Shoichi Nagayoshi

Studies on the role of M protein in virus assembly using a is mutant of HVJ (Sendai virus)

Abstract A temperature-sensitive mutant of HVJ, HVJ cl.151, was isolated from BHK cells persistently infected with HVJ and characterized. HVJ c1.151 virion had an M polypeptide different in apparent molecular weight from that of HVJ wild-type, that is 36,000 and 34,000 daltons, respectively. HVJ c1.151 was blocked in a late function required for virus maturation. M protein antigen of HVJ c1.151 was detected in infected cells by immunofluorescent microscopy only at permissive temperature but not at nonpermissive temperature, although GP and NP antigens were detected at both temperatures. Further, analysis of the infected cells by SDS-polyacrylamide gel electrophoresis showed that viral structural polypeptides P, F 0 , NP, and F and nonstructural polypeptide C were synthesized in infected cells at nonpermissive temperature and these structural polypeptides were incorporated into virions upon temperature shiftdown, whereas polypeptides HN and M, which may be synthesized at nonpermissive temperature, were not able to be incorporated into virions upon temperature shift down. Thus, the temperature-sensitive lesion of HVJ c1.151 is considered to be in HN and M proteins. Membrane fluorescense, immunoferritin electron microscopy, and SDS-polyacrylamide gel electrophoresis of plasma membranes showed that migration of F 0 , and F to the cell surface occurred normally even at nonpermissive temperature. Immunoferritin electron microscopy also demonstrated that the viral glycoproteins which arrived at the plasma membrane were dispersed on the entire surface of the membrane at the nonpermissive temperature and that viral components synthesized at this temperature could not assemble at the plasma membrane. In addition, it was found that antibody-induced redistribution of viral glycoproteins on the surface of cells infected with HVJ c1.151 and incubated at nonpermissive temperature occurred very rapidly; in contrast, such redistribution of viral glycoproteins occurred more slowly and less completely in cells incubated at permissive temperature, suggesting that viral glycoproteins on the plasma membrane of cells at nonpermissive temperature have a high degree of mobility in the plane of the membrane as compared with those on the plasma membrane of cells at permissive temperature. These results suggest strongly that a function which fixes the viral glycoproteins at restricted areas of plasma membrane to form a viral envelope is blocked in HVJ c1.151-infected cells at nonpermissive temperature. Analysis of plasma membrane by SDS-polyacrylamide gel electrophoresis showed that viral glycopolypeptides but no NP were present on membranes isolated from HVJ cl.151-infected cells at nonpermissive temperature in spite of the presence of a large amount of NP in the whole cells, whereas plasma membranes isolated from cells at permissive temperature contained all viral structural polypeptides. The possible roles of M protein of HVJ in formation of the viral envelope and association of nucleocapsid with the envelope during assembly are discussed based on these results.

Class distribution of immunoglobulin-containing plasma cells in the stroma of medullary carcinoma of breast

SummaryA class distribution of plasma cells associated with the stroma in twenty-eight cases of medullary carcinoma of the breast was investigated by an unlabeled immunoperoxidase method. The stroma of the medullary carcinomas tested was found to contain predominantly IgG plasma cells except in two cases. Stroma of the other types of breast carcinoma, including ten cases of papillo-tubular carcinoma, five cases of scirrhous carcinoma, and six cases of medullary tubular carcinoma, contained predominantly IgG plasma cells, although few plasma cells were associated with carcinoma tissues in the latter group. Plasma cells associated with control specimens, including normal breast, fibroadenoma, cystic disease, and intraductal papilloma, were found to be predominantly of IgA type. Few carcinomatous epithelial cells contained secretory components in the cytoplasm, while a number of cells positive for secretory components were observed in acinar and ductular epithelia of normal breast tissues and in benign proliferative lesions of the breast. It is suggested that the lymphoid cells infiltrating the stroma of medullary carcinoma represent a sign of host immune response against the carcinoma cells which is related to the well-known favorable prognosis associated with this tumor.

A new retrovirus produced by tissue culture cell line from mammary tumor of a house musk shrew, Suncus murinus

A new type of retrovirus (Sm-MTV) released by cultured cells of a spontaneous mammary tumor from a house musk shrew, Suncus murinus, is described. The Sm-MTV is distinct morphologically from type C particles. In spite of certain morphological similarities to type B and type D retroviruses, the Sm-MTV is readily distinguishable. The extracellular virions had a spikeless envelope containing a centrally located nucleoid with a small electron-dense core surrounded by an inner membrane. The budding particles contained a doughnut-shaped nucleoid. Intracytoplasmic type A particles similar in profile to those associated with mouse mammary tumor cells were also found, and tended to form a small cluster of several particles in the cytoplasm. The virus banded at 1.169 g/cm3 in isopycnic centrifugation and possessed constitutive Mg2+-dependent reverse transcriptase. The viral RNA had a molecular size ranging from 50 to 70 S in its native form and 30 to 40 S in its denatured form by a glycerol gradient ultracentrifugation. Major viral polypeptides were 72K, 69K, 47K, 44K/43K, 27K, 20.5K, and 15K.

Sandwich enzyme immunoassay of mouse mammary tumor virus

A highly sensitive sandwich enzyme immunoassay for the mouse mammary tumor virus (MMTV) is described. The assay can detect 3 ng/ml of MMTV. The enzyme used is ß-D-galactosidase fromEscherichia coli and the solid phase used is a piece of silicon rubber.

Acute infantile gastroenteritis caused by rotavirus in Japan

We had an outbreak of acute infantile gastroenteritis accompanied by milky-white stool (called Hakuri in Japanese) during the winters of 1976 and 1977. Stool specimens collected from 72 cases of Hakuri were studied by negative-staining electron microscopy. Rotavirus was detected with a very high frequency (89%).Rotavirus obtained from one of the patients was isolated and passaged in cultures of primary human embryonic kidney cells. Viral antigens could be detected in the cytoplasm of the cells by indirect immuno-fluorescence. The fluorescence-positive cells increased in number with repeated passage.Serum anti-viral activities in 11 patients were titrated by indirect immuno-fluorescence, using the cells infected with the passaged rotavirus. All 11 patients developed IgM responses in the convalescent phase. However, in 4 of the 11 patients, no IgG responses were detected even 2–3 weeks after the onset of illness. The reinfection which has occasionally be seen in our country may be related to these poor IgG responses.

Genetic resistance to mammary tumorigenesis in a mouse strain with high murine mammary tumor virus expression

Although II-TES mice release large amounts of murine mammary tumor virus (MMTV) in milk, they are resistant to mammary tumorigenesis. High mammary tumor incidence was observed in (BALB/ca X II-TES)F1 and (C57BL/6N X II-TES)F1, whereas no mammary tumors developed in BALB/ca X OZ-F)F1. Mammary tumors developed in 68% of (OZ-F X (OZ-F X II-TES)F1 and 45% of (II-TES X (OZ-F X II-TES)F1). These results suggest that the II-TES mouse carries a recessive gene for mammary tumor resistance which does not inhibit MMTV release, and two independent dominant mammary tumor promoting genes which are inhibited by the resistant gene.

The enhanced expression of cellular fibronectin in mouse-mouse somatic cell hybrids☆

Abstract The expression of cellular fibronectin in mouse-mouse somatic cell hybrids was compared with that in the parental cells by indirect immunofluorescence and enzyme-linked sandwich immunoassay. The hybrid clones were all found to express much larger amounts of cellular fibronectin than the parental cells. This indicates that somatic cell hybridization does not necessarily suppress the expression of cellular fibronectin.

Simple sandwich enzyme immunoassay for quantification of mouse mammary tumor virus in mouse milk

We have developed a sandwich enzyme immunoassay in order to measure quantitatively mouse mammary tumor virus (MMTV) in mouse milk. In this assay, the antibody-beta-D-galactosidase complex and antibody-bound silicon rubber pieces pieces as solid phase are used. The assay is able to detect 10 ng/ml of MMTV in the milk sample.

Characterization of cell fusion in XC cells induced by Suncus murinus mammary tumor virus

SummarySyncytium formation in a rat tumor cell line (XC) induced bySuncus murinus mammary tumor virus (Sm-MTV) was studied. Multinucleate giant cells containing 20–30 nuclei were formed in a monolayer of XC cells by cocultivation with X-ray-irradiated Sm-MTV producing cells (Sm-MT-1). By fluorescent antibody staining, Sm-MTV antigens were demonstrated in the cytoplasm of syncytia, and budding particles and intracytoplasmic A particles were found in syncytial giant cells by electron microscopy. Cell-free supernatant of Sm-MT-1 was capable of inducing syncytia at a much lower incidence than cocultivation of Sm-MT-1 cells. Syncytium formation was completely inhibited when anti-Sm-MTV bovine serum was added to the coculture medium. Pretreatment of XC cells with actinomycin-D caused a partial reduction of Sm-MTV-induced cell fusion, but syncytium formation did occur at a reduced rate even when cellular RNA synthesis was completely inhibited. Dexamethasone increased virus production in Sm-MT-1 cells, resulting in the enhancement of Sm-MTV mediated syncytium formation. Sm-MTV was found to have a unique characteristic of cell fusion activity on XC cells with striking enhancement by dexamethasone.