RESUMO
D-serine and D-aspartate are important regulators of mammalian physiology. D-aspartate is found in nervous and endocrine tissue, specifically in hypothalamic supraoptic and paraventricular nuclei, pituitary, and adrenal medullary cells. Endogenous D-aspartate is selectively degraded by D-aspartate oxidase. We previously reported that adult male mice lacking the gene for D-aspartate oxidase (Ddo(-/-) mice) display elevated concentrations of D-aspartate in several neuronal and neuroendocrine tissues as well as impaired sexual performance and altered autogrooming behaviour. In the present study, we analyzed behaviours relevant to affect, cognition, and motor control in Ddo(-/-) mice. Ddo(-/-) mice display deficits in sensorimotor gating and motor coordination as well as reduced immobility in the forced swim test. Basal corticosterone concentrations are elevated. The Ddo(-/-) mice have D-aspartate immunoreactive cells in the cerebellum and adrenal glands that are not observed in the wild-type mice. However, no differences in anxiety-like behaviour are detected in open field or light-dark preference tests. Also, Ddo(-/-) mice do not differ from wild-type mice in either passive avoidance or spontaneous alternation tasks. Although many of these behavioural deficits may be due to the lack of Ddo during development, our results are consistent with the widespread distribution of D-aspartate and the hypothesis that endogenous D-aspartate serves diverse behavioural functions.
Assuntos
Ansiedade/enzimologia , D-Aspartato Oxidase/metabolismo , Ácido D-Aspártico/metabolismo , Comportamento Exploratório/fisiologia , Reflexo de Sobressalto/fisiologia , Estimulação Acústica , Glândulas Suprarrenais/enzimologia , Animais , Cerebelo/enzimologia , D-Aspartato Oxidase/genética , Resposta de Imobilidade Tônica/fisiologia , Ativação do Canal Iônico/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Inibição Neural/fisiologia , Fenótipo , Teste de Desempenho do Rota-Rod , Natação/fisiologiaRESUMO
Apolipoprotein B (apoB) is the major protein component of large lipoprotein particles that transport lipids and cholesterol. We have developed a detailed model of the first 1000 residues of apoB using standard sequence alignment programs (ClustalW and MACAW) and the MODELLER6 package for three-dimensional homology modeling. The validity of the apoB model was supported by conservation of disulfide bonds, location of all proline residues in turns and loops, and conservation of the hydrophobic faces of the two C-terminal amphipathic beta-sheets, betaA (residues 600-763) and betaB (residues 780-1000). This model suggests a lipid-pocket mechanism for initiation of lipoprotein particle assembly. In a previous model we suggested that microsomal triglyceride transfer protein might play a structural role in completion of the lipid pocket. We no longer think this likely, but instead propose a hairpin-bridge mechanism for lipid pocket completion. Salt-bridges between four tandem charged residues (717-720) in the turn of the hairpin-bridge and four tandem complementary residues (997-1000) at the C-terminus of the model lock the bridge in the closed position, enabling the deposition of an asymmetric bilayer within the lipid pocket.
Assuntos
Apolipoproteínas B/química , Biofísica/métodos , Lipoproteínas/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Galinhas , Colesterol/química , Biologia Computacional/métodos , Cisteína/química , Bases de Dados de Proteínas , Dissulfetos/química , Fundulidae , Humanos , Lemur , Bicamadas Lipídicas/química , Lipídeos/química , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Prolina/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Ranidae , Sais/farmacologia , Homologia de Sequência de Aminoácidos , Software , Tilápia/metabolismo , TrutaRESUMO
BACKGROUND & AIMS: This study aimed to determine the role of the RNA binding protein apobec-1 in radioprotection of the intestine. METHODS: Apobec-1-deleted mice (APOBEC-1(-/-)) and wild-type controls were treated with 12 Gy of whole-body gamma-irradiation in a cesium irradiator. The number of surviving intestinal crypts was assessed 3.5 days after irradiation by using a clonogenic assay. Cyclooxygenase-2 messenger RNA and protein expression were determined by real-time polymerase chain reaction and Western blot, respectively. RNA stability was studied by examining the turnover of a chimeric transcript containing the cyclooxygenase-2 3' untranslated region cloned downstream of luciferase complementary DNA. Apobec-1 binding to the cyclooxygenase-2 3' untranslated region was studied by electrophoretic mobility shift and UV crosslinking assays. RESULTS: After gamma-irradiation, the survival of intestinal stem cells decreased significantly in APOBEC-1(-/-) mice. In wild-type mice treated with lipopolysaccharide before gamma-irradiation, intestinal stem cells were protected by marked increases in prostaglandin E 2 mediated by cyclooxygenase-2. No such effect was observed in the APOBEC-1(-/-) mice. The mechanism of this radioprotective effect involves the binding of apobec-1 to AU-rich sequences in the first 60 nucleotides of the 3' untranslated region of cyclooxygenase-2. Upon binding to the AU-rich sequences, apobec-1 stabilizes cyclooxygenase-2 messenger RNA. This stabilization process does not seem to be mediated by p38 mitogen-activated protein kinase pathways. CONCLUSIONS: Lipopolysaccharide increases intestinal stem cell survival through apobec-1-mediated regulation of cyclooxygenase-2 messenger RNA stability.
Assuntos
Citidina Desaminase/fisiologia , Regulação Enzimológica da Expressão Gênica , Intestinos/efeitos da radiação , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Lesões por Radiação/prevenção & controle , Regiões 3' não Traduzidas , Desaminase APOBEC-1 , Animais , Ciclo-Oxigenase 2 , Raios gama , Imidazóis/farmacologia , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Piridinas/farmacologia , RNA Mensageiro/análise , Células-Tronco/efeitos da radiação , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Phosphatidylserine synthase 1 (Pss1) and phosphatidylserine synthase 2 (Pss2) produce phosphatidylserine by exchanging serine for the head groups of other phospholipids. Pss1 and Pss2 are structurally similar (approximately 32% amino acid identity) but differ in their substrate specificities, with Pss1 using phosphatidylcholine for the serine exchange reaction and Pss2 using phosphatidylethanolamine. Whether Pss1 and Pss2 are both required for mammalian growth and development is not known, and no data exist on the relative contributions of the two enzymes to serine exchange activities in different tissues. To address those issues and also to define the cell type-specific expression of Pss2, we generated Pss2-deficient mice in which a beta-galactosidase marker is expressed from Pss2 regulatory sequences. Histologic studies of Pss2-deficient mice revealed very high levels of beta-galactosidase expression in Sertoli cells of the testis and high levels of expression in brown fat, neurons, and myometrium. The ability of testis extracts from Pss2-deficient mice to catalyze serine exchange was reduced by more than 95%; reductions of approximately 90% were noted in the brain and liver. However, we found no perturbations in the phospholipid content of any of these tissues. As judged by Northern blots, the expression of Pss1 was not up-regulated in Pss2-deficient cells and tissues. Testis weight was reduced in Pss2-deficient mice, and some of the male mice were infertile. We conclude that Pss2 is responsible for the majority of serine exchange activity in in vitro assays, but a deficiency in this enzyme does not cause perturbations in phospholipid content or severe developmental abnormalities.