RESUMEN
A herpes simplex virus type 1 (HSV-1) containing a thymidine (TK) gene with an amber mutation at the 8th position counted from the first AUG codon was isolated from a child with acute gingivostomatitis. The virus was predicted to express a mutant viral translated from the 2nd AUG codon at the 46th amino acid position and consisting of 331 amino acids. The virus was as sensitive to acyclovir (ACV), 5-bromovinyl-2'-deoxyuridine (BVdU), 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), and 1-beta-D-arabinofuranosylthymine (araT) as a wild-type HSV-1. The mutant TK showed the same level of TK activity as the wild-type TK at reaction temperatures of 34 degrees C, 37 degrees C and 39 degrees C. ACV, BVdU, BVaraU, and araT inhibited the replication of the TK-deficient and drug-resistant HSV-1 and HSV-2 in 293T cells in which the mutant TK was expressed to the same extent as in cells in which intact HSV-1-TK was expressed, whereas BVdU and BVaraU inhibited the replication of these viruses less strongly in cells in which HSV-2-TK was expressed. It can be concluded that the mutant HSV-1 exists in nature as a variant and possesses the necessary phosphorylation activities to form ACV-monophosphate from ACV, to form BVdU-diphosphate through BVdU-monophosphate from BVdU, and to form BVaraU-diphosphate through BVaraU-monophosphate from BVaraU. These results indicate that the mutant HSV-1-TK with a deletion of the first 45 amino acid residues is phenotypically the same as that of wild-type HSV-1-TK in terms of the phosphorylation activity of TK-associated anti-herpes virus drugs.
Asunto(s)
Antivirales/farmacología , Codón de Terminación/genética , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/genética , Timidina Quinasa/genética , Proteínas Virales/genética , Secuencia de Bases , Línea Celular , Niño , Herpesvirus Humano 1/aislamiento & purificación , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 2/genética , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Eliminación de Secuencia , Estomatitis Herpética/virología , Temperatura , Timidina Quinasa/metabolismo , Ensayo de Placa Viral , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
A system for rapid determination of viral RNA sequences, RDV, was improved for detection of avian RNA virus in allantoic fluids. We detected avian paramyxovirus nucleotide sequences using RDV method ver 2.0.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Virus ARN/genética , ARN Viral/genética , Animales , Aves , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
We previously reported that cells with persistent severe acute respiratory syndrome coronavirus (SARS-CoV) infection were established after apoptotic events. In the present study, we investigated the cytopathic effects of dual infection with SARS-CoV and Mycoplasma fermentans on Vero E6 cells. Dual infection completely killed cells and prevented the establishment of persistent SARS-CoV infection. M. fermentans induced inhibition of cell proliferation, but the cells remained alive. Apoptosis was induced easily in M. fermentans-infected cells, indicating that they were primed for apoptosis. These results indicated that M. fermentans enhances apoptosis in surviving cells that have escaped from SARS-CoV-induced apoptosis.
Asunto(s)
Infecciones por Mycoplasma/microbiología , Mycoplasma fermentans/fisiología , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Animales , Apoptosis , Chlorocebus aethiops , Efecto Citopatogénico Viral , Infecciones por Mycoplasma/complicaciones , Síndrome Respiratorio Agudo Grave/complicaciones , Células Vero/microbiología , Células Vero/patologíaRESUMEN
Serious vascular leakage is central to the pathogenesis of hantavirus infections. However, there is no evidence suggesting the hantavirus infection of endothelial cells directly causes obvious cell damage or morphological alteration either in vivo or in vitro. In this study, we examined whether Hantaan virus (HTNV) infection modifies the barrier function of endothelial cell monolayers upon the exposure to pro-inflammatory cytokines. Low levels (1 ng/ml) of tumor necrosis factor-alpha initially increased the permeability in both HTNV-infected and uninfected monolayers similarly. Thereafter, however, these monolayers showed significant difference. The HTNV-infected monolayers remained irreversibly hyper-permeable during the experimental period up to 4 days, while the uninfected monolayers completely recovered the barrier function. The prolonged hyper-permeability of HTNV-infected monolayers was not associated with cell death or gap formation in the monolayers, and was independent from their nitric oxide or prostaglandin production. These results are the first evidence that hantavirus infection modifies barrier function of endothelial cell monolayers and suggest that HTNV-infection of endothelial cells may contribute to the increased vascular leakage through the prolonged response to cytokines.
Asunto(s)
Células Endoteliales/metabolismo , Células Endoteliales/virología , Endotelio Vascular/citología , Endotelio Vascular/virología , Virus Hantaan/patogenicidad , Factor de Necrosis Tumoral alfa/farmacología , Compuestos de Boro , Células Cultivadas , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Humanos , Indometacina/farmacología , Interleucina-6/metabolismo , Metacrilatos , Metilmetacrilatos , Óxido Nítrico/biosíntesis , Permeabilidad , Prostaglandinas/biosíntesis , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Venas Umbilicales , omega-N-Metilarginina/farmacologíaRESUMEN
We developed an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), using partial recombinant nucleoproteins (rNP) of Reston Ebola virus (EBO-R) and Zaire Ebola virus (EBO-Z). We examined the reaction of 10 sera from cynomolgus macaques naturally infected with EBO-R to each of the partial rNP in the IgG ELISA. All the sera reacted to the C-terminal halves of the rNP of both EBO-R and EBO-Z. Most of the sera reacted to the RdeltaC (amino acid (aa) 360-739), and Rdelta6 (aa 451-551) and/or Rdelta8 (aa 631-739) at a higher dilution than to the corresponding truncated rNPs of EBO-Z. The results indicate that this IgG ELISA is useful for detecting EBO-R specific antibody, and may have a potential to discriminate EBO-R infection from other subtypes.
Asunto(s)
Ebolavirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Fiebre Hemorrágica Ebola/veterinaria , Inmunoglobulina G/análisis , Macaca fascicularis , Enfermedades de los Monos/diagnóstico , Animales , Anticuerpos Antivirales/análisis , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/diagnóstico , Humanos , Nucleoproteínas/inmunología , Sensibilidad y EspecificidadRESUMEN
Anti-TSH receptor antibodies (TRAbs) have been known to be involved in Graves' disease and primary hypothyroidism. We previously isolated and reconstituted immunoglobulin (Ig) genes of Epstein-Barr virus-transformed B cell clones producing monoclonal TRAbs obtained from Graves' patients. In the present study, we performed a similar experiment using a B cell clone, 32A-5, derived from a patient with primary hypothyroidism. The variable region genes of Ig heavy (H) and light (L) chains were isolated and sequenced from the 32A-5 clone. A significant number of somatic mutations were found in variable regions of H and L chain gene segments. Each pair of H and L chain cDNAs was ligated into an expression vector for IgG1 production and stably introduced into myeloma cells. The transfectants were injected ip into BALB/c mice to yield ample volume of the antibody for following applications. Interactions of recombinant 32A-5 with Graves' sera with varying thyroid-stimulating antibody (TSAb) activities were studied. The recombinant antibody tended to suppress TSAb activities in 10 of 15 Graves' sera, in which four were significantly inhibited. In summary, this is the first study to analyze human monoclonal TSH-stimulation blocking antibodies (TSBAb) at the molecular level. Use of human recombinant monoclonal TSBAb may be an analytical tool for molecular-basis etiology and an alternative therapeutic path for Graves' disease.
Asunto(s)
Anticuerpos Monoclonales/sangre , Hipotiroidismo/inmunología , Inmunoglobulinas Estimulantes de la Tiroides/sangre , Mixedema/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Cricetinae , Enfermedad de Graves/sangre , Enfermedad de Graves/inmunología , Humanos , Hipotiroidismo/sangre , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Inmunoglobulinas Estimulantes de la Tiroides/química , Inmunoglobulinas Estimulantes de la Tiroides/farmacología , Linfocitos/química , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mixedema/sangre , Proteínas Recombinantes , Análisis de Secuencia de ADN , Tirotropina/inmunologíaRESUMEN
Although antineutrophil antibodies are thought to be involved in drug-induced neutropenia, neither the precise mechanisms nor the particular antigens on the neutrophil surface have yet been clarified. Recently, we examined a patient with Graves' disease who developed antineutrophil cytoplasmic antibodies (ANCA) after propylthiouracil treatment and exhibited neutropenia. Because several target antigens of ANCA are expressed on the surface of neutrophils, it was suggested that ANCA might contribute to neutropenia. The patient's serum bound specifically to neutrophils and HL-60 cells differentiated into granulocytes, and lysed the HL-60 cells via a complement-mediated mechanism. Furthermore, two representative ANCA antigens, proteinase 3 and myeloperoxidase, significantly inhibited both the binding and cytotoxicity of the serum. Finally, tumour necrosis factor-alpha, which is known to up-regulate cell surface expression of several ANCA antigens, enhanced both the binding and cytotoxicity of the serum. These findings suggest that ANCA induced by propylthiouracil contributed to leucopenia through a complement-mediated mechanism.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Antitiroideos/efectos adversos , Autoinmunidad/inmunología , Activación de Complemento/inmunología , Granulocitos/inmunología , Neutropenia/inducido químicamente , Neutropenia/inmunología , Propiltiouracilo/efectos adversos , Antitiroideos/uso terapéutico , Autoinmunidad/efectos de los fármacos , Citotoxicidad Inmunológica , Femenino , Enfermedad de Graves/tratamiento farmacológico , Células HL-60 , Humanos , Persona de Mediana Edad , Propiltiouracilo/uso terapéuticoRESUMEN
We determined the complete genome sequence of Ebola virus subtype Reston (EBO-R) in the Philippines in 1996. The deduced transcriptional signals were highly conserved among Ebola viruses except for the stop signal of L genes. The intergenic regions were composed of 4 to 7 nucleotides, and of 2 characteristic overlaps and a long intergenic region. The glycoprotein (GP) had several amino acid differences from EBO-R isolated in 1989 and 1992. The variety of GP sequences strongly suggests the independent introduction of EBO-R from unknown natural reservoirs in 1996.
Asunto(s)
Ebolavirus/genética , Genoma Viral , Nucleoproteínas , Secuencia de Aminoácidos , Animales , Ebolavirus/clasificación , Glicoproteínas/química , Haplorrinos , Proteínas de la Nucleocápside , ARN Viral/química , Transcripción Genética , Proteínas del Núcleo Viral/genéticaRESUMEN
Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a novel acylated peptide produced in the gastrointestinal endocrine cells as well as neuroendocrine cells in the hypothalamus. The Ser(3) residue of ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. Human medullary thyroid carcinoma is known to produce a variety of gastrointestinal and neuroendocrine peptides. In the present study we investigated ghrelin production in the thyroid gland, especially in human medullary thyroid carcinoma. PCR amplification demonstrated prepro-ghrelin gene transcripts in normal human thyroid tissue and two medullary thyroid carcinoma cell lines (human TT cells and rat 6-23 cells), but not in a rat thyroid follicular cell line. TT cells showed the expression of prepro-ghrelin mRNA of about 0.6 kb by Northern blot analysis. Furthermore, production of ghrelin in TT cells was demonstrated by RIA and immunocytochemistry. Accumulation of des-n-octanoyl ghrelin in the cultured medium of the cells was confirmed. Finally, human medullary thyroid carcinoma surgical specimens showed significantly higher des-n-octanoyl ghrelin contents than normal thyroid tissues. In conclusion, we revealed that ghrelin was produced by the human thyroid parafollicular carcinoma cell line, TT cells. These findings suggest that ghrelin is produced in the thyroid C cells as well as in medullary thyroid carcinoma and may provide opportunities to investigate its physiological role in the thyroid gland.
Asunto(s)
Carcinoma Medular/metabolismo , Hormonas Peptídicas , Péptidos/metabolismo , Neoplasias de la Tiroides/metabolismo , Ghrelina , Humanos , Inmunohistoquímica , Péptidos/análisis , Precursores de Proteínas/genética , ARN Mensajero/análisis , Radioinmunoensayo , Glándula Tiroides/metabolismo , Células Tumorales CultivadasRESUMEN
The synergistic relationship between GH-releasing secretagogue (GHS) and GH-releasing hormone (GHRH) with respect to GH secretion is well known. In the present study, we report a similar relationship between GHRH and ghrelin, a recently identified endogenous ligand for the GHS receptor. In normal male adults, various doses of ghrelin were intravenously administered alone or together with 1.0 microg/kg GHRH. At small doses of 0.08 and 0.2 microg/kg ghrelin, combined administration of the two peptides significantly stimulated GH release in a synergistic manner; the mean GH response values of the two peptide combinations were more than the summed mean GH response values of each peptide alone (P < 0.05). In addition, at 1.0 microg/kg ghrelin, the tendency of the synergistic effect was observed, although the comparison was not statistically significant probably due to a submaximal dose ceiling effect. No synergistic effects with respect to ACTH or prolactin secretion were observed. In conclusion, the synergistic interaction between ghrelin and GHRH was clearly shown and might be useful for a provocation test to diagnose GH deficiency.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona de Crecimiento Humana/metabolismo , Hormonas Peptídicas , Péptidos/farmacología , Hormona Adrenocorticotrópica/sangre , Adulto , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Ghrelina , Humanos , Hidrocortisona/sangre , Masculino , Persona de Mediana Edad , Hormonas Adenohipofisarias/sangre , Prolactina/sangreRESUMEN
With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope defined by a 26-amino-acid stretch near the C terminus of NP. In a sandwich ELISA system with the MAb, as little as 30 ng of purified recombinant NP (rNP) was detected. Although this MAb was prepared by immunization with rNP of subtype Zaire, it also reacted to the corresponding region of NP derived from the Reston and Sudan subtypes. These results suggest that our ELISA system should work with three of four Ebola subtypes. Furthermore, our ELISA system detected the NP in subtype Reston-infected monkey specimens, while the background level in noninfected specimens was very low, suggesting the usefulness of the ELISA for laboratory diagnosis with clinical specimens.
Asunto(s)
Anticuerpos Monoclonales , Antígenos Virales/análisis , Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/diagnóstico , Nucleoproteínas/inmunología , Proteínas del Núcleo Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Ebolavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Mapeo Epitopo , Fiebre Hemorrágica Ebola/veterinaria , Fiebre Hemorrágica Ebola/virología , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Enfermedades de los Monos/diagnóstico , Enfermedades de los Monos/virologíaRESUMEN
Thyrotropin receptor (TSHR) is a member of the glycoprotein hormone receptor family and an autoantigen of Graves' disease. Various attempts have been made to obtain a large amount of soluble ectodomain of TSHR in insect or mammalian cells, but most of them failed to secrete the overexpressed ectodomain. In the present study, we observed that about one-third of the ectodomain protein (sTSHR-gp), in which the signal peptide of TSHR was replaced by the baculovirus-encoded glycoprotein 67-signal peptide, was secreted into the culture medium and the remainder stayed within cells in the recombinant baculovirus system. Microsequencing the N-terminal of the purified protein confirmed that the baculovirus signal peptide was cleaved at the expected site. Carbohydrate studies using several glycosidases and lectins revealed that the secreted form of the ectodomain had biantennary carbohydrate, whereas the non-secreted form had high-mannose. Moreover, the secreted form of sTSHR-gp exhibited high-affinity ligand binding, whereas the non-secreted form did not show any significant ligand binding. Regarding the interactions of TSHR ectodomains with anti-TSHR antibodies, both the secreted and non-secreted forms of sTSHR-gp, almost completely neutralized the stimulatory and inhibitory anti-TSHR antibody activities. In conclusion, we succeeded in secreting the ectodomain of TSHR into culture medium, which was capable of binding to TSH and neutralizing anti-TSHR antibody activities.
Asunto(s)
Baculoviridae/genética , Receptores de Tirotropina/química , Receptores de Tirotropina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Baculoviridae/fisiología , Células Cultivadas , Cromatografía de Afinidad , Cromatografía en Gel , ADN Recombinante , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/metabolismo , Glicosilación , Enfermedad de Graves/inmunología , Lectinas/metabolismo , Pruebas de Neutralización , Estructura Terciaria de Proteína , Receptores de Tirotropina/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Spodoptera , Tirotropina/metabolismoRESUMEN
Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Neoplasias Inducidas por Radiación , Neoplasias Cutáneas/etiología , Rayos Ultravioleta , Xerodermia Pigmentosa/complicaciones , Animales , Ciclo Celular , Reparación del ADN , Proteínas de Unión al ADN/genética , Genes p53 , Ratones , Ratones Noqueados , Mutación , Neoplasias Inducidas por Radiación/complicaciones , Neoplasias Inducidas por Radiación/genética , Unión Proteica , Proteínas Ribosómicas/genética , Neoplasias Cutáneas/complicaciones , Neoplasias Cutáneas/genética , Xerodermia Pigmentosa/genética , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
A novel recombinant baculovirus which expresses Ebola virus (EBO) nucleoprotein (NP) under the control of the cytomegalovirus immediate-early promoter was constructed. HeLa cells abortively infected with the baculovirus expressed EBO NP, and this was used as an immunofluorescent (IF) antigen to detect EBO immunoglobulin G (IgG) antibody. This IF method has high efficacy in detecting EBO IgG antibody in clinical specimens, indicating its usefulness in the diagnosis of EBO infections and seroepidemiological studies.
Asunto(s)
Anticuerpos Antivirales/sangre , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/diagnóstico , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Baculoviridae , Citomegalovirus/genética , Ebolavirus/genética , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Células HeLa , Fiebre Hemorrágica Ebola/sangre , Fiebre Hemorrágica Ebola/inmunología , Humanos , Nucleoproteínas/inmunología , Regiones Promotoras Genéticas , Proteínas Recombinantes/inmunología , Transfección , Proteínas del Núcleo Viral/inmunologíaRESUMEN
The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione S-transferase-tagged recombinant proteins in an Escherichia coli system. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Ebolavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Fiebre Hemorrágica Ebola/diagnóstico , Enfermedad del Virus de Marburg/diagnóstico , Marburgvirus/inmunología , Proteínas de Unión al ARN , Ribonucleoproteínas , Proteínas Virales , Animales , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Fiebre Hemorrágica Ebola/epidemiología , Humanos , Inmunoglobulina G/sangre , Enfermedad del Virus de Marburg/epidemiología , Ratones , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Conejos , Proteínas Recombinantes/inmunología , Sensibilidad y EspecificidadRESUMEN
The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER). To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system. Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1). The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65. Sequence analysis revealed that XAB1 has four sequence motifs G1-G4 of the GTP-binding protein family in the N-terminal half. XAB1 also contains an acidic region in the C-terminal portion. Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm. Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity. Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA. The deletion of five amino acids, residues 30-34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1. These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.
Asunto(s)
Citoplasma/enzimología , Proteínas de Unión al ADN/metabolismo , GTP Fosfohidrolasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Células HeLa , Humanos , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Unión Proteica , Transporte de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Eliminación de Secuencia/genética , Testículo/metabolismo , Técnicas del Sistema de Dos Híbridos , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
In a previous study we identified a microsatellite marker near the thyrotropin receptor (TSHR) gene. Studies with this marker, TSHR-CA, revealed a significant association between autoimmune thyroid disease (AITD) in Japanese patients and one specific allele (allele 1; 180 base pair [bp]) of the microsatellite sequence. In addition, weak evidence for association of AITD with two alleles of the CTLA-4 gene was observed. In the present study, TSHR-CA has been mapped to approximately 600 kb of the TSHR gene using radiation hybrid mapping. TSHR-CA and another TSHR microsatellite marker, TSHR-AT, which is located in intron 2 of TSHR gene, were genotyped in a set of 349 unrelated Japanese AITD patients and 218 Japanese controls. The TSHR-AT marker showed association in this Japanese AITD population with a significant increase in allele 5 (294 bp; p < 0.05) and a significant decrease in allele 7 (298 bp; p < 0.05). The association of allele 5 of TSHR-AT was also significant in hypothyroid patients (thyrotropin-binding inhibitory immunoglobulin-positive [TBII+], P < 0.01; thyrotropin-binding inhibitory immunoglobulin-negative [TBII-], p < 0.05). The association of allele 7 of TSHR-AT were also significant for the hypothyroid TBII+ patients (p < 0.05). The CTLA-4 gene was also genotyped in this expanded set of Japanese AITD patients and controls. Association between AITD susceptibility and allele 2 (102 bp; p < 0.01) and allele 4 (106 bp; p < 0.01) were observed. These associations were also observed with GD patients (allele 2, p < 0.01; allele 4, p < 0.01). Associations with TSHR-CA were observed for Hashimoto's thyroiditis (HT) patients with respect to alleles 3 (179 bp; p < 0.05) and 5 (175 bp; p < 0.05) and with hypothyroid TBII- patients for allele 4 (177 bp; p < 0.05). The presence of specific alleles of TSHR-CA, TSHR-AT, and CTLA-4 contribute significant increase in risk of development of AITD. These results confirm and expand on our previous study suggesting that alleles of the TSHR and CTLA-4 genes, or genes near them contribute to AITD susceptibility and set the stage for future studies of interactions between these genes and AITD.
Asunto(s)
Antígenos de Diferenciación/genética , Inmunoconjugados , Repeticiones de Microsatélite , Receptores de Tirotropina/genética , Tiroiditis Autoinmune/genética , Abatacept , Alelos , Antígenos CD , Antígeno CTLA-4 , Salud de la Familia , Femenino , Predisposición Genética a la Enfermedad , Humanos , Japón , Masculino , Polimorfismo Genético , Mapeo de Híbrido por Radiación , Tiroiditis Autoinmune/diagnósticoRESUMEN
We computed the spectrum of gravitational waves from a dust disk star of radius R inspiraling into a Kerr black hole of mass M and specific angular momentum a. We found that when R is much larger than the wave length of the quasinormal mode, the spectrum has several peaks and the separation of peaks Deltaomega is proportional to R-1 irrespective of M and a. This suggests that the radius of the star in coalescing binary black hole-star systems may be determined directly from the observed spectrum of gravitational waves. This also suggests that the spectrum of the radiation may give us important information in gravitational wave astronomy as in optical astronomy.
RESUMEN
Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.