RESUMO
We investigated the prevalence of measles-sensitive pregnant women and the clinical usefulness of measles vaccination in postpartum women. Measles antibody levels were measured in 751 pregnant women. Forty-four women were vaccinated postpartum, and screened for antibody levels and adverse effects 1 month after vaccination. The prevalence of measles-sensitive pregnant women was 10-20%, with the highest prevalence in those under 24 years of age. Almost all (97.7%) vaccinated women acquired immunity, and did not show any adverse effects. Serum measles antibody levels should be determined in all pregnant women as a screening tool,and sensitive women should be vaccinated immediately after delivery.
Assuntos
Vacina contra Sarampo , Sarampo/prevenção & controle , Adolescente , Adulto , Anticorpos Antivirais/sangue , Feminino , Humanos , Testes Imunológicos , Japão/epidemiologia , Sarampo/epidemiologia , Sarampo/imunologia , Vírus do Sarampo/imunologia , Cuidado Pós-Natal , Gravidez , Prevalência , Resultado do Tratamento , Vacinação/métodos , Adulto JovemRESUMO
BACKGROUND: Disopyramide, an antiarrhythmia drug, has been reported to cause hypoglycaemia. Pre-existing factors that increase the concentration of the drug in the blood increase the risk of hypoglycaemia. Furthermore, other factors can also increase the risk of hypoglycaemia even when disopyramide levels are in the therapeutic range. It has been proposed that disopyramide-induced hypoglycaemia is caused by inhibition of the pancreatic B-cell K(ATP) channels. CASE REPORT: We report a case of severe disopyramide-induced hypoglycaemia in a 62-year-old woman with Type 2 diabetes taking low-dose glimepiride treatment. She had not experienced hypoglycaemia prior to the start of disopyramide therapy. No further hypoglycaemic episodes occurred following withdrawal of disopyramide therapy. FUNCTIONAL STUDY: Current recordings of K(ATP) channels expressed in Xenopus oocytes showed that at their estimated therapeutic concentrations, disopyramide and glimepiride inhibited K(ATP) channels by about 50-60%. However, when both drugs were applied together, K(ATP) channels were almost completely closed (approximately 95%). Such dramatic inhibition of K(ATP) channels is sufficient to cause B-cell membrane depolarization and stimulate insulin secretion. CONCLUSIONS: Disopyramide therapy is not recommended for patients treated with K(ATP) channel inhibitors.
Assuntos
Antiarrítmicos/efeitos adversos , Arritmias Cardíacas/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Disopiramida/efeitos adversos , Hipoglicemia/induzido quimicamente , Arritmias Cardíacas/complicações , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/complicações , Relação Dose-Resposta a Droga , Feminino , Humanos , Bloqueadores dos Canais de Potássio/metabolismoRESUMO
Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Microssomos Hepáticos/metabolismo , Proteínas Ribossômicas/biossíntese , Animais , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/imunologia , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Membranas Intracelulares/metabolismo , Masculino , Microssomos Hepáticos/ultraestrutura , Puromicina/farmacologia , Ratos , Propriedades de SuperfícieRESUMO
Induction of cytochrome P-450s by 3-methylcholanthrene (MC) and phenobarbital (PB) and distribution of P-450s in the rat liver nuclear envelope were investigated by biochemical analyses and ferritin immunoelectron microscopy using specific antibodies against the major molecular species of MC- and PB-induced cytochrome P-450. It was found, in agreement with Kasper (J. Biol. Chem., 1971, 246: 577-581), that the total amount of cytochrome P-450s determined by biochemical analysis was markedly increased by MC, but not by PB, treatment. Immunoelectron microscopic analysis, however, showed marked and slight increases in ferritin labeling by MC and PB treatment, respectively. The latter finding was interpreted as resulting from the induction of a particular molecular species of PB-induced cytochrome P-450s. Ferritin immunoelectron microscopic analysis of intact isolated nuclei, naked nuclei from which the outer membrane of the nuclear envelope was partially detached (mechanically), and isolated nuclear envelopes have shown that the ferritin particles are found exclusively on the cytoplasmic face of the outer nuclear envelopes. Neither the nucleoplasmic face of the inner membrane of the nuclear envelope nor the cisternal face of both membranes of the nuclear envelope showed any labeling with ferritin. This indicates that cytochrome P-450 is located only on the outer membrane of the nuclear envelope and does not diffuse laterally into the domain of the inner membrane of the nuclear envelope across the nuclear pores. Our results suggest that a marked heterogeneity exists in the enzyme distribution between the outer and inner membrane of the nuclear envelope and that microsomal marker enzymes such as cytochrome P-450 exist exclusively in the outer membrane. In addition, it appears that cytochrome P-450 is probably not a transmembrane protein but an intrinsic protein located on the cytoplasmic face of the outer membrane of the nuclear envelope.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Membrana Nuclear/enzimologia , Animais , Indução Enzimática/efeitos dos fármacos , Ferritinas , Complexo de Golgi/enzimologia , Fígado/ultraestrutura , Masculino , Metilcolantreno/farmacologia , Membrana Nuclear/ultraestrutura , Fenobarbital/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Mice lacking the gene encoding the receptor for prostaglandin F2alpha (FP) developed normally but were unable to deliver normal fetuses at term. Although these FP-deficient mice showed no abnormality in the estrous cycle, ovulation, fertilization, or implantation, they did not respond to exogenous oxytocin because of the lack of induction of oxytocin receptor (a proposed triggering event in parturition), and they did not show the normal decline of serum progesterone concentrations that precedes parturition. Ovariectomy at day 19 of pregnancy restored induction of the oxytocin receptor and permitted successful delivery in the FP-deficient mice. These results indicate that parturition is initiated when prostaglandin F2alpha interacts with FP in ovarian luteal cells of the pregnant mice to induce luteolysis.
Assuntos
Corpo Lúteo/metabolismo , Dinoprosta/metabolismo , Trabalho de Parto , Receptores de Prostaglandina/metabolismo , Animais , Feminino , Marcação de Genes , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Ocitocina/biossíntese , Ocitocina/farmacologia , Gravidez , Progesterona/sangue , Receptores de Ocitocina/biossíntese , Receptores de Prostaglandina/genética , Contração Uterina/efeitos dos fármacos , Útero/metabolismoRESUMO
PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Esteroide Hidroxilases , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Receptor Constitutivo de Androstano , Família 2 do Citocromo P450 , Dimerização , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenobarbital/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores X de Retinoides , Fatores de Tempo , Transativadores/genética , Células Tumorais CultivadasRESUMO
The Cyp 2d-9 gene encodes the male-specific steroid 16 alpha-hydroxylase in mouse liver and shares a conserved regulatory element (-100TTCCGGGC-93) with another male-specific Slp promoter. As shown with the Slp promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad. Sci. USA 92:1302-1306, 1995), the male-preferential demethylation also occurs at CpG/-97 in the Cyp 2d-9 promoter. The transcription factor which specifically binds to the demethylated element has been purified. The peptide sequences reveal that the factor consists of GABP alpha and GABP beta 1 with Ets and Notch motifs, respectively. Both DNase I footprinting and gel shift assays indicate that the bacterially expressed glutathione S-transferase-GABP fusion proteins bind to the regulatory element only when CpG/-97 is demethylated. Moreover, Cyp 2d-9 promoter is trans-activated by coexpression of GABP proteins in HepG2 cells. Given the additional results that CpG/-50 of the female-specific steroid 15 alpha-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Regiões Promotoras Genéticas/genética , Esteroide 16-alfa-Hidroxilase , Esteroide Hidroxilases/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/isolamento & purificação , Fosfatos de Dinucleosídeos/metabolismo , Feminino , Fator de Transcrição de Proteínas de Ligação GA , Humanos , Fígado/química , Masculino , Metilação , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Diferenciação Sexual/genética , Fatores de Transcrição/isolamento & purificação , Ativação TranscricionalRESUMO
In response to phenobarbital (PB) and other PB-type inducers, the nuclear receptor CAR translocates to the mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318-6322, 1999). To define the translocation mechanism, fluorescent protein-tagged human CAR (hCAR) was expressed in the mouse livers using the in situ DNA injection and gene delivery systems. As in the wild-type hCAR, the truncated receptor lacking the C-terminal 10 residues (i.e., AF2 domain) translocated to the nucleus, indicating that the PB-inducible translocation is AF2 independent. Deletion of the 30 C-terminal residues abolished the receptor translocation, and subsequent site-directed mutagenesis delineated the PB-inducible translocation activity of the receptor to the peptide L313GLL316AEL319. Ala mutations of Leu313, Leu316, or Leu319 abrogated the translocation of CAR in the livers, while those of Leu312 or Leu315 did not affect the nuclear translocation. The leucine-rich peptide dictates the nuclear translocation of hCAR in response to various PB-type inducers and appears to be conserved in the mouse and rat receptors.
Assuntos
Núcleo Celular/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Xenobióticos/farmacologia , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Receptor Constitutivo de Androstano , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenobarbital/farmacologia , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Fatores de Transcrição/genéticaRESUMO
The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive enhancer module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its binding to PBREM in livers of PB-treated mice. We have investigated the cellular mechanism of PB-induced increase of CAR binding. Western blot analyses of mouse livers revealed an extensive nuclear accumulation of CAR following PB treatment. Nuclear contents of CAR perfectly correlate with an increase of CAR binding to PBREM. PB-elicited nuclear accumulation of CAR appears to be a general step regulating the induction of CYP2B genes, since treatments with other PB-type inducers result in the same nuclear accumulation of CAR. Both immunoprecipitation and immunohistochemistry studies show cytoplasmic localization of CAR in the livers of nontreated mice, indicating that CAR translocates into nuclei following PB treatment. Nuclear translocation of CAR also occurs in mouse primary hepatocytes but not in hepatocytes treated with the protein phosphatase inhibitor okadaic acid. Thus, the CAR-mediated transactivation of PBREM in vivo becomes PB responsive through an okadaic acid-sensitive nuclear translocation process.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Fenobarbital/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Esteroide Hidroxilases , Transativadores/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Receptor Constitutivo de Androstano , Família 2 do Citocromo P450 , Primers do DNA/genética , Elementos Facilitadores Genéticos , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ácido Okadáico/farmacologiaRESUMO
The levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) are controlled by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element (ERSE), the consensus sequence of which is CCAAT-N(9)-CCACG. We recently proposed that ER stress response factor (ERSF) binding to ERSE is a heterologous protein complex consisting of the constitutive component NF-Y (CBF) binding to CCAAT and an inducible component binding to CCACG and identified the basic leucine zipper-type transcription factors ATF6alpha and ATF6beta as inducible components of ERSF. ATF6alpha and ATF6beta produced by ER stress-induced proteolysis bind to CCACG only when CCAAT is bound to NF-Y, a heterotrimer consisting of NF-YA, NF-YB, and NF-YC. Interestingly, the NF-Y and ATF6 binding sites must be separated by a spacer of 9 bp. We describe here the basis for this strict requirement by demonstrating that both ATF6alpha and ATF6beta physically interact with NF-Y trimer via direct binding to the NF-YC subunit. ATF6alpha and ATF6beta bind to the ERSE as a homo- or heterodimer. Furthermore, we showed that ERSF including NF-Y and ATF6alpha and/or beta and capable of binding to ERSE is indeed formed when the cellular UPR is activated. We concluded that ATF6 homo- or heterodimers recognize and bind directly to both the DNA and adjacent protein NF-Y and that this complex formation process is essential for transcriptional induction of ER chaperones.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Fatores de Transcrição/metabolismo , Fator 6 Ativador da Transcrição , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação/genética , Fator de Ligação a CCAAT/química , Sequência Consenso , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Proteínas de Ligação a DNA/química , Dimerização , Fatores de Ligação G-Box , Células HeLa , Humanos , Substâncias Macromoleculares , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Mutação , Estresse Oxidativo , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , Fatores de Transcrição/químicaRESUMO
Transcription of genes encoding molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) is induced by accumulation of unfolded proteins in the ER. This intracellular signaling, known as the unfolded protein response (UPR), is mediated by the cis-acting ER stress response element (ERSE) in mammals. In addition to ER chaperones, the mammalian transcription factor CHOP (also called GADD153) is induced by ER stress. We report here that the transcription factor XBP-1 (also called TREB5) is also induced by ER stress and that induction of CHOP and XBP-1 is mediated by ERSE. The ERSE consensus sequence is CCAAT-N(9)-CCACG. As the general transcription factor NF-Y (also known as CBF) binds to CCAAT, CCACG is considered to provide specificity in the mammalian UPR. We recently found that the basic leucine zipper protein ATF6 isolated as a CCACG-binding protein is synthesized as a transmembrane protein in the ER, and ER stress-induced proteolysis produces a soluble form of ATF6 that translocates into the nucleus. We report here that overexpression of soluble ATF6 activates transcription of the CHOP and XBP-1 genes as well as of ER chaperone genes constitutively, whereas overexpression of a dominant negative mutant of ATF6 blocks the induction by ER stress. Furthermore, we demonstrated that soluble ATF6 binds directly to CCACG only when CCAAT exactly 9 bp upstream of CCACG is bound to NF-Y. Based on these and other findings, we concluded that specific and direct interactions between ATF6 and ERSE are critical for transcriptional induction not only of ER chaperones but also of CHOP and XBP-1.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico , Proteínas Nucleares/genética , Dobramento de Proteína , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator 6 Ativador da Transcrição , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Transporte/genética , Extratos Celulares , Núcleo Celular/metabolismo , Retículo Endoplasmático , Chaperona BiP do Retículo Endoplasmático , Expressão Gênica , Células HeLa , Humanos , Mamíferos , Chaperonas Moleculares/genética , Mutagênese , Fatores de Transcrição de Fator Regulador X , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição CHOP , Transcrição Gênica , Proteína 1 de Ligação a X-BoxRESUMO
The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.
Assuntos
GTP Fosfo-Hidrolases/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos/metabolismo , Células PC12/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/genética , Genes Dominantes , Mutagênese Sítio-Dirigida , Fatores de Crescimento Neural/farmacologia , Células PC12/efeitos dos fármacos , Células PC12/ultraestrutura , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Transfecção , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
Expression of the Cyp 2d-9 (steroid 16 alpha-hydroxylase) gene in mouse liver is male specific in such Mus musculus domesticus strains as FVB/N, whereas the corresponding P450 genes in the wild mouse species Mus spretus are not sex specific in their expression. These parental differences in the gene expressions were independently inherited in F1 offspring from crosses of FVB/N and M. spretus. A 5' flanking sequence (-110CTC CTCCCTATTCCGGGCC-92) was defined as a regulatory element (named SDI-A1) for the domestic Cyp 2d-9 promoter. The nucleotide which corresponds to T at position -99 within SDI-A1 was found to be substituted with C in the wild mouse P450 genes. The placing of C at position -99 abolished the transcriptional activity of SDI-A1 in HepG2 cells as well as the binding of SDI-A1 to a nuclear factor. This factor (designated NF2d9) was purified from mouse nuclear extracts, and its cDNA cloned. The purified NF2d9 bound to SDI-A1 but not to the mutated SDI-A1 with C at position -99. The deduced amino acid sequence revealed that NF2d9 is 72 and 94% identical to mouse CP2 and human LBP-1a, respectively. NF2d9 thus belongs to the CP2 family and is the mouse homolog of human LBP-1a, which modulates human immunodeficiency virus type 1 transcription. Anti-NF2d9, which was raised against the bacterially expressed protein, supershifted the SDI-A1 complex with the liver nuclear extract. Both the bacterially expressed and in vitro-translated NF2d9 inhibited SDI-A1 complex formation, although they did not bind to SDI-A1 directly. The results, therefore, indicate that the domestic Cyp 2d-9 gene can be regulated through a specific association of NF2d9 with SDI-A1.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Ligação a DNA/genética , Fígado/metabolismo , Caracteres Sexuais , Esteroide 16-alfa-Hidroxilase , Esteroide Hidroxilases/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Cruzamentos Genéticos , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Masculino , Dados de Sequência Molecular , Muridae , Mutação , Filogenia , Ligação Proteica , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Distribuição TecidualRESUMO
Increasing evidence suggests that disruption of metal homeostasis contributes to the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease, prion diseases, Lewy body diseases, and vascular dementia. Conformational changes of disease-related proteins (amyloidogenic proteins), such as ß-amyloid protein, prion proteins, and α-synuclein, are well-established contributors to neurotoxicity and to the pathogenesis of these diseases. Recent studies have demonstrated that these amyloidogenic proteins are metalloproteins that bind trace elements, including zinc, iron, copper, and manganese, and play significant roles in the maintenance of metal homeostasis. We present a current review of the role of trace elements in the functions and toxicity of amyloidogenic proteins, and propose a hypothesis integrating metal homeostasis and the pathogenesis of neurodegenerative diseases that is focused on the interactions among metals and between metals and amyloidogenic proteins at the synapse, considering that these amyloidogenic proteins and metals are co-localized at the synapse.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Sinapses/fisiologia , Oligoelementos/metabolismo , Humanos , Doenças Neurodegenerativas/etiologiaRESUMO
Alteration of DNA demethylation in five CpG sites (-547, -533, -475, -464, and -454) immediately upstream from the estrogen response element of lactoferrin promoter was determined in the uteri of immature (17-day-old) and mature (21- and 30-day-old) mice treated neonatally with DES. Only the CpG/-464 was found to be abnormally demethylated by diethylstilbestrol (DES) treatment in the mature uteri. This abnormal demethylation occurred in specific response to DES in neonatal mice, because DES injected into the 30-day-old mature mice did not demethylate CpG/-464. This site, however, remained methylated in the neonatally DES-treated/ovariectomized mice, indicating that this DES-elicited demethylation is under hormonal control. Thus, neonatal DES treatment appeared to imprint an abnormal, site-specific demethylation of CpG/-464, which requires ovarian hormones to occur in adult mice. Moreover, the demethylation was maintained in uterine tumors of the neonatally DES-treated mice. This mode of demethylation is reminiscent of uterine tumor formation, which also depends on both neonatal DES exposure and ovarian hormone stimulation in adulthood. Thus, neonatal DES treatment may induce tumor formation as well as demethylation through a common cellular process.
Assuntos
Carcinógenos/toxicidade , Ilhas de CpG , Dietilestilbestrol/toxicidade , Estrogênios/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lactoferrina/genética , Útero/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Metilação de DNA/efeitos dos fármacos , Feminino , Genes , Lactoferrina/biossíntese , Camundongos , Ovariectomia , Regiões Promotoras Genéticas , Neoplasias Uterinas/induzido quimicamente , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Útero/crescimento & desenvolvimento , Útero/metabolismoRESUMO
As a family of serine-dependent enzymes, the carboxylesterases (EC 3.1.1.1) demonstrate a broad substrate specificity. Mouse carboxylesterases comprise at least 20 genetically distinct loci. We cloned a full-length cDNA for a novel mouse carboxylesterase, Es-male which was expressed predominantly in male livers. This carboxylesterase consisted of 554 amino acid residues, and exhibited 43% and 42% similarities to the known mouse esterases Es-22 and pEs-N, respectively. Es-male contained a C-terminal ER-retention signal PEEL, indicating that it may be a microsomal carboxylesterase.
Assuntos
Hidrolases de Éster Carboxílico/genética , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Hidrolases de Éster Carboxílico/biossíntese , Clonagem Molecular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Fatores SexuaisRESUMO
This review summarizes recent advances in the molecular characterization of prostanoid receptors. Prostanoids exert versatile actions in diverse tissues and cells through specific cell surface receptors. Molecular biological studies revealed the primary structure of eight types and subtypes of prostanoid receptor from various species. These include the thromboxane A2 receptor, prostacyclin receptor, prostaglandin (PG) F receptor, PGD receptor and four subtypes of PGE receptors. They are coupled to different signal transduction systems. In addition, multiple isoforms of PGE receptor EP3 subtype have been identified in various species. They are produced through alternative RNA splicing from a single gene and differ only in their carboxy-terminal tails. These isoforms differ in the efficiency of G protein activation, in the specificity of coupling to G proteins or in sensitivity to desensitization. This molecular characterization is useful for understanding the diverse physiological roles of prostanoids.
Assuntos
Receptores de Prostaglandina/química , Receptores de Prostaglandina/fisiologia , Receptores de Tromboxanos/química , Receptores de Tromboxanos/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Prostaglandinas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Kinetic data of 45Ca2+ exchange in exponentially growing mastocytoma P-815 cells at 37 degree C indicated the presence of at least two calcium compartments, fast and slowly exchangeable. The fast phase of calcium exchange at the cell surface had a rate constant of 36.1 min-1 and a compartment size of 8.27 nmol/10(6) cells, while the slow phase, the intracellular calcium exchange, showed a rate constant of 2.4 min-1 and a compartment size of 0.53 nmolS/10(6) cells. The fast phase comprised two calcium binding sites of high and low affinities with dissociation constants of 0.59 and 4.88 mM, respectively, and binding maxima of 5.15 and 34.4 nmol/10(6) cells, respectively. The calcium uptake at both binding sites was inhibited by 80% on the addition of ruthenium red (0.4 mM) or lanthanum chloride (0.5 mM), but not by tetracaine (5 mM). The high-affinity site had two negative reaction enthalpy changes, delta H 0 = -1.55 (from 0 to 22 degree C) and -4.27 (from 22 degree C to 37 degree C) kcal . mol-1, while the low-affinity site showed two positive reaction enthalpy changes, delta H0 = +6.53 (below 22 degree C) and +2.05 (above 22 degree C) kcal . mol-1, suggesting the effect of membrane fluidity on calcium binding. The slow phase of calcium exchange was also inhibited by ruthenium red and lanthanum chloride resulting in a decrease of the Hill's coefficient from 1.70 to 1.28 and 1.13, respectively, but was not affected by tetracaine. Calcium efflux from 45 Ca-loaded cells was also inhibited by ruthenium red and lanthanum chloride and was also temperature sensitive. The efflux rate constant was reduced by 80% and 100% at 20 degree C and 0 degree C, respectively. As calcium uptake fluctuated during the cell cycle of synchronous mastocytoma P-815 cells with a peak at the mid S phase and the lowest level during the late M phase, the compartment size of fast phase also fluctuated, being highest at the S phase and lowest at the M phase. In contrast, the compartment size of the slow phase was highest at the G1 phase and lowest at the M phase. In parallel with these changes, the binding maximum of the high-affinity site was highest at the S phase and lowest at the M phase, while that of the low-affinity site fluctuated little throughout the cell cycle.
Assuntos
Cálcio/metabolismo , Plasmocitoma/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , Cinética , Camundongos , Neoplasias Experimentais/metabolismo , Zinco/farmacologiaRESUMO
Glycyrrhetinic acid bound specifically to a particulate fraction of rat liver. The binding was dependent on time, temperature and pH, equilibrium being reached after 10 min at 37 degrees C. The equilibrium dissociation constant and the maximal concentration of the binding site, as determined by Scatchard plot analysis, were 31 nM and 43 pmol/mg protein, respectively, indicating a single binding site entity. The binding site was highly specific for glycyrrhetinic acid, glycyrrhizin, various steroids, various fatty acids and retinoids showing no or only very low affinity. The binding was inhibited by boiling or treatment with trypsin or phospholipases. The specific activity of glycyrrhetinic acid binding was the highest in the liver, followed by in the kidney. The results suggest that glycyrrhetinic acid plays a significant role in the rat liver through its specific binding protein.
Assuntos
Ácido Glicirretínico/metabolismo , Fígado/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Centrifugação , Concentração de Íons de Hidrogênio , Cinética , Fígado/ultraestrutura , Masculino , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Reagentes de Sulfidrila/farmacologia , TemperaturaRESUMO
Recently, we reported that in mouse mastocytoma P-815 cells the cytosol contains some factor(s) which promotes the release of GTP-activated Gi2 alpha from the membrane, and that thrombin induces the translocation of Gi2 alpha from the membrane to the cytosol (Takahashi, S., Negishi, M. and Ichikawa, A. (1991) J. Biol. Chem. 266, 5367-5370). Here we investigated the mechanism underlying the thrombin-induced translocation of Gi2 alpha in mastocytoma cells. Thrombin induced a rapid and transient increase in the intracellular Ca2+ concentration ([Ca2+]i) within 1 min, attenuated pertussis toxin-catalyzed ADP-ribosylation of Gi2 in the membrane, and caused the subsequent translocation of Gi2 alpha. Thrombin induced the translocation of protein kinase C from the cytosol to the membrane, and a protein kinase C inhibitor, staurosporine, completely inhibited the thrombin-induced translocation of Gi2 alpha. When cells were treated with thrombin, the ability of the cytosol to release Gi2 alpha from the membrane in the presence of GTP gamma S markedly increased. This stimulatory effect of thrombin on the ability of the cytosol was mimicked by 12-O-tetradecanoylphorbol 13-acetate (TPA), but not by the Ca2+ ionophore, ionomycin. The thrombin- and TPA-induced potentiation of the ability of the cytosol to release Gi2 alpha was completely abolished by staurosporine. Furthermore, phosphorylation of the cytosol by protein kinase C markedly potentiated the ability of the cytosol to release Gi2 alpha. These results together demonstrate that the thrombin-induced translocation of Gi2 alpha is due to enhancement of the ability of the cytosol to release Gi2 alpha via activation of protein kinase C.