The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.

Animals↗

PubMedDate not supplied

Co-localization of receptor and transducer proteins in the glycosphingolipid-enriched, low density, detergent-insoluble membrane fraction of sea urchin sperm.

The low density, detergent-insoluble membrane fraction (LD-DIM), where gangliosides are likely to be highly enriched, was prepared from sperm of two sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus. Immunoblotting showed the presence in the LD-DIM of two receptors for egg ligands, a glycosylphosphatidylinositol (GPI)-anchored protein, and four proteins which may be involved in signal transduction. Co-immunoprecipitation revealed that at least three proteins, the speract receptor, the 63kDa GPI-anchored protein and the alpha subunit of a heterotrimeric Gs protein, are localized in the LD-DIM. This suggests that the LD-DIM fraction may be a membrane microdomain for speract-speract receptor interaction, as well as the subsequent signal transduction pathway involved in induction of sperm respiration, motility and possibly the acrosome reaction.

Animals↗

PubMedDate not supplied

Sudden deafness with severe loss of cochlear neurons.

We report our postmortem findings in the temporal bones of a patient who, at the age of 13, after having had a cold, experienced a sudden and profound hearing loss in her right ear. She died of nasopharyngeal carcinoma at 41. The patient's right temporal bone showed a complete loss of cochlear neurons, but the organ of Corti was well-preserved. These findings indicate a primary degeneration of the cochlear nerve. The authors speculated that the possible cause of this sudden deafness was severe neuronitis of the cochlear nerve.

Adult↗

PubMedDate not supplied

Histopathological study of otitis media with effusion.

Thirteen temporal bones with otitis media with effusion (OME) taken from ten patients were studied. The effusions in the tympanum were histologically classifed into serous and mucoid types. In the serous effusion, neutrophils were dominantly distributed and the mucosa and submucosa showed inflammatory signs. Few epithelial cells and lymphocytes were observed in the effusion of the mucoid type. Eosinophils were rarely seen in the effusions of both types. The presence of inflammatory process was a common finding in OME, particularly with serous effusion. A case, with an immunologic disorder suspected, showed an extensive proliferation of mucosa and unusually thick liquid in the middle ear. This histological feature of the case did not resemble those of other ears with mucoid type of effusion.

Aged↗

PubMedDate not supplied

Cholesteatoma debris as an activator of human monocytes. Potentiation of the production of tumor necrosis factor.

Tumor necrosis factor (TNF) is a cytokine which stimulates osteoclastic bone resorption and inhibits collagen synthesis in vitro. In this study the effect of human cholesteatoma debris and its constituents on the production of TNF-alpha by human monocytes in vitro was studied. Cultured human peripheral monocytes secreted TNF into the culture medium when exposed to cholesteatoma debris in a dose-dependent manner. The TNF production, however, was partially inhibited by the treatment of the debris with polymyxin B which inhibits biological activities of lipopolysaccharide (LPS). When individual constituents of cholesteatoma debris, i.e. keratin, cholesterol, lauric acid and LPS, were added to the cultured monocytes at concentrations equivalent to those in the debris, significant production of TNF was observed only with the keratin and LPS. These data suggest that cholesteatoma debris is a potent activator of the TNF production of human monocytes in vitro, and that LPS and keratin are responsible for the production.

Bone Resorption↗

PubMedDate not supplied

Activation of peritoneal macrophages with human cholesteatoma debris and alpha-keratin.

The effect of human cholesteatoma debris on mouse peritoneal macrophages was studied in vivo. The number of macrophages and lymphocytes increased 5 days after injection of the debris into the peritoneal cavity. A similar increase in peritoneal cells was observed when an urea-extracted fraction of the cholesteatoma debris or alpha-keratin, a major component of the debris, was injected. Both cholesteatoma debris- and alpha-keratin-elicited macrophages exhibited a greater response of luminol-dependent chemiluminescence upon exposure to zymosan, suggesting that the elicited macrophages were activated. In contrast, other constituents of the debris, such as cholesterol or fatty acid--with the exception of lipopolysaccharide (LPS)--failed to elicit or activate peritoneal macrophages at the similar doses detected in the debris. The chemiluminescent response of macrophages obtained by injecting LPS was, however, much lower than that of alpha-keratin-induced macrophages. These results indicate that cholesteatoma debris is capable of eliciting and activating macrophages, and that alpha-keratin is responsible for the activation.

Animals↗