Search PubMed⌕ Search

Biomedical subjects

J Nunez

Publications and source records attributed to J Nunez.

At least 91 records · Page 5Linked to original sources
PubMed1980

Changes in composition and activity of microtubule-associated proteins during brain development.

The onsert of neuronal differentiation is characterised by intensive neurite growth; because microtubule formation is strictly required during this process, in vitro assembly of the tubulin present in the rat brain has been studied at different stages of development: the rate of assembly is very slow in the early stages and increases progressively with age from birth until adulthood. Other data also suggested that the limiting factor in the young brain is the amount or activity of one or several of the minor components which co-polymerise into microtubules with tubulin. We show here that both the composition and the activity of the microtubule-associated proteins change during the time course of rat brain development.

Age Factors↗
PubMed1980

Cytosolic thyroxine-binding protein and brain development.

The properties of cytosolic thyroxine binding protein were studied in the cortex and cerebellum of the rat at different stages of postnatal development: (1) Polyacrylamide-gel electrophoretic analysis showed that rat-brain cortex and cerebellum contain the same cytosolic thyroxine-binding protein which is very similar to the liver-corresponding entity. No changes in the electrophoretic mobility were seen during development in the 2 brain regions. In contrast, no defined triiodothyronine-binding component could be observed by the same technique. (2) Kinetic analysis studies revealed that the equilibrium of binding is reached in approximately 10 min whatever the brain region, the concentration of cytosolic protein and the stage of development. In all these cases saturation was obtained with the same thyroxine concentration (approximately 5 x 10(-7) M). Scatchard analysis also showed that whatever the experimental conditions, brain cytosolic protein contains a single class of thyroxine-binding sites with a K A of approximately 8 x 10(7) M-1. (3) Comparison of the K A during development showed that this constant remains unchanged from day 3 after birth until day 35 in both the cortex and the cerebellum. In contrast the number of binding sites significantly decreases in the cortex (approximately 2-fold; p less than 0.001) from day 3 to 35 with an already significant decline from day 3 to 6 (p less than 0.001). In the cerebellum this decline was even more marked since almost no binding activity was left at adulthood. Comparison of cortex and cerebellum binding activities also showed that this latter region contains approximately half the binding sites (p less than 0.001) at every stage of development studied.

Aging↗
PubMed1980

Opposite effects of thiocyanate on tyrosine iodination and thyroid hormone synthesis.

The effect of a pseudohalide, SCN-, an anion with the same molecular size as iodide, was studied on two reactions: thyroglobulin iodination and thyroid hormone synthesis (coupling reaction) catalyzed by peroxidases. The coupling reaction was studied separately from the iodination reaction by using labelled thyroglobulin samples previously iodinated but containing little or no hormones. 1. SCN- inhibits iodide oxidation (I- leads to I2) whatever the enzyme, thyroid, lactoperoxidase or horseradish peroxidase. The amount of SCN- required to completely inhibit this reaction varies depending on the enzyme. Similarly tyrosine iodination is inhibited by SCN- with large variations, depending on the peroxidase, in the concentration of this anion required for inhibition. 2. In contrast SCN- stimulates the coupling reaction: (a) this affect is seen with the thyroid and lactoperoxidases but not with horseradish peroxidase; (b) the concentration of SCN- required for half-maximal stimulation of the coupling reaction is much lower (0.5-1 microM) than that required for the inhibition of iodide oxidation (60-80 microM); (c) ClO4(-), an anion with the same molecular size as SCN- and I-, has no effect on the coupling reaction; (d) this stimulatory effect of SCN- does not depend on a modification of the thyroglobulin molecule since it is not seen with horseradish peroxidase or in purely chemical coupling conditions. 3. The stimulatory effect of SCN- is therefore seen as resulting from the binding of this anion to a limited number of high-affinity sites present at the surface of both thyroid and lactoperoxidases. The inhibitory effect depends, in contrast, on the binding of SCN- to the substrate site with lower affinities. Since iodide also behaves both as a substrate for the iodination reaction and as a stimulatory ligand for the coupling reaction, these data provide further support in favour of the existence of an enzyme-iodide (or SCN-) complex with catalytic properties different from those of the native peroxidase.

Dose-Response Relationship, Drug↗
PubMed1980

Regulatory effects of iodide and thiocyanate on tyrosine oxidation catalyzed by thyroid peroxidase.

the effects of iodide, thiocyanate and perchlorate, three anions with the same molecular size, on the oxidation of tyrosine to 3,3'-bityrosine by several peroxidases were evaluated at pH 8.8, i.e. in conditions in which iodide is not oxidized. The following results were obtained: 1. Iodide greatly stimulates the rate of bityrosine formation in the presence of thyroid peroxidase. No effect was seen with horseradish peroxidase or lactoperoxidase. Maximal iodide effects were obtained with about 0.5 mM iodide and Km for iodide was equal to about 0.028 mM. These results suggest that thyroid peroxidase contains a simple class of regulatory binding sites for iodide. 2. SCN- mimics iodide effects; maximal stimulatory effects were seen with about 0.5 mM thiocyanate and Km for SCN- was equal to 0.1 mM. The effects of SCN- and those of iodide were not additive. These results suggest that SCN- binds to the same regulatory site as iodide but with a slightly lower affinity. No effect of SCN- was seen with horseradish peroxidase or lactoperoxidase. 3. ClO-4, another anion with the same molecular size as iodide and SCN-, had neither an effect on the oxidation of tyrosine to bityrosine nor did it prevent the stimulatory effect of iodide on this reaction. Bromide was without effect on the same reaction.

Horseradish Peroxidase↗
PubMed1980

Rat, mouse, and guinea pig brain development and microtubule assembly.

The development of in vitro microtubule assembly and of tubulin concentration have been studied during brain maturation in the mouse and the rat, two species which have postnatal brain development, and in one species which is mature at birth, the guinea pig. (a) The rat of tubulin assembly is very slow soon after birth in both the mouse and rat; it increases progressively with age until adulthood. In contrast, in the guinea pig this rate is maximal at birth and slower rates are seen only at foetal stages. (b) Postnatal changes in the lag period of assembly and in the minimal concentration of tubulin (Cc) required to obtain in vitro assembly are seen in the mouse and the rat; in contrast these parameters are constant at all postnatal stages in the guinea pig with longer lag periods and lower Cc values being seen only at foetal stages. (c) Maximal rates of assembly, minimal lag periods, and minimal Cc values are restored after addition of microtubule-associated proteins to foetal guinea pig or young mouse and rat preparations, suggesting that the difference in the kinetic parameters of assembly between these species depends on differences in the concentration or activity of these proteins. (d) Maximal tubulin concentrations are observed before birth in the guinea pig and approximately at day 10 in the rat and mouse.

Aging↗
PubMed1979

The effects of thioureylene compounds (goitrogens) on lactoperoxidase activity.

The rates of oxidation of several goitrogens by lactoperoxidase and the rates of inactivation of lactoperoxidase by the same goitrogens have been measured. The influence of iodide on both reactions has also been evaluated. It has been shown by us that iodide acts catalytically in regulating lactoperoxidase activity at pH 8.8. The rate data have been analyzed by a computer program which solves the differential equations for the above mentioned reactions. From this computer analysis we have been able to obtain binding constants of the goitrogens and inactivation rate constants of lactoperoxidase. Iodide was shown to inhibit goitrogenic activity either by increasing the rate of drug oxidation or by reducing the rate of enzyme inactivation, or both, depending on the particular drug. Iodide had little or no effect on the goitrogen-binding constants. We have also shown that the relative rates of enzyme inactivation can be correlated with the potency of the goitrogen as an antithyroid drug.

Antithyroid Agents↗
PubMed1979

Regulation of lipogenesis in adipocytes. Independent effects of thyroid hormones, cyclic AMP and insulin on the uptake of deoxy-D-glucose.

Thyroidectomy is known to enhance fat cell phosphodiesterase activity; as a result, the response to lipolytic hormones is markedly reduced. Thyroidectomy also stimulates overall lipogenesis and the uptake of glucose: the present experiments investigated whether there was a correlation between cyclic AMP and glucose uptake. The parameter measured was the transport and phosphorylation (uptake) of deoxy-D-glucose in the presence of two modifiers of the cyclic AMP pool: phosphodiesterase inhibitors and the analogue, dibutyryl cyclic AMP. The inhibition by methylxanthines and dibutyryl cyclic AMP of deoxy-D-glucose uptake observed, was the same in fat cells from normal and thyroidectomized rats: the latter nonetheless still maintained their enhanced glucose uptake. It was therefore concluded that thyroid hormones and cyclic AMP control this step by different, separate pathways. Insulin, well known for its lipogenic effect, enhanced deoxy-D-glucose uptake in fat cells from both normal and thyroidectomized rats to the same extent (about 40%). An additive effect of thyroidectomy and insulin on glucose uptake was thus demonstrated. These results imply that glucose uptake in the adipocyte is controlled by at least three factors: thyroid hormones, cyclic AMP and insulin, each of which can act independently. Maximum glucose uptake is achieved in the presence of a combination of low concentrations of cyclic AMP, of insulin, and in the absence of thyroid hormones.

Adipose Tissue↗
PubMed1979

Iodide binding and regulation of lactoperoxidase activity toward thyroid goitrogens.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.

Iodides↗
PubMed1979

Cyclic AMP and lipogenesis in fat cells from thyroidectomized rats.

Thyroid hormones regulate lipid metabolism by affecting lipogenesis as well as lipolysis. The present paper discusses the way thyroidectomy induced an enhancement in lipogenesis in rat fat cells. The doubling in the conversion of glucose to CO2 and fatty acids seen after thyroidectomy was found to be due to a modification in the actual pathway of glucose metabolism: there was a preferential stimulation of the conversion of glucose to CO2 by the pentose cycle (utilisation of [1-14C]glucose) while the production of fatty acids and glyceride-glycerol proceeded, respectively, much more, or only slightly more, via the pathway of [6-14C]glucose metabolism. Studies employing the phosphodiesterase inhibitor MIX, or the cyclic AMP analogue, DBcAMP showed that the lipogenic process depends on cyclic AMP. As the stimulatory effect of thyroidectomy was not abolished, however, lipogenesis must be under the independent control of both cyclic AMP and absence of thyroid hormones. Insulin, a further mediator of lipogenesis was found to further enhance the already preexisting high conversion of glucose to CO2 in fat cells from thyroidectomized rats. It is concluded that at least three factors modify lipogenesis: thyroidectomy, cyclic AMP and insulin; each achieving its effect in an independent manner.

1-Methyl-3-isobutylxanthine↗
PubMed1979

Competitive inhibition of colchicine binding to tubulin by microtubule-associated proteins.

Microtubule-associated proteins (MAPs) promote tubulin polymerization, whereas colchicine inhibits this process. In this paper, MAPs have been shown to inhibit colchicine binding to tubulin in a competitive manner. Attempts were made to identify which of the MAPs fraction(s) was responsible; both tau protein (a thermostable molecule with a molecular weight of approximately 70,000) and a high molecular weight fraction (HMW) were able to compete with colchicine. In contrast, Mg2+, which also induces microtubule assembly in vitro, had no effect on colchicine binding to tubulin.

Animals↗
PubMed1979

Thyroid hormones and neurotubule assembly in vitro during brain development.

A new model has been used to evaluate the effects of thyroid hormones on brain development. This model is based on the assumption that the major effect of thyroid hormones is in regulating the rate of neurite growth of the rat brain at early stages of postnatal development. Microtubules were chosen as markers of neurite growth. We tested, therefore, whether the rate of microtubule assembly in vitro is under thyroid hormone control. The following results were obtained: The rate of tubulin assembly into microtubules in vitro seems to be thyroid hormone dependent: (a) in 15-day-old hypothyroid rats the rates of tubulin assembly in vitro are low, comparable to those levels found in normal rats on day 3; (b) normal rates of assembly in vitro are restored upon addition of very small amounts of microtubule fragments which act as nucleating centers in the process of microtubule formation; (c) addition of microtubule-associated proteins to a hypothyroid preparation restores maximal assembly rates; similar results were obtained on adding one of the microtubule-associated proteins (purified tau protein); (d) physiological amounts of thyroid hormones completely restore normal assembly rates provided that they are administered very early after birth; (e) the ability of tubulin to assemble maximally does not seem to be permanently impaired, since normal assembly rates are spontaneously restored when hypothyroidism is maintained until an adult stage; (f) normal microtubule assembly is observed when hypothyroidism is produced at an adult stage. The model which may be constructed from these results implies that thyroid hormones are required briefly after birth to accelerate the rate of microtubule assembly thus allowing intensive neurite growth during the critical period of brain development.

Aging↗
PubMed1978

[In vitro synthesis of 3,5,3'-triiodothyronine catalyzed by thyroid peroxidase (author's transl)].

3,5,3'-triiodotyronine formation was studied in vitro after iodination of goiter thyroglobulin catalyzed by purified thyroid peroxidase. A fractionnal number of T3 is always obtained per mole of thyroglobulin in both in vitro and in vivo. This result cannot be explained by a heterogeneity in thyroglobulin iodination or in a partial conversion of T3 to T4. It is suggested that thyroglobulin is heterogenous either in its primary sequence or in its teritiary configuration. Thyroglobulin contains tyrosine residues which are specific for the T3 formation and moreover T3 is not the precursor of T4. The efficiency of T3 formation was studied: the maximal number of T3 molecules is obtained with 30 iodine atoms per mole of thyroglobulin. In addition the results suggest that the tyrosines which are coupled with a high efficiency are iodinated sequentially.

Catalysis↗
PubMed1977

Microtubule assembly in vitro. Purification of assembly-promoting factors.

The role of microtubule-associated proteins in the assembly of tubulin to microtubules in vitro has been studied. 1. It has been confirmed that pure tubulin obtained by phosphocellulose column chromatography does not significantly assemble in vitro in the absence of minor components which co-polymerize with tubulin. Although tubulin aggregates in a morpholino-ethanesulfonate buffer containing high Mg2+ concentrations, this process was neither inhibited by Ca2+ or colchicine, nor reversed by cold exposure. 2. Microtubule-associated proteins were prepared, either by phosphocellulose column chromatography or by a direct method based on boiling reassembled microtubules in the presence of 2 mM dithiothreitol and 0.75 M NaCl. From each of these preparations two protein fractions were purified, either by Ultrogel ACA34 chromatography or by sucrose gradient ultracentrifugation. The first one, with a high molecular weight, did not promote tubulin assembly; ageing of this material did not induce any activity. On the other hand, the second fraction, with an apparent molecular weight of 70 000 (tau protein), when almost completely purified, was active in promoting assembly. Thus a single specific protein is able to promote assembly of pure tubulin.

Animals↗