RESUMEN
Povidone-iodine solutions prepared to various concentrations (0.01, 0.1, 1 and 10%) with 0.2M phosphate buffer (pH 7.0) (PVP-I PB) were analyzed to determine their free iodine concentrations using membrane permeation cells, and their inactivation effects on three viruses (influenza A virus, poliovirus type 1 and adenovirus type 3) were examined. The free iodine concentrations in the 0.01-10% PVP-I PB were determined to be 1.84, 4.88, 1.58 and 0.17 ppm (approximate values), respectively, with the maximum obtained for the 0.1% solution. The virucidal efficacy of these PVP-I PB against poliovirus type 1 and adenovirus type 3 was found to be generally dependent on free iodine concentration, with the 0.1% solution being the most effective. Influenza A virus was inactivated with an action time of 15 s at all four concentrations examined. The results of this study suggested an association between free iodine concentration and virucidal efficacy for the 0.01-10% PVP-I PB.
Asunto(s)
Antivirales/farmacología , Yodo/farmacología , Povidona Yodada/farmacología , Tampones (Química) , Yodo/química , Viabilidad Microbiana/efectos de los fármacos , Povidona Yodada/química , Virus/efectos de los fármacosRESUMEN
HIV-1 CRF01_AE and subtype B (B) have dominated and their different circulating recombinant forms (CRFs) have emerged in East and Southeast Asian countries. Here, we report a novel drug-resistant HIV-1 CRF. Five independent recombinant specimens exhibiting discordant subtype results for the gag, pol, and env sequences were isolated. These recombinants had the CRF01_AE (gag p17)/B (pol PR-RT and IN)/CRF01_AE (env C2-V3) pattern similar to CRF69_01B. Sequence analysis of four near full-length HIV-1 genomes revealed a unique phylogenetic cluster distinct from previously reported CRFs. Of the four recombinants, three shared an identical mosaic structure including seven breakpoints in the gag, pol, vif, and env regions, designated CRF76_01B. The one remaining recombinant had additional recombination breakpoints in the vpu region and exhibited another unique recombinant form composed of CRF76_01B and B. These findings provide important insight into the transmission dynamics of HIV-1 in Asia that may be important for its effective prevention.
Asunto(s)
Farmacorresistencia Viral , Genoma Viral , Genotipo , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/aislamiento & purificación , Recombinación Genética , Asia , Estudios Transversales , Variación Genética , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Filogenia , Análisis de Secuencia de ADN , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G1-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.
Asunto(s)
Ciclo Celular , Ciclina D1/genética , Regulación hacia Abajo , Corazón/crecimiento & desarrollo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Lysine methylation of the histone tail is involved in a variety of biological events. G9a and GLP are known as major H3-K9 methyltransferases and contribute to transcriptional silencing. The functions of these genes in organogenesis remain largely unknown. Here, we analyzed the phenotypes of cardiomyocyte specific GLP knockout and G9a knockdown (GLP-KO/G9a-KD) mice. The H3-K9 di-methylation level decreased markedly in the nuclei of the cardiomyocytes of GLP-KO/G9a-KD mice, but not single G9a or GLP knockout mice. In addition, GLP-KO/G9a-KD mice showed neonatal lethality and severe cardiac defects (atrioventricular septal defects, AVSD). We also showed that hypoplasia in the atrioventricular cushion, which is a main part of the atrioventricular septum, caused AVSD. Expression analysis revealed downregulation of 2 AVSD related genes and upregulation of several non-cardiac specific genes in the hearts of GLP-KO/G9a-KD mice. These data indicate that G9a and GLP are required for sufficient H3-K9 di-methylation in cardiomyocytes and regulation of expression levels in multiple genes. Moreover, our findings show that G9a and GLP have an essential role in normal morphogenesis of the atrioventricular septum through regulation of the size of the atrioventricular cushion.
Asunto(s)
Tabique Interatrial/enzimología , Defectos de los Tabiques Cardíacos/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Morfogénesis/genética , Animales , Tabique Interatrial/embriología , Tabique Interatrial/patología , Embrión de Mamíferos , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Ingeniería Genética , Defectos de los Tabiques Cardíacos/embriología , Defectos de los Tabiques Cardíacos/enzimología , Defectos de los Tabiques Cardíacos/patología , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Recombinación Homóloga , Masculino , Ratones , Ratones Transgénicos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Transducción de SeñalRESUMEN
In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.
Asunto(s)
Diferenciación Celular/genética , Proliferación Celular , Ciclina D1/fisiología , Corazón/embriología , Miocitos Cardíacos/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Ciclina D1/genética , Embrión de Mamíferos , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Corazón/fisiología , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Proteínas del Tejido Nervioso/genética , Complejo Represivo Polycomb 2 , Transducción de Señal , Factores de TiempoRESUMEN
To address the regulation and evolution of precursor protein cleavability in caliciviruses, we examined constraints on diversity of upstream regions of calicivirus precursor cleavage sites. We performed alanine scanning and supplementary mutagenesis of amino acids at P1, P2, P3, and P4 sites using four viruses representing the four major genera of the family Caliciviridae. This study complements previous mutagenesis studies and shows strong restrictions in mutations at the P1 and P4 sites for effective cleavage reactions. By contrast, such restrictions were less frequently observed at the P2 and P3 sites. Shannon entropy analysis of the reported sequences showed that the P2, P3, and P4 sites allow variations in amino acid size within a calicivirus genus whereas the P1 sites do not. Notably, the human sapovirus precursor protein exceptionally retains a basic rather than aromatic amino acid at the P4 site of the NS4/NS5 cleavage site in reported strains, and a substitution from basic to aromatic amino acid significantly enhanced cleavability at this site. Taken together, these data suggest the existence of (i) structural constraints on the P1 site that restrict size changes within each calicivirus genus, (ii) plastic substrate surfaces that accommodate size variation at the P2, P3, and P4 sites and modulate their own cleavabilities, and (iii) biological constraints on the P4 site that maintain the lower cleavability of the NS4/NS5 site in sapovirus.
Asunto(s)
Caliciviridae/genética , Caliciviridae/fisiología , Polimorfismo Genético , Precursores de Proteínas/genética , Proteínas Virales/genética , Replicación Viral , Sustitución de Aminoácidos , Humanos , Lactante , Mutagénesis Sitio-Dirigida , Péptido Hidrolasas/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Virales/metabolismoRESUMEN
The SaV genome is a positive-sense, non-segmented single-strand RNA molecule of approximately 7.5 kb that is polyadenylated at its 3' terminus. The major capsid (VP1) of SaV is thought to be produced as the ORF1 polyprotein followed by cleavage, or translation from subgenomic RNA (3'-coterminal with the virus genome), or both. We have recently reported the formation of SaV VLP from subgenomic-like RNA in mammalian cells. In the present study, we demonstrated that the VP1 cleaved from a part of ORF1 polyprotein self-assembled into VLP in mammalian cells when a transient expression system using a recombinant vaccinia virus encoding T7 RNA polymerase was used.
Asunto(s)
Proteínas de la Cápside/metabolismo , Poliproteínas/metabolismo , Sapovirus/fisiología , Virión/fisiología , Ensamble de Virus , Animales , Células COS , Proteínas de la Cápside/genética , Chlorocebus aethiops , Poliproteínas/genética , Sapovirus/genética , Virión/genéticaRESUMEN
Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.
Asunto(s)
Ciclina D1/metabolismo , Histonas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Ciclina D1/genética , Regulación de la Expresión Génica , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina , Histonas/química , Histonas/genética , Humanos , Metilación , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/metabolismoRESUMEN
Norovirus (NoV) is a causative agent of acute gastroenteritis. NoV binds to histo-blood group antigens (HBGAs), namely, ABH antigens and Lewis (Le) antigens, in which type 1 and type 2 carbohydrate core structures constitute antigenically distinct variants. Norwalk virus, the prototype strain of norovirus, binds to the gastroduodenal junction, and this binding is correlated with the presence of H type 1 antigen but not with that of H type 2 antigen (S. Marionneau, N. Ruvoen, B. Le Moullac-Vaidye, M. Clement, A. Cailleau-Thomas, G. Ruiz-Palacois, P. Huang, X. Jiang, and J. Le Pendu, Gastroenterology 122:1967-1977, 2002). It has been unknown whether NoV distinguishes between the type 1 and type 2 chains of A and B antigens. In this study, we synthesized A type 1, A type 2, B type 1, and B type 2 pentasaccharides in vitro and examined the function of the core structures in the binding between NoV virus-like particles (VLPs) and HBGAs. The attachment of five genogroup I (GI) VLPs from 5 genotypes and 11 GII VLPs from 8 genotypes, GI/1, GI/2, GI/3, GI/4, GI/8, GII/1, GII/3, GII/4, GII/5, GII/6, GII/7, GII/12, and GII/14, to ABH and Le HBGAs was analyzed by enzyme-linked immunosorbent assay-based binding assays and Biacore analyses. GI/1, GI/2, GI/3, GI/4, GI/8, and GII/4 VLPs were more efficiently bound to A type 2 than A type 1, and GI/8 and GII/4 VLPs were more efficiently bound to B type 2 than B type 1, indicating that NoV VLPs distinguish between type 1 and type 2 carbohydrates. The dissociation of GII/4 VLPs from B type 1 was slower than that from B type 2 in the Biacore experiments; moreover, the binding to B type 1 was stronger than that to B type 2 in the ELISA experiments. These results indicated that the type 1 carbohydrates bind more tightly to NoV VLPs than the type 2 carbohydrates. This property may afford NoV tissue specificity. GII/4 is known to be a global epidemic genotype and binds to more HBGAs than other genotypes. This characteristic may be linked with the worldwide transmission of GII/4 strains. GI/2, GI/3, GI/4, GI/8, GII/4, and GII/7 VLPs bound to Le(a) expressed by nonsecretors, suggesting that NoV can infect individuals regardless of secretor phenotype. Overall, our results indicated that HBGAs are important factors in determining tissue specificity and the risk of transmission.
Asunto(s)
Antígenos de Grupos Sanguíneos/metabolismo , Virus Norwalk/fisiología , Receptores Virales/metabolismo , Acoplamiento Viral , Adulto , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oligosacáridos/síntesis química , Oligosacáridos/metabolismo , Unión Proteica , Resonancia por Plasmón de Superficie , VirosomasRESUMEN
A common feature of caliciviruses is the proteolytic processing of the viral polyprotein catalyzed by the viral 3C-like protease encoded in open reading frame 1 (ORF1). Here we report the identification and structural characterization of the protease domains and amino acid residues in sapovirus (SaV) and feline calicivirus (FCV). The in vitro expression and processing of a panel of truncated ORF1 polyproteins and corresponding mutant forms showed that the functional protease domain is 146 amino acids (aa) in SaV and 154 aa in FCV. Site-directed mutagenesis of the protease domains identified four amino acid residues essential to protease activities: H(31), E(52), C(116), and H(131) in SaV and H(39), E(60), C(122), and H(137) in FCV. A computer-assisted structural analysis showed that despite high levels of diversity in the primary structures of the protease domains in the family Caliciviridae, the configurations of the H, E, C, and H residues are highly conserved, with these residues positioned closely along the inner surface of the potential binding cleft for the substrate. These results strongly suggest that the H, E, C, and H residues are involved in the formation of a conserved catalytic surface of the SaV and FCV 3C-like proteases.
Asunto(s)
Modelos Moleculares , Péptido Hidrolasas/química , Sapovirus/enzimología , Proteínas de la Matriz Viral/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Calicivirus Felino/enzimología , Calicivirus Felino/genética , Dominio Catalítico/genética , Gatos , Línea Celular , Humanos , Imagenología Tridimensional , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Sistemas de Lectura Abierta , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Sapovirus/genética , Especificidad por Sustrato/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
Sapovirus (SaV), a member of the family Caliciviridae, is a causative agent of acute gastroenteritis in humans and swine and is currently divided into five genogroups, GI-GV. The proteolytic processing of the SaV open reading frame 1 (ORF1) polyprotein with a human GII SaV Mc10 strain has recently been determined and the products are arranged in the following order: NH(2)-p11-p28-p35 (NTPase)-p32-p14 (VPg)-p70 (Pro-Pol)-p60 (VP1)-COOH. The cleavage site between p14 (VPg) and p70 (Pro-Pol) was identified as E(1055)/A(1056) by N-terminal amino acid sequencing. To identify other cleavage sites, a series of GII SaV Mc10 full-length clones containing disrupted potential cleavage sites in the ORF1 polyprotein were constructed and used to generate linear DNA templates for in vitro coupled transcription-translation. The translation products were analysed by SDS-PAGE or by immunoprecipitation with region-specific antibodies. N-terminal amino acid sequencing with Escherichia coli-expressed recombinant proteins was also used to identify the cleavage site between p32 and p14. These approaches enabled identification of the six cleavage sites of the Mc10 ORF1 polyprotein as E(69)/G(70), Q(325)/G(326), Q(666)/G(667), E(940)/A(941), E(1055)/A(1056) and E(1722)/G(1723). The alignment of the SaV full-length ORF1 amino acid sequences indicated that the dipeptides used for the cleavage sites were either E or Q at the P1 position and A, G or S at the P1' position, which were conserved in the GI, GII, GIII, GIV and GV SaV ORF1 polyprotein.
Asunto(s)
Poliproteínas/metabolismo , Sapovirus/metabolismo , Proteínas Virales/metabolismo , Sitios de Unión/genética , Infecciones por Caliciviridae/virología , Escherichia coli/metabolismo , Gastroenteritis/virología , Humanos , Datos de Secuencia Molecular , Mutagénesis , Sistemas de Lectura Abierta , Poliproteínas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Sapovirus/genéticaRESUMEN
Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI-GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 x 10(7) to 2.5 x 10(1) copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses.
Asunto(s)
Infecciones por Caliciviridae/diagnóstico , Gastroenteritis/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sapovirus/aislamiento & purificación , Australia , Secuencia de Bases , Heces/virología , Genoma Viral , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Sistemas de Lectura Abierta/genética , Sapovirus/genética , Sensibilidad y Especificidad , Alineación de SecuenciaRESUMEN
Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1-14 and GII/1-17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.
Asunto(s)
Variación Antigénica , Variación Genética , Norovirus/genética , Norovirus/inmunología , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Reacciones Cruzadas , Genotipo , Humanos , Datos de Secuencia Molecular , Norovirus/clasificación , Filogenia , Alineación de Secuencia , Virión/inmunologíaRESUMEN
Estrogen receptor (ER) is a hormone-inducible transcription factor as a member of the nuclear receptor gene superfamily. Unliganded ER is transcriptionally silent and capable of DNA binding; however, it is unable to suppress the basal activity of the target gene promoters, unlike non-steroid hormone receptors that associate with corepressors in the absence of their cognate ligands. To study the molecular basis of how unliganded human ERalpha is maintained silent in gene regulation upon the target gene promoters, we biochemically searched interactants for hERalpha, and identified heat shock protein 70 (Hsc70). Hsc70 appeared to associate with the N-terminal hormone binding E domain, that also turned out a transcriptionally repressive domain. Competitive association of Hsc70 with a best known coactivator p300 was observed. Thus, these findings suggest that Hsc70 associates with unliganded hERalpha, and thereby deters hERalpha from recruiting transcriptional coregulators, presumably as a component of chaperone complexes.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Ligandos , Proteínas Represoras/metabolismo , Células Cultivadas , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica/genética , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Transcripción Genética , Activación Transcripcional/genética , TransfecciónRESUMEN
The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in (1171)Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH(2)-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Sistemas de Lectura Abierta , Poliproteínas/metabolismo , Sapovirus/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Poliproteínas/química , Sapovirus/enzimología , Sapovirus/genética , Proteínas Virales/químicaRESUMEN
Stool specimens collected between November 2002 and April 2003 from hospitalized infants with acute gastroenteritis from four distinct geographical regions in Thailand were examined for norovirus (NoV) and sapovirus (SaV) by reverse transcription-PCR and sequence analysis. Of the 80 specimens examined, we identified 11 NoV and 9 SaV single infections, and 3 NoV/SaV mixed infections. The majority of NoV strains (64%) belonged to genogroup II/ genotype 4 (GII/4; Lordsdale cluster). Other NoV strains co-circulating belonged to GII/1, GII/3, GII/6, and one new genotype cluster (GII/New). The majority of SaV strains (83%) were from the Manchester cluster. One isolated SaV strain represented a recently discovered novel genogroup within the SaV genus (SG-V), and another isolated SaV strain represented a novel SaV genogroup II cluster.
Asunto(s)
Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , Gastroenteritis/virología , Norovirus/aislamiento & purificación , Sapovirus/aislamiento & purificación , Gastroenteritis/epidemiología , Humanos , Lactante , Norovirus/clasificación , Norovirus/genética , Filogenia , Sapovirus/clasificación , Sapovirus/genética , Tailandia/epidemiologíaRESUMEN
We identified a human multiprotein complex (WINAC) that directly interacts with the vitamin D receptor (VDR) through the Williams syndrome transcription factor (WSTF). WINAC has ATP-dependent chromatin-remodeling activity and contains both SWI/SNF components and DNA replication-related factors. The latter might explain a WINAC requirement for normal S phase progression. WINAC mediates the recruitment of unliganded VDR to VDR target sites in promoters, while subsequent binding of coregulators requires ligand binding. This recruitment order exemplifies that an interaction of a sequence-specific regulator with a chromatin-remodeling complex can organize nucleosomal arrays at specific local sites in order to make promoters accessible for coregulators. Furthermore, overexpression of WSTF could restore the impaired recruitment of VDR to vitamin D regulated promoters in fibroblasts from Williams syndrome patients. This suggests that WINAC dysfunction contributes to Williams syndrome, which could therefore be considered, at least in part, a chromatin-remodeling factor disease.
Asunto(s)
Núcleo Celular/genética , Cromatina/genética , Células Eucariotas/metabolismo , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Receptores de Calcitriol/genética , Síndrome de Williams/genética , Transporte Activo de Núcleo Celular/genética , Animales , Sitios de Unión/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Replicación del ADN/genética , Feto , Regulación de la Expresión Génica/genética , Genes Reguladores/genética , Humanos , Sustancias Macromoleculares , Ratones , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Estructura Terciaria de Proteína/genética , Receptores de Calcitriol/metabolismo , Fase S/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Síndrome de Williams/metabolismoRESUMEN
Pluripotent mesenchymal stem cells in bone marrow differentiate into adipocytes, osteoblasts and other cells. Balanced cytodifferentiation of stem cells is essential for the formation and maintenance of bone marrow; however, the mechanisms that control this balance remain largely unknown. Whereas cytokines such as interleukin-1 (IL-1) and tumour-necrosis factor-alpha (TNF-alpha) inhibit adipogenesis, the ligand-induced transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma), is a key inducer of adipogenesis. Therefore, regulatory coupling between cytokine- and PPAR-gamma-mediated signals might occur during adipogenesis. Here we show that the ligand-induced transactivation function of PPAR-gamma is suppressed by IL-1 and TNF-alpha, and that this suppression is mediated through NF-kappaB activated by the TAK1/TAB1/NF-kappaB-inducing kinase (NIK) cascade, a downstream cascade associated with IL-1 and TNF-alpha signalling. Unlike suppression of the PPAR-gamma transactivation function by mitogen-activated protein kinase-induced growth factor signalling through phosphorylation of the A/B domain, NF-kappaB blocks PPAR-gamma binding to DNA by forming a complex with PPAR-gamma and its AF-1-specific co-activator PGC-2. Our results suggest that expression of IL-1 and TNF-alpha in bone marrow may alter the fate of pluripotent mesenchymal stem cells, directing cellular differentiation towards osteoblasts rather than adipocytes by suppressing PPAR-gamma function through NF-kappaB activated by the TAK1/TAB1/NIK cascade.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Adipocitos/citología , División Celular/fisiología , Citocinas/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , Quinasas Quinasa Quinasa PAM/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Recombinantes de Fusión/fisiología , Tiazolidinedionas , Factores de Transcripción/fisiología , Adipocitos/efectos de los fármacos , Animales , Northern Blotting , Western Blotting , Línea Celular , Cromanos/farmacología , Ensayo de Cambio de Movilidad Electroforética , Ratones , Plásmidos , Pruebas de Precipitina , Transducción de Señal , Tiazoles/farmacología , Troglitazona , Quinasa de Factor Nuclear kappa BRESUMEN
The rat mineralocorticoid receptor (MR) has two activation functions in distinct regions of the A/B domain, designated activation function 1a (AF-1a; amino acids 1 to 169) and AF-1b (amino acids 451 to 600). Since the p160 family protein TIF2, a known component of the AF-2 coactivator complex, potentiates the transactivation function of AF-1b but not that of AF-1a, it is likely that some other, novel protein complex interacts with the AF-1a region. Therefore, we attempted to identify such coactivator complexes from HeLa nuclear extracts by biochemical purification using a glutathione S-transferase-MR AF-1a fusion protein. Purified AF-1a region-interacting proteins were found to contain RNA helicase A (RHA) and CBP. Further analysis showed that RHA interacted with the AF-1a region directly and then recruited a complex with histone acetyltransferase (HAT) activity that contained CBP. For full-length MR, aldosterone, but not hydrocortisone, was found to induce the binding of RHA/CBP complexes to the AF-1a region, as well as to allow the cooperative potentiation of MR transcriptional activity by RHA and CBP. In addition, a chromatin immunoprecipitation assay showed that aldosterone-bound MR, but not hydrocortisone-bound MR, recruited RHA/CBP complexes to native MR target gene promoters. Our results suggested that an altered conformation of the A/B region induced by aldosterone, but not hydrocortisone, might determine the accessibility of MR AF-1a to RHA/CBP complexes.