Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly

A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.     

Thyroid hormones and adipose tissue development

The effect of thyroid hormones on the cellularity of the retroperitoneal adipose tissue (R.P.A.T.) was investigated in rats that were 3, 6 and 12 weeks old. Two groups of rats were respectively made hypothyroid by the antithyroid compound propylthiouracil, or hyperthyroid by thyroxine. The number of adipocytes was less in the hypothyroid rats than in the controls; it was higher in the hyperthyroid rats without any concomitant increase in the weight of their R.P.A.T. Moreover, there was no significant correlation between adipose cell number and adipose tissue weight within any group of T4 or control rats. In all groups of rats, the number of adipose cells in the R.P.A.T. was larger in males than in females; the difference was highly significant in 12 week old control rats.     

Thyroid hormones and mitochondria

Because of their central role in the regulation of energy-transduction, mitochondria, the major site of oxidative processes within the cell, are considered a likely subcellular target for the action that thyroid hormones exert on energy metabolism. However, the mechanism underlying the regulation of basal metabolic rate (BMR) by thyroid hormones still remains unclear. It has been suggested that these hormones might uncouple substrate oxidation from ATP synthesis, but there are no clear-cut data to support this idea. Two iodothyronines have been identified as effectors of the actions of thyroid hormones on energy metabolism: 3',3,5-triiodo-L-thyronine (T3) and 3,5-diiodo-L-thyronine (T2). Both have significant effects on BMR, but their mechanisms of action are not identical. T3 acts on the nucleus to influence the expression of genes involved in the regulation of cellular metabolism and mitochondria function; 3,5-T2, on the other hand, acts by directly influencing the mitochondrial energy-transduction apparatus. A molecular determinant of the effects of T3 could be uncoupling protein-3 (UCP-3), while the cytochrome-c oxidase complex is a possible target for 3,5-T2. In conclusion, it is likely that iodothyronines regulate energy metabolism by both short-term and long-term mechanisms, and that they act in more than one way in affecting mitochondrial functions.     

On the role of the tubulin nonexchangeable GTP site in bovine neurotubule assembly.

A procedure for radiolabeling the terminal phosphoryl group of the tubulin nonexchangeable GTP site using bacterial acetate kinase and acetyl-32P is described. Warming such samples to 37 degrees results in microtubule assembly and hydrolysis of the nonexchangeable site GTP in a parallel fashion. Removal of the microtubule-associated protein fraction from lebeled tubulin prevents hydrolysis and assembly, and recombination of these components restores both processes again in a parallel fashion. These and other experiments indicate that the nonexchangeable site GTP hydrolysis and assembly are intimately linked. The experiments also demonstrate that GRP is not required at the exchangeable nucleotide site for assembly to occur.     

Thyroid hormone receptors in brain and liver during ageing

The binding properties--binding capacity (MBC) and affinity (Ka)--of T3 nuclear receptors were analyzed in cortex, cerebellum and liver of rats aged 3, 6, 12 and 24 months. A slight but not significant decrease of Ka was observed in different tissues of normal rats. In hypothyroid animals the Ka in cortex at 24 months was significantly lower than at 3 months. During ageing the MBC of brain receptors decreased whereas hepatic receptors were not altered. Hypothyroidism did not further affect the MBC of the receptors. The data indicate that during ageing the T3 nuclear receptors behave differently in brain and liver. The difference in MBC suggests selectivity in organ sensitivity to thyroid hormones.     

Thyroid hormones and lipid metabolism

Some major pathways of lipid metabolism are under control of thyroid hormones. Thyroxine changes the lipid composition of different cell membranes. Modification of thyroid hormone metabolism during ontogenesis is one of the reasons of changes in lipid composition and function of cell nuclei and its other structures. Atherosclerosis and obesity may be a result of the thyroid dysfunction and modulation of the cellular lipid metabolism.     

Thyroid hormones and the cardiomyocytes

The authors present the current knowledge on the intracellular mechanisms of thyroid hormone action in the cardiomyocytes. Many of the clinical manifestations of thyroid diseases are due to the ability of thyroid hormone to alter cardiovascular hemodynamics. Triiodothyronine affects the hemodynamic state mainly by its influence on the expression of cardiomyocyte genes. These genes encode both structural and regulatory proteins in the heart (myosin heavy chains, sarcoplasmic reticulum calcium-activated ATP-ase, phospholamban). The impaired myocardium contractile activity in hypothyreosis reminds findings in heart failure and may warrant further exploration of therapeutic approaches using thyroid hormone to improve cardiac function in heart failure.     

Thyroid hormones and muscle metabolism in dogs

Muscle contents of ATP, ADP, AMP, creatine phosphate and creatine as well as glycogen, some glycolytic intermediates, pyruvate and lactate were compared in the intact, thyroidectomized and triiodothyronine (T3) treated dogs under resting conditions. After thyroidectomy muscle glycogen, glucose 1-phosphate and glucose 6-phosphate contents were significantly elevated while in T3-treated animals these variables were decreased in comparison with control dogs. Muscle free glucose was not altered by thyroidectomy but T3 treatment significantly increased its content. Muscle lactate content was elevated both in hypo- and hyperthyroid animals. Muscle ATP and total adenine nucleotide contents were significantly increased in hyperthyroid dogs while no differences were found between the three groups in the muscle creatine phosphate content. It is assumed that in T3-treated animals carbohydrate catabolism is enhanced in the resting skeletal muscle in spite of high tissue ATP content. Muscle metabolite alterations in hypothyroid dogs seem to reflect the hypometabolism accompanied by a diminished rate of glycogenolysis with inhibited rate of pyruvate oxidation or decreased rate of lactate removal from the cells.     

Thyroid hormones, learning and memory

Thyroid hormones (THs), T3 and T4, have many physiological actions and are essential for normal behavioral, intellectual and neurological development. THs have a broad spectrum of effects on the developing brain and mediate important effects within the CNS throughout life. Insufficient maternal iodine intake during gestation and TH deficiency during human development are associated to pathological alterations such as cretinism and mental retardation. In adulthood, thyroid dysfunction is related to neurological and behavioral abnormalities, including memory impairment. Analysis of different experimental models suggests that most of the effects on cognition as a result of thyroid dysfunction rely on hippocampal modifications. Insufficiency of THs during development thus alters hippocampal synaptic function and impairs behavioral performance of hippocampal-dependent learning and memory tasks that persist in euthyroid adult animals. In the present review, we summarize the current knowledge obtained by clinical observations and experimental models that shows the importance of THs in learning and mnemonic processes.     

Thyroid hormone regulates alpha and alpha + isoforms of Na,K-ATPase during development in neonatal rat brain

The brain contains two molecular forms of Na,K-ATPase designated alpha found in non-neuronal cells and neuronal soma and alpha + found in axolemma. Previously we have shown that the abundance of both forms (determined by immunoblots) as well as Na,K-ATPase activity increases 10-fold between 4 days before and 20 days after birth (Schmitt, C. A., and McDonough, A. A. (1986) J. Biol. Chem. 261, 10439-10444). Hypothyroidism in neonates blunts these increases. Neonatal, but not adult brain Na,K-ATPase is thyroid hormone (triiodothyronine, T3) responsive. This study defines the period during which brain Na,K-ATPase responds to T3. The start of the critical period was defined by comparing Na,K-ATPase activity and alpha and alpha + abundance in hypothyroid and euthyroid neonates (birth to 30 days of age). For all parameters, euthyroid was significantly higher by 15 days of age. The end of the critical period was defined by dosing hypothyroid neonates with T3 daily (0.1 micrograms/g body weight) beginning at increasing days of age, and sacrificing all at 30 days then assaying enzyme activity and abundance. Those starting T3 treatment on or before day 19 were restored to euthyroid levels of Na,K-ATPase activity and abundance, while those starting T3 treatment on or after day 22 remained at hypothyroid levels of enzyme activity and abundance. We conclude that brain Na,K-ATPase alpha and alpha + isoforms are sensitive to T3 by as late as 15 days of age and that the period of thyroid hormone responsiveness is over by 22 days.     

Microtubules and brain development: The effects of thyroid hormones

The data accumulated during the past twenty years suggest that thyroid hormones have a direct effect on the differentiation of both the neurons and the glial cell during the critical period of brain development. A fast survey of the available data (which is presented in the introduction of this article) on the mechanism of action of thyroid hormones and on their different effects during brain development suggests that the most dramatic effect of hypothyroidism is a hypoplastic neuropile. Both in vivo, during the critical period of nerve cell differentiation and in vitro, when added to primary cultures of embryonic nerve cells thyroid hormones stimulate neurite outgrowth. Since neurite outgrowth requires massive microtubule assembly the assumption was made that thyroid hormones stimulate nerve cell differentiation by changing the concentration and/or activity of the different proteins (tubulin and “microtubule associated proteins”, MAPs) which co-polymerize to form microtubules.

Preliminary information was obtained by following the kinetics of microtubule assembly in crude brain supernatants. The data showed that: (1) the rate of in vitro microtubule assembly increases with age during brain development; (2) hypothyroidism, when produced in the rat at late pregnancy, slows this evolution; (3) early replacement therapy with thyroid hormones restores normal rates of assembly; (4) the addition of purified MAPs to normal young or 15-day-old hypothyroid brain preparations restores normal rates of polymerization. These and other data suggested that thyroid hormones regulate microtubule assembly by changing the concentration and/or activity of one or more of the MAPs.

Further analysis revealed that striking qualitative changes in MAPs composition occur during brain development. For instance, the TAU fraction, a group of 4–5 proteins with a molecular weight of 60–68 K which is present in adult brain, is absent at early stages of postnatal development: two other entities are present, TAU slow and TAU fast, with different molecular weights, lower activity and different peptide mapping. This latter observation suggests that different TAU genes are expressed during brain development; a conclusion which has been confirmed by cell-free translation of the mRNas coding for these proteins. Analysis of the TAU fraction prepared from hypothyroid rat brains also revealed that a group of TAU proteins. “TAU₃”, is almost missing, whereas thyroid hormone administration markedly increases its concentration. Two-dimensional gel electrophoresis showed that the TAU fraction is composed with more than 15 entities, with at least five of them being under thyroid hormone control.

The precise physiological significance of the heterogeneity of MAPs and of the changes in MAPs composition seen during development and in hypothyroid rat brain remains to be determined. The assumption is made that these changes might be of utmost importance to regulate the number and length of the microtubules, and therefore the number and length of the neurites which are formed during the differentiation process of the different neurons. Thyroid hormones would be in these respects one of the epigenic factors required to synchronize sequentially the expression of the genes coding for these proteins in the different nerve cells.     

Chromatin assembly during SV40 DNA replication in vitro

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.     

Studies on cystathionase activity in rat liver and brain during development. Effects of hormones and amino acids in vivo

High activity of cystathionase was present in rat liver but only low amounts of activity in rat brain during development. Triamcinolone had no effect on liver cystathionase activity in foetuses but increased the enzyme activity significantly in postnatal rats. l-Thyroxine decreased liver cystathionase activity significantly in newborn rats; administration of pyridoxal 5'-phosphate did not prevent this effect. l-Methionine significantly increased liver cystathionase activity in newborn rats.     

Ciboulot regulates actin assembly during Drosophila brain metamorphosis

A dynamic actin cytoskeleton is essential for the remodeling of cell shape during development, but the specific roles of many actin partners remain unclear. Here we characterize a novel actin binding protein, Ciboulot (Cib), which plays a major role in axonal growth during Drosophila brain metamorphosis. Loss of Cib function leads to axonal growth defects in the central brain, while overexpression of the gene during development leads to overgrown projections. The Cib protein displays strong sequence similarity to beta-thymosins but has biochemical properties like profilin: the Cib-actin complex participates in actin filament assembly exclusively at the barbed end, and Cib enhances actin-based motility in vitro. Genetic experiments show that Cib and the Drosophila profilin protein Chickadee (Chic) cooperate in central brain metamorphosis.