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J Nunez

Publications and source records attributed to J Nunez.

At least 73 records · Page 4Linked to original sources
PubMed1985

Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture.

Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins. The cells were cultured for 4 days in serum-free and thyrotropin-free conditions. Thyrotropin was then added for varying periods of time (6-96 h). In the absence of thyrotropin, the cells have an elongated flattened aspect. Exposure to thyrotropin for 6-24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape. After varying periods of culture the cells were labelled with [35S]methionine for 6 h and then analyzed by one- and two-dimensional gel electrophoresis, followed by autoradiography. The results were as follows. After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased. After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively. Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified. Actin synthesis was markedly decreased with a maximum 24-48 h after the addition of thyrotropin. A modification in the ratio between alpha and beta tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30 000-40 000) and the isoelectric points of tropomyosins. Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by cAMP. In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and cAMP) triggers the changes in cell shape which occur during thyroid cell culture. They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.

Actins↗
PubMed1985

Microtubule-associated proteins and in vitro astrocyte differentiation.

Primary cultures of mouse brain astrocytes have been used to identify the microtubule-associated proteins (MAPs) present in this cell type at different stages of in vitro differentiation. The MAPs of the astrocyte have been identified by polyacrylamide gel electrophoresis and immunological detection. Two antisera were raised against two brain MAPs, tau and MAP-2. These antisera were also used to label the microtubular network in the intact astrocytes at different stages of the culture. The mature astrocyte contains a variety of MAP-like proteins. Anti-MAP-2 serum detected several proteins of high molecular weight (380,000, 260,000, 205,000 and 165,000 mol wt) and one microheterogeneous peak of 83,000 mol wt. Anti-tau also detected high molecular weight components (380,000 to approximately 200,000 mol wt) but not the 165,000-mol-wt peak; in addition two microheterogeneous peaks of 83,000 and 62,000 mol wt were detected by the anti-tau serum. The 62,000-mol-wt peak was therefore detected only by the anti-tau serum whereas the 83,000-mol-wt component cross-reacted with both antisera. At early stages of the culture the immature cell contained about two times less immunoreactive material than at mature stages. Qualitative changes of the high molecular weight components were also observed. In the intact cell both antisera revealed a dense fibrous network. At early stages of the culture the astroblasts were stained by the antisera but the reaction was very diffuse in the cytoplasm; few fibrous cells were intensively stained. Morphological differentiation, which began after serum deprivation and which was accelerated by forskolin (a drug that induces cyclic AMP accumulation), led to high labeling of both the cell body and the cellular processes. In the presence of colchicine the staining regressed, the processes shortened, and the cell returned to a less-apparently differentiated state.

Animals↗
PubMed1984

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin.

Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.

Animals↗
PubMed1984

Interaction between calmodulin and microtubule-associated proteins prepared at different stages of brain development.

Rat brain microtubules were prepared at the adult stage and from immature (i.e., 4-day-old) animals. At an early stage of development, the composition of microtubule-associated proteins is qualitatively different from that found at the adult stage [(1982) Eur. J. Biochem. 129, 465-471]. The influence of calmodulin on the time course of assembly of second cycle microtubules was compared at both stages of brain development (i.e., microtubules originating from 4-day-old and adult animals). In the presence of Ca2+ the inhibition of microtubule assembly was more pronounced at a young stage of brain development than at the adult stage. Cross-linking studies with 125I-labeled calmodulin further established that the two major microtubule-associated proteins, MAP2 and TAU were able to bind to calmodulin at both stages of brain development but with different intensities. The labeling with 125I-labeled calmodulin was Ca2+-dependent, specific, displaced by unlabeled calmodulin and trifluoperazine.

Aging↗
PubMed1984

Thyroid hormones and fat cell phosphorylation.

The phosphorylation of cytosolic and plasma membrane proteins was studied in isolated fat cells from euthyroid and thyroidectomized rats. The analysis, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, of subcellular fractions of 32P-labelled fat cells revealed the presence of 10-12 phosphoprotein bands in the cytosol. The washed plasma membrane fraction contained 4 major phosphoproteins with estimated molecular weights of 70-67, 60, 42-40 and 26-22 kDa. Two-dimensional analysis of the 32P-labelled phosphoproteins showed that their isoelectric points were between 6.3 and 4.1. The profiles and the isoelectric points were similar in fat cells from euthyroid and thyroidectomized rats. The state of hypothyroidism did not affect the basal phosphorylation of fat cell proteins of the cytosolic or plasma membrane fractions. The incubation of fat cells from euthyroid rats in the presence of isoproterenol or dibutyryl adenosine cyclic monophosphate led to (a) an increase in the 32P labelling of cytosolic proteins which may be subunits of acetyl CoA carboxylase, ATP citrate lyase, hormone-sensitive lipase and other proteins, with apparent molecular weights between 50 and 42 kDa, and (b) an increase in the 32P labelling of plasma membrane proteins of 26-22 kDa. In the case of fat cells from hypothyroid rats, the dibutyryl adenosine cyclic monophosphate increased the 32P labelling of plasma membrane proteins, whereas in the presence of isoproterenol these reactions did not occur. These results show that thyroid hormones control the 32P labelling of proteins of the cytosol and plasma membrane fractions of rat fat cells and therefore, at least in some cases, the lipolytic and lipogenic pathways.

5'-Nucleotidase↗
PubMed1983

Multisite phosphorylation of tau proteins from rat brain.

tau proteins from adult and young rat brains were phosphorylated in vitro by protein kinases present in microtubule preparations. Several phosphates were incorporated in each molecular species of this group of proteins. Cyclic AMP dependent protein kinases and casein kinase (type I) phosphorylated tau proteins on different sites. These observations indicate that tau proteins are an example of multisite phosphorylation.

Adenosine Triphosphate↗
PubMed1982

Heterogeneity of microtubule-associated proteins and brain development.

Developmental changes in the composition of brain microtubule-associated proteins have been studied in three species: the rat and the mouse, which are characterized by post-natal brain development, and the guinea-pig, whose brain is mature at birth. 1. At an adult stage, and whatever the species, two major microtubule-associated proteins, which have been referred to MAP2 and tau, have been identified by polyacrylamide gel electrophoresis. Rat tau is composed of four closely spaced bands; mouse tau contains only three components with one of them being present in higher proportion than the others; adult guinea-pig tau is essentially present as a single band. 2. Microtubule-associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the tau region at immature stages of development (fast tau and slow tau). However adult tau factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2. 3. Peptide mapping analysis of the purified tau entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat tau proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different. 4. The activities of young tau proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast tau and young slow tau were purified and both show to be active in promoting pure tubulin polymerization. 5. These data demonstrate the existence of two types of heterogeneity of microtubule-associated proteins: plurality of protein species at every stage of brain development and changes in composition and activity dependent on development.

Animals↗
PubMed1982

A biochemical model for neurite outgrowth during brain development.

Maximal amounts of tubulin in rat brain are observed during the 3 to 10-day postnatal period. The rates of in vitro tubulin polymerization are very low at these stages of development; they increase thereafter during the second postnatal week, reaching a maximum at adulthood. The increased rate of polymerization could depend either on modifications in the concentration and activity of microtubule-associated proteins (MAPs), which play a crucial role in microtubule assembly in vitro, or on changes in their composition. The results show that the composition and activity of TAU proteins (MW: 58-68000) change during development. Analysis of "young" and "adult" TAU protein peptide mapping suggests that their amino acid sequence is different. Our data indicate a good correlation between tubulin capacity to polymerize in vitro and changes in the composition and activity of TAU proteins which occur during the critical period when the neuronal network is constructed.

Aging↗
PubMed1981

Thyroid hormone synthesis in thyroglobulin. The mechanism of the coupling reaction.

[U-14C]Tyrosine-labeled noniodinated hog thyroglobulin was iodinated enzymatically and nonenzymatically (iodine, iodide-chloramine-T, pH 7.4, or iodine monochloride, pH 8.1). This led to similar levels of iodine incorporation as well as of thyroid hormone synthesis. Iodine monochloride at pH 5.5 formed "hormonogenic" iodotyrosine residues, but no hormone residues. The latter were formed when the iodinated thyroglobulin was brought to pH 8.5 and then treated with horseradish peroxidase and glucose-glucose oxidase in the absence of iodide and iodine monochloride. Enzymatic hydrolysates contained labeled hormone and pyruvic acid; acid hydrolysates labeled thyronine and acetic acid. (Treatment with acid converts hormone to thyronine and pyruvic to acetic acid.) After borohydride treatment, labeled alanine was present instead of pyruvic or acetic acid. The pyruvic acid/hormone, acetic acid/thyronine, alanine/hormone, and alanine/thyronine molar ratios always were 1, independently of the method of iodination. The "coupling reaction" consists of an oxidation step and nonoxidative coupling and decomposition steps. The oxidation step may be either enzymatic or nonenzymatic. The decomposition step always leads to 1 dehydroalanine residue for each hormone residue synthesized. (Dehydroalanine residues appear in the various hydrolysates as acetic acid, pyruvic acid, and alanine, respectively.) Since proper alignment of 2 iodotyrosine residues is a prerequisite for coupling, a model is proposed according to which oxidation of hormonogenic iodotyrosine residues leads to a charge transfer complex which is the same zwitterion-biradical resonance hybrid no matter whether it resulted from a free radical (enzymatic) or an ionic (nonenzymatic) oxidation.

Animals↗
PubMed1981

Effect of tau on the vinblastine-induced aggregation of tubulin.

Two microtubule-associated proteins, tau and the high molecular weight microtubule-associated protein 2 (MAP 2), were purified from rat brain microtubules. Addition of either protein to pure tubulin caused microtubule assembly. In the presence of tau and 10 microM vinblastine, tubulin aggregated into spiral structures. If tau was absent, or replaced by MAP 2, little aggregation occurred in the presence of vinblastine. Thus, vinblastine may be a useful probe in elucidating the individual roles of tau and MAP 2 in microtubule assembly.

Microtubule-Associated Proteins↗