transport in the rat sciatic motor axons were radiolabeled by injecting 35S-methionine into the spinal cord, and the distribution of their solubility through the 2 main components of slow transport (SCa and SCb) was considered. For this purpose, a cytoskeleton-stabilizing buffer was designed in which a pellet enriched in macromolecular and polymeric structures was separated from the solubilized proteins. The monomer/polymer ratios for tubulin were quantified in the 2 rate components. Our results indicate that 90% of the total tubulin was carried with SCa. Of this, 75% was in a polymeric state, versus only 50% of the tubulin carried with SCb. The monomeric tubulin recovered in the soluble fraction was concomitantly transported with the polymerized microtubules, suggesting that it might represent metastable regions of these microtubules. The insoluble and soluble fractions of the transported actin were measured. Actin was mostly (70%) transported with SCb. Of this, more than 80% was recovered in the soluble fraction, but we cannot say whether it was in a monomeric or polymeric state, nor if it was transported free or bound to a structure solubilized during fractionation. The other 30% of the actin, most of it transported with SCa, was recovered in the polymer-enriched fraction, probably bound to a stabilized polymer, such as the microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Axônios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Polímeros/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Transporte Biológico , Fracionamento Químico , Ratos , Ratos Endogâmicos , Fatores de Tempo , Distribuição Tecidual
14.
J Neurosci Res ; 27(1): 55-64, 1990 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2254956

RESUMO

Analysis of glial fibrillary acidic protein (GFAP) and vimentin in mouse lens epithelial cells (MLEC) during ontogenesis revealed a two-step developmental expression similar to that observed in astrocytes. Vimentin was first immunostained at E11 corresponding with the closure of the lens vesicle, whereas GFAP was detected only after a further 7 days (E18); this protein appeared simultaneously in the mouse lens and CNS. In the latter case, it was present in the hypothalamic tanycytes and spinal cord. This similarity in the timing of appearance of GFAP in the non-neural MLEC and in fetal astrocytes suggests a common mechanism for its expression in tissues of different embryological origin. However, it has previously been observed that, in contrast to the situation in astrocytes, GFAP disappears from differentiating MLEC in vivo. We have shown that in vitro this protein also disappears rapidly from MLEC in the presence of fetal calf serum (FCS). However, the use of mouse serum instead of FCS inhibited the migration of MLEC out of the explant, and in these cells GFAP persisted.


Assuntos
Sistema Nervoso Central/metabolismo , Cristalinas/biossíntese , Proteína Glial Fibrilar Ácida/biossíntese , Cristalino/metabolismo , Vimentina/biossíntese , Animais , Astrócitos/metabolismo , Diferenciação Celular , Células Cultivadas , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Vimentina/genética
15.
Biol Cell ; 65(2): 109-17, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2736326

RESUMO

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture, neurons were shown to express a high degree of tubulin heterogeneity (8 alpha and 10 beta isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 alpha and 4 beta isoforms. After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with alpha-tubulin and a minor one with beta-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 alpha and 7 beta isoforms, while those of astroglia were resolved into only 2 alpha and 2 beta isoforms. The same alpha isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated form(s) of alpha-tubulin. Whether acetate-labeling of alpha-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii, is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phosphorylation of alpha-tubulin isoforms was detected. A single beta-tubulin isoform (beta'2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells.


Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/biossíntese , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , Camundongos , Fosforilação
16.
Biol Cell ; 81(1): 11-6, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7987237

RESUMO

Polyglutamylation, a posttranslational modification which consists of the sequential addition of one to six glutamyl units in the carboxy-terminal domain of both tubulin subunits, is a major event in neurons. Its structure has been investigated by using monoreactive polyclonal antibodies directed against distinct glutamylation motifs, ie alpha- and gamma-linkages between glutamyl units. It is shown that, beside alpha-linkages previously characterized, gamma-linkages also occur in glutamyl chains of brain tubulin. The co-existence of these two basic motifs leads to a conception of the polyglutamyl chain with a very sophisticated structure which could, through its complexity, help the microtubule to reach its structure and fulfil its functions.


Assuntos
Ácido Glutâmico/química , Peptídeos/síntese química , Tubulina (Proteína)/química , Sequência de Aminoácidos , Animais , Anticorpos , Química Encefálica , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/imunologia , Conformação Proteica , Tubulina (Proteína)/imunologia
17.
J Cell Sci ; 107 ( Pt 10): 2909-18, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7533173

RESUMO

Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional characteristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organization of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn't interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantitatively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcellular distribution of cytokeratin by changing their phosphorylation status and therefore to study their potential cellular functions.


Assuntos
Colforsina/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Queratinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Queratinas/metabolismo , Queratinas/ultraestrutura , Fosforilação , Células Tumorais Cultivadas
18.
Cell Motil Cytoskeleton ; 27(4): 337-49, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7520839

RESUMO

Microtubular networks are extensively developed in many ciliate species. In several of them, we investigate the occurrence of the post-translational glutamylation of tubulin [Eddé et al., 1990: Science 247:82-85; Eddé et al., 1991: J. Cell. Biochem. 46:134-142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425-432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy-terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold-resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules. Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies.


Assuntos
Cílios/química , Eucariotos/química , Glutamatos , Tubulina (Proteína)/química , Animais , Anticorpos Monoclonais , Citoplasma/química , Epitopos , Eucariotos/imunologia , Eucariotos/ultraestrutura , Ácido Glutâmico , Microtúbulos/química , Sondas Moleculares , Paramecium/imunologia
19.
Eur J Biochem ; 210(1): 359-64, 1992 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-1332866

RESUMO

Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could not detect significative change in topoisomerase I content or activity (magnesium stimulation and inhibition by ATP) during the course of liver regeneration. Topoisomerase I can be resolved into two species of 97 kDa and 100 kDa, with the same pI of 8.2-8.6 as shown by two dimensional gel electrophoresis. The two polypeptides contained a non-phosphorylated precursor and others forms with variable degrees of phosphorylation. In-vitro dephosphorylation with alkaline phosphatase leads to the disappearance of the phosphorylated forms and inactivation of the enzyme. The affinity of topoisomerase I for chromatin (measured by salt elution) differs markedly between normal and regenerating liver: nearly 50% of topoisomerase I remained bound to the chromatin from normal liver at 250 mM NaCl whereas it was completely eluted from 24-h-regenerating-liver nuclei. The biological significance of these results is discussed.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Regeneração Hepática , Fígado/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Cromatina/metabolismo , Eletroforese em Gel Bidimensional , Masculino , Fosforilação , Ratos , Ratos Wistar , Especificidade por Substrato
20.
Mol Chem Neuropathol ; 16(3): 273-88, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1418220

RESUMO

Calcium-activated neutral protease (CANP) in normal and dysmyelinating mutant, paralytic tremor (PT) rabbit myelin and premyelin fractions was studied using immature (4-5 wk) or adult animals. The enzyme was estimated by determination of its catalytic activity as well as by using immunoblot analysis after SDS-PAGE separation. The presence of two forms of CANP--one activated by calcium in the micromolar concentration (mu CANP) range and the other exhibiting low calcium sensitivity in the millimolar concentration range (m-CANP)--was found in the myelin and premyelin fractions. The developmental pattern of the enzyme activity was different for each of these two enzyme isoforms depending on the fraction studied. The higher activity on CANP (both isoforms) found in PT myelin and premyelin could be related to delayed myelination and/or to the higher turnover rate of already formed myelin. These results suggest complex and specific roles for these isoenzymes during myelin formation as is discussed further in this article. Our results confirm the extensive degradation of myelin basic protein (MBP), proteolipid protein (PLP), and, to a lesser extent, the other myelin proteins by endo- and exogenous CANP. This degradation process was significantly elevated in PT rabbit myelin. Moreover as was shown by two-dimensional gel electrophoresis, calcium-controlled proteolysis in nonmutant rabbits affected the net-charge of MBP in a manner similar to that reported for PT myelin, suggesting the possible involvement of CANP in the generation of charge isomers of MBP.


Assuntos
Calpaína/análise , Doenças Desmielinizantes/metabolismo , Isoenzimas/análise , Proteínas Musculares/análise , Bainha de Mielina/enzimologia , Coelhos/metabolismo , Animais , Química Encefálica , Doenças Desmielinizantes/genética , Mutação , Proteínas da Mielina/metabolismo , Paralisia/enzimologia , Paralisia/genética , Coelhos/genética , Tremor/enzimologia , Tremor/genética
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