RESUMO
The peripheral and cerebral metabolism of thyroid hormones in 10-day-old rats was evaluated by measuring the kinetics of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) fluxes. Labeled iodo-compounds were measured in the plasma, cerebellum and brain (without cerebellum) for 24 hours after the intravenous injection of [125I]T4 plus [131I]T3. Data were interpreted by compartment analysis. T4 was produced at 8.93 pmol x h(-1) and T3 at 2.26 pmol x h(-1) for 22.7 g body weight. The T4 and T3 distribution volumes were 4.26 and 22.7 ml, whereas the extra-cellular fluid volume was 9.42 ml. T4 was therefore considered to be mostly extra-cellular and T3 mostly intracellular. This was confirmed in the brain and cerebellum, where the extra-cellular fluid (ECF) fraction was 0.021 ml/g organ and the tissue-to-plasma ratio of labeled and endogenous hormones was 0.54-6.54 ml plasma/g tissue for T3 and 0.048-0.136 ml plasma/g tissue for T4. The T3 in the brain and cerebellum was distributed in several pools. The first, representing 11% of the cerebellum and 8% of the brain (without cerebellum) T3, was quickly exchanged with circulating T3. The second pool, derived from the local T4 5'-deiodination, represented 48% of the cerebellum and 94% of the brain (without cerebellum) hormone; a possible third pool (41% of the hormonal content) in the cerebellum appeared to be unlabeled by radioactive T3, and motionless. The in vivo T4 to T3 conversion, as a function of weight, accounted for 21% of cerebellum needs and 43% of brain needs. The rest was provided by T3 uptake.
Assuntos
Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Tiroxina/farmacocinética , Tri-Iodotironina/farmacocinética , Animais , Animais Lactentes , Biotransformação , Compartimentos de Líquidos Corporais , Cerebelo/crescimento & desenvolvimento , Córtex Cerebral/crescimento & desenvolvimento , Injeções Intravenosas , Iodeto Peroxidase/metabolismo , Radioisótopos do Iodo/análise , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Wistar , Tiroxina/administração & dosagem , Tri-Iodotironina/administração & dosagemRESUMO
The cerebellum of young rats contains significant 5'-deiodinase (5'-D) activity, but technical difficulties have made it impossible to identify the enzyme in cultured cerebellar astrocytes. We have developed a culture method which allows cerebellar astrocytes from 6-day-old rats to grow and develop 5'-D activity. Astrocytes cultured for 2 weeks in medium containing 3.25 microM reduced glutathione (GSH) and 0.21 microM vitamin E (VitE) as alpha-tocopherol had 5'-D activity which was stimulated by 1 mM dibutyryl cyclic adenosine monophosphate (dBcAMP) given 16 hours before measuring enzyme activity. Cells cultured without GSH and VitE showed little 5'-D activity, which was not stimulated by dBcAMP Primary cultures of cerebellar astrocytes were cultured for four weeks with or without GSH+VitE, and stimulated by dBcAMP had high 5'-D activity, but were also sometimes contaminated with fibroblasts. The effect of such contamination on the astrocyte 5'-D activity was assessed by preparing primary cultures of fibroblasts from the meninges surrounding 6-day-old rat cerebella. They were grown in the same media and under the same conditions as the astrocytes. The cultured fibroblasts had 5'-D activity independent of GSH+VitE or culture time. The 5'-D activity of both cell populations could be type II 5'-deiodinase (5'-DII) because it was not inhibited by 6-n-propylthiouracil (PTU). Thus, cerebellar astrocytes cultured for 2 weeks in medium containing GSH and VitE have 5'-DII activity. Prolonged cultures favor enzyme activity, but also enhance contamination with fibroblasts, which may also show 5'-DII activity.
Assuntos
Astrócitos/enzimologia , Cerebelo/citologia , Iodeto Peroxidase/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/química , Astrócitos/citologia , Bucladesina/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Fibroblastos/química , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibronectinas/análise , Proteína Glial Fibrilar Ácida/análise , Glutationa/farmacologia , Neurônios/citologia , Neurônios/enzimologia , Gravidez , Ratos , Ratos Wistar , Vitamina E/farmacologiaRESUMO
As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.
Assuntos
RNA Polimerase II/química , Baculoviridae , Clonagem Molecular , Cisteína/análise , Glutationa/metabolismo , Humanos , Metionina/análise , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , RNA Polimerase II/genéticaRESUMO
The transcription/DNA repair factor TFIIH consists of nine subunits, several exhibiting known functions: helicase/ATPase, kinase activity and DNA binding. Three subunits of TFIIH, cdk7, cyclin H and MAT1, form a ternary complex, cdk-activating kinase (CAK), found either on its own or as part of TFIIH. In the present work, we demonstrate that purified human CAK complex (free CAK) and recombinant CAK (rCAK) produced in insect cells exhibit a strong preference for the cyclin-dependent kinase 2 (cdk2) over a ctd oligopeptide substrate (which mimics the carboxy-terminal domain of the RNA polymerase II). In contrast, TFIIH preferentially phosphorylates the ctd as well as TFIIE alpha, but not cdk2. TFIIH was resolved into four subcomplexes: the kinase complex composed of cdk7, cyclin H and MAT1; the core TFIIH which contains XPB, p62, p52, p44 and p34; and two other subcomplexes in which XPD is found associated with either the kinase complex or with the core TFIIH. Using these fractions, we demonstrate that TFIIH lacking the CAK subcomplex completely recovers its transcriptional activity in the presence of free CAK. Furthermore, studies examining the interactions between TFIIH subunits provide evidence that CAK is integrated within TFIIH via XPB and XPD.