RESUMO
Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.
Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ovinos , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: The aim was to considerably enhance the sensitivity of hepatitis B virus (HBV) surface antigen (HBsAg) detection and investigate whether the window period for HBV detection could be reduced. STUDY DESIGN AND METHODS: A high-sensitivity chemiluminescent enzyme immunoassay (CLEIA) was developed for quantitative HBsAg detection by a combination of monoclonal antibodies, each one for a specific epitope of HBsAg, and by improving the conjugation technique. The sensitivity of the assay was compared with that of the existing chemiluminescent immunoassay (CLIA). Commercially available seroconversion panels and samples of HBV-infected chimpanzees were tested with the developed prototype to assess whether the window period for HBsAg detection could be reduced to that for DNA detection. RESULTS: Compared to the existing CLIA, the CLEIA prototype detected HBsAg with approximately 230-fold higher sensitivity and showed a reduced window period. HBsAg detection by the CLEIA prototype and HBV DNA detection by polymerase chain reaction (PCR) occurred simultaneously. The mean time for the CLEIA prototype to first detect HBsAg was approximately 17.4 days less than that for the existing systems. Further, CLEIA prototype enabled HBsAg detection even in anti-HBs-positive seroconversion samples. In the inoculated chimpanzees the HBsAg and HBV DNA became detectable simultaneously and concentrations increased in parallel, whereas HBsAg remained detectable longer than HBV DNA in the declining phase of viremia. CONCLUSION: The CLEIA prototype yielded results comparable with those of HBV DNA PCR. This novel high-sensitivity assay may be useful for early detection of HBV infection and monitoring patients with a history of infection.
Assuntos
DNA Viral/genética , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Hepatite B/imunologia , Hepatite B/virologia , Imunoensaio/métodos , Animais , DNA Viral/metabolismo , Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Técnicas de Diluição do Indicador , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Pan troglodytes , Especificidade por Substrato , Fatores de TempoRESUMO
Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular domains, designated HD1 (residues 41-118) and HD2 (residues 171-252), that are homologous to histone H5 from chicken erythrocytes. We have determined the three-dimensional structure of globular domain HD1 with high precision by heteronuclear magnetic resonance spectroscopy. The structure had a winged helix-turn-helix motif composed of an alphabetaalphaalphabetabeta fold and closely resembled the structure of the globular domain of histone H5. Interestingly, the second globular domain, HD2, in Hho1p was unstructured under physiological conditions. Gel mobility assay demonstrated that Hho1p preferentially binds to supercoiled DNA over linearized DNA. Furthermore, NMR analysis of the complex of a deletion mutant protein (residues 1-118) of Hho1p with a linear DNA duplex revealed that four regions within the globular domain HD1 are involved in the DNA binding. The above results suggested that Hho1p possesses properties similar to those of linker histones in higher eukaryotes in terms of the structure and binding preference towards supercoiled DNA.
