Duox2 (and probablyDuox1Reactive oxygen species (ROS) 1 have emerged as important molecules involved in regulating essential cell functions, such as growth and differentiation (1). NAD(P)H oxidases are a major source of ROS. Phagocyte oxidase is the oxidase that has been investigated most thoroughly (2). It catalyzes the production of superoxide by the one-electron reduction of oxygen, using NADPH as the electron donor. The catalytic moiety of the phagocyte NADPH oxidase is gp91phox , a plasma membraneassociated flavohemoprotein. Recently, it was discovered that gp91 phox belongs to a family consisting of several very similar oxidases. Seven NOX (NADPH oxidase) and DUOX/ThOX (dual oxidase/thyroid oxidase) genes have been identified that encode different NADPH oxidases with differing mRNA tissue expression. The Nox family comprises gp91phox , now known as Nox2; Nox1, which is predominantly expressed in the colon (3); Nox3, cloned from fetal kidney (4); Nox4 found in the kidney cortex (5, 6); and Nox5, expressed in the testis, spleen, and lymph nodes (7). In addition to the basic structure of gp91 phox , Nox5 has a long intracellular N-terminal domain with four calcium binding sites implicated in its Ca 2ϩ -dependent activation (8).The biological functions of these Nox proteins are now under investigation. They are involved in signal transduction related to cell growth and cancer (9 -11) and to angiogenesis (12).Duox1 and Duox2 are large homologues of Nox2 with an N-terminal extension comprising two EF-hand motifs, an additional transmembrane helix, and a peroxidase homology ectodomain (see Fig. 4A) (13,14). DUOX genes have been identified in the thyroid gland, where they are strongly expressed (13,14). However, the DUOX are also expressed on the mucosal surfaces of the trachea and the bronchi (15) and in the airway epithelial cells (16,17), where it has been suggested that Duox1 is the isoform responsible for acid production and secretion in airways (16) and plays a critical role in mucin expression (18). DUOX2 was also expressed throughout the digestive tract, where it was found to be functional (19,20), in addition to the salivary gland and rectum (15).It has been suggested that Duox2, which was identified by purifying thyroid NADPH oxidase, may constitute the catalytic core of this enzyme and generate the H 2 O 2 used by Tpo to catalyze the biosynthesis of thyroid hormones at the apical surface of the thyrocytes (13). Although no functional Duoxbased H 2 O 2 -generating system has yet been reconstituted (21), this proposal is corroborated by a recent report of permanent and severe congenital hypothyroidism in a patient with a bial-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ Recipient of a fellowship from the Commissariat à l'Energie Atomique (Paris, France).ʈ Recipients of a fellowship from the National Education, Research, an...
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 ACAW 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 70000 (z protein), when almost completely purified, was active in promoting assembly.Thus a single specific protein is able to promote assembly of pure tubulin.Increasing evidence is available showing that in several systems where the microtubules are not stable as in cilia, specific signals induce a rapid massive polymerization of microtubules from a preexisting store of their basic subunit 6-S tubulin [l -41.For instance, in previous preliminary publications [5,6] we have shown that the amount of tubulin is not a limiting factor during rat brain development. However, the rate of polymerization in vitro and the overall extent of microtubule assembly is much lower with crude supernatants prepared from young rat brain than with control adult rat brain preparations. These results suggested that a 'factor' or 'signal' is needed for microtubule assembly and that the amount of this factor is limiting in the young brain.The nature of these signals or factors remains controversial.
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 z, have been identified by polyacrylamide gel electrophoresis. Rat z is composed of four closely spaced bands; mouse z contains only three components with one of them being present in higher proportion than the others; adult guinea-pig z 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 z region at immature stages of development (fast z and slow z). However adult z 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 z entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat z 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 z proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast z and young slow z 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.
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.
The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T3) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na(+)-independent, and efficiently inhibited by T3 (half-inhibition at approximately 2 microM). Two Na(+)-independent L-like systems (L1 and L2), common to leucine and aromatic amino acids, were characterized kinetically. System L2 had a low affinity for leucine and tryptophan (Km = 0.3-0.9 mM). The high-affinity system L1 (Km approximately 10 microM for both amino acids) was competitively inhibited by T3 with a Ki of 2-3 microM (close to the T3 transport Km). Several T3 analogues inhibited system L1 and the T3 transport system similarly. Glutamine uptake and alpha-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T3 had little effect on the uptakes of glutamine and alpha -(methylamino)isobutyric acid. The results indicate that the T3 transport system and system L1 are related.
The uptake of 3,3',5-[3'-125I]triiodo-L-thyronine ([125I]L-T3) and of L-[3',5'-125I]thyroxine ([125I]L-T4) by cultured rat glial cells was studied under initial velocity (Vi) conditions. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The following respective values of Km (microM) and Vmax (fmol/min/microgram of DNA) were obtained at 25 degrees C: 0.52 +/- 0.09 and 727 +/- 55 for L-T3 and 1.02 +/- 0.21 and 690 +/- 85 for L-T4. Ki values (microM) for the inhibition of [125I]L-T3 uptake by unlabeled analogues were as follows: L-T4, 0.88; 3,3',5'-triiodo-L-thyronine, 1.4; 3,3'-diiodo-L-thyronine, 2.9; 3,3',5-triiodo-D-thyronine, 4.8; and triiodothyroacetic acid, 5.3. These values indicate that the uptake system is stereospecific. Unlabeled L-T3 was a better competitor than unlabeled L-T4 for the uptake of [125I]L-T4, an observation suggesting that both hormones were taken up by a common carrier system. L-T3, and L-T4 uptake was pH dependent, a finding suggesting that the phenolic unionized form of the hormones was preferentially taken up. L-T3 uptake was studied in the presence of various inhibitors; the results suggest that uptake was independent of the transmembrane Na+ gradient and of the cellular energy. Compounds that inhibited cellular uptake but were without effect on L-T3 binding to isolated nuclei also inhibited L-T3 nuclear binding in intact cells, an observation suggesting that uptake could be rate limiting for the access of L-T3 to nuclear receptors when transport is severely inhibited.
Cultured astroglial cells were found to contain a type II 5'-deiodinase (5'D) activity which was increased by 10(-3) M (Bu)2cAMP but not by 2 X 10(-3) M n-butyrate. 8-Bromo-cAMP (8-Br-cAMP) (10(-3) M) also increased this enzyme activity. Cycloheximide (2 micrograms/ml) inhibited the 8-Br-cAMP effect on 5'D activity. Forskolin (10(-5) M), cholera toxin (5 micrograms/ml), 10(-5) M isoproterenol, and 3 X 10(-6) M norephinephrine also increased the 5'D activity of astroglial cells. After a 4-h incubation these agents or cAMP analogs had maximal effect, and enzyme activities were 6- to 14-fold above control value. The stimulatory effects of isoproterenol and norepinephrine were almost completely reversed after 8 h incubation. The induction of 5'D activity by isoproterenol or norepinephrine was inhibited by the beta-adrenergic antagonist alprenolol (5 X 10(-6) M). The effect of norepinephrine was not significantly affected by the alpha 1-adrenergic antagonist, prazosin (10(-5) M). Thus, 5'D activity is controlled by agents increasing cAMP in astroglial cells, and in particular by the neurotransmitter, norephinephrine, via a beta-adrenergic mechanism.
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