RESUMO
Cyclin-dependent kinase 5 (cdk5) is essential for brain development and p35 and p67 are the regulatory molecules for cdk5. In this study, we have investigated the expression of cdk5, p35, and p67 mRNAs in the developing rat brain with in situ hybridization histochemistry. The expression of cdk5 mRNA was already observed in embryonic day 12 (E12), start point of neurogenesis in rat brain, throughout the brain and gradually increased until postnatal day 3 (P3). At this period, strong expression of cdk5 mRNA was observed in the cerebral cortex, hippocampus, dentate gyrus, thalamus, hypothalamus, and inferior colliculus. High level of cdk5 expression was maintained in the postnatal rat brain and prominent expression was observed in the hippocampus, dentate gyrus, cerebellum, and choroid plexus of adult rat brain. Strong expression of p35 mRNA was observed between E16 and E20 in the cerebral cortex, hippocampus, dentate gyrus, thalamus, hypothalamus, and inferior colliculus as like as cdk5. After birth, the expression of p35 mRNA was gradually decreased and significant differences in the expression of cdk5 and p35 were observed in the thalamus, hypothalamus, midbrain, and cerebellum. In the embryonic period, the expression pattern of p67 was very similar with that of p35 but expression level was lower than p35. After birth, strong expression of p67 was observed in the areas where the expression of cdk5 was high. From these results, it is suspected that p35 may function in neuronal migration, and p67 in differentiation and maturation, as a major regulator for cdk5 in developing rat brain.
Assuntos
Adulto , Animais , Humanos , Ratos , Encéfalo , Cerebelo , Córtex Cerebral , Plexo Corióideo , Quinase 5 Dependente de Ciclina , Giro Denteado , Expressão Gênica , Hipocampo , Hipotálamo , Hibridização In Situ , Colículos Inferiores , Mesencéfalo , Neurogênese , Neurônios , Parto , RNA Mensageiro , TálamoRESUMO
An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system [10] To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored. Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect. A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction. The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558). The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction. Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.
Assuntos
Humanos , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/metabolismo , Sistema Livre de Células , Primers do DNA , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , NADH Desidrogenase/metabolismo , NADPH Oxidases , Neutrófilos/enzimologia , Oxirredução , Fragmentos de Peptídeos/química , Fosfoproteínas/química , Reação em Cadeia da Polimerase , Deleção de SequênciaRESUMO
Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.