Shinsuke Saga

Characterization of a novel transformation-sensitive heat-shock protein (HSP47) that binds to collagen

The synthesis of a major collagen-binding glycoprotein of molecular weight 47,000 was previously shown to be regulated by malignant transformation as well as by heat shock in chick embryo fibroblasts. The 47-kDa protein purified from chick embryos was characterized biochemically, and was found to exist as a monomer in native form. Its composition was enriched in basic amino acids and glycine, with fewer acidic residues and virtually no cysteine. N-terminal amino acid sequencing covering 36 residues revealed a single, novel sequence with an internal tandem repeat of Asp-Lys-Ala-Thr-Thr-Leu-Ala and Asp-Arg-Ser-Thr-Thr-Leu-Ala.

Expression of meta-vinculin associated with differentiation of chicken embryonal muscle cells☆

We confirmed the existence of meta-vinculin, which cross-reacts immunologically with vinculin and has a slightly higher molecular weight than the latter. The immunological cross-reactivity between meta-vinculin and vinculin was confirmed with monoclonal antibodies against vinculin. Furthermore, we found that this protein is present only in either smooth or striated muscle, but is absent in non-muscular tissues and that the expression of this protein is associated with a differentiation of muscle cells either in vivo or in vitro. Microinjection experiments of fluorescently labelled vinculin and/or meta-vinculin into the cytoplasm of cultured myotubes suggested that meta-vinculin may be localized at sites similar to vinculin in muscle cells.

The transformation-sensitive heat shock protein (HSP47) binds specifically to fetuin

The transformation-sensitive heat shock protein of Mr = 47,000 (hsp47) has been shown to bind to collagen and gelatin. We examined the binding specificity of hsp47. The binding of hsp47 to gelatin-Sepharose 4B was competitively inhibited by fetuin as effectively as by gelatin or collagen, whereas a variety of other proteins tested had no effect. Fetuin-coupled Sepharose 4B was found to bind hsp47 even at high ionic strength, but the complex was dissociated at pH less than or equal to 5.5.)

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.

Age-related attenuation of HSP47 heat response in fibroblasts

The collagen-binding heat shock protein of molecular weight 47,000 (HSP47), resident in the endoplasmic reticulum (ER), is assumed to play a specific role as a molecular chaperon in the processing of procollagen molecules. The present investigation of age-related alteration in the HSP47 heat response in cultured murine and human fibroblasts revealed expression in cells with a low population doubling level (PDL) derived from young mice and people more inducible by heat treatment than those from older mice and people. On the other hand, cells with a high PDL showed a very low heat response in terms of HSP47 expression regardless of the donor age. Northern blot analysis of HSP47 m-RNA indicated that the age related attenuation of HSP47 expression was regulated by transcriptional mechanisms. Furthermore, immunofluorescent analysis using a monoclonal antibody against the carboxylterminal propeptide of type I procollagen revealed far greater retention of procollagen molecules in the ER lumen of cells from old persons than in those from young persons. This was particularly prominent in heat-treated cells from old persons, indicating the possibility that the observed decrease in HSP47 heat response might cause blockage of procollagen transport to the Golgi and therefore secretion.

Establishment of a cell line producing basement membrane components from an adenoid cystic carcinoma of the human salivary gland

SummaryA new cell line has been established from an adenoid cystic carcinoma arising in the submandibular gland of a 63-year-old woman. The cultured epithelial-like cells grew vigorously and adhered together to form a sheet. Immunohistochemical stainings for type IV collagen, laminin and fibronectin were clearly positive in the intercellular matrix and on the surface of the culture cells. Chondroitin 6-sulfate proteoglycan and heparan sulfate were also detected. Ultrastructural studies showed that the cells had abundant rough endoplasmic reticulum and a well-developed Golgi apparatus. Rough endoplasmic reticulum near the cell surface was markedly dilated, and contained material of low electron density. This cell line would be useful for biological and biochemical studies on the mechanisms by which the stromal component is formed.

Pleomorphic carcinoma with osteoclastic giant cells of the breast: Immunohistochemical differentiation between coexisting neoplastic and reactive giant cells

Herein is described a unique case of breast carcinoma with two different types of giant cells noted in both cytological and histological specimens. A 51‐year‐old Japanese woman noticed a hard mass in the upper outer quadrant of her left breast. Aspiration cytology exhibited numerous anaplastic giant cells; the cytological diagnosis was high‐grade ductal carcinoma, although a few osteoclastic giant cells were also observed. A left simple mastectomy and sentinel lymph node biopsy were performed. Histologically, approximately 90% of the tumor was composed of giant cells; conventional invasive ductal carcinoma and ductal carcinoma in situ were found focally at the periphery of the tumor. The main part of the tumor contained both anaplastic, neoplastic giant cells and non‐neoplastic, osteoclastic giant cells that were distinguishable from nuclear atypism. The presence of the two types of giant cells was also confirmed on immunohistochemistry using a histiocytic marker (CD68) and two epithelial markers (AE1/AE3 and CAM5.2). Based on the latest World Health Organization classification, the diagnosis was pleomorphic carcinoma with osteoclastic giant cells. To the authors knowledge there has been no previous report on this subject except for a single case mentioned in Rosens Breast Pathology.

Immunohistochemical characteristics of chicken spleen ellipsoids using newly established monoclonal antibodies

Ellipsoids, the extra-vasculature sites surrounding penicilliary capillaries of the chicken spleen, play critical roles in the immune response and also in the clearance of pathogens or other particles. The meshwork of ellipsoids is formed by fibroblastic reticular cells. To characterize ellipsoidal reticular cells, a series of monoclonal antibodies against the chicken spleen have been developed. Of these antibodies, CSA-1 antibody reacts with fibroblastic reticular cells in ellipsoids and with endothelial cells. The reticular nature of positive cells in ellipsoids is indicated by immuno-electron microscopy, and by double-staining with anti-heat-shock protein 47 kDa (hsp47) antibody. The reaction of CSA-1 with reticular cells is limited in ellipsoids; CSA-1 does not react with reticular cells in other lymphoid organs. These findings indicate that ellipsoidal reticular cells share the antigen with endothelial cells. Ontogenic studies reveal that, on embryonic day 18, the development of ellipsoids is completed, penicilliary capillaries become fenestrated, and CSA-1 expression in ellipsoids begins. These findings suggest that CSA-1 is expressed on the cell surface of ellipsoidal reticular cells once they are exposed to blood flow.

Tissue distribution of mouse mammary tumor virus (MMTV) antigens and new endogenous MMTV loci in Japanese laboratory mouse strains

The distribution of mouse mammary tumor virus (MMTV) antigens was studied by the immunoper‐oxidase method in the II‐TES and I‐TES mouse strains as well as their progenitors, CS and DBA/2 strains. In the II‐TES, I‐TES and CS strains, and BALB/c mice foster‐nursed with these strains, MMTV antigens were found not only in epithelial cells of the mammary glands but also in those of other tissues including the seminal vesicle, vas deferens, epididymis, prostate, parotid, submandibular, lacrimal, sebaceous, and urethral glands. In DBA/2 and BALB/cfDBA/2 mice, however, the MMTV antigens were found only in the mammary glands. Electron microscopic examination showed MMTV particles in these organs. When we examined the presence of Mtv‐1 and 2 proviruses, which are known to be responsible for MMTV expression, in the genomes of the II‐TES, I‐TES, CS and DBA/2 strains by Southern blotting, Mtv‐2 was not found in any of the mice and Mtv‐1 was found in the II‐TES and DBA/2 mice but not in the I‐TES and CS mice. Instead, four new endogenous MMTV loci, which have never previously been reported in laboratory mouse strains, were detected in the genomes of the II‐TES, I‐TES and CS strains. One (designated Mtv‐42) was common in the three strains and the other three (designated Mtv‐43, 44 and 45) were common in the II‐TES and I‐TES strains or the II‐TES and CS strains. These results thus suggest that new endogenous MMTV loci may be responsible for MMTV expression in a variety of tissues of these three strains.

Preparation and characterization of monoclonal antibodies against occluded virions of Bombyx mori nuclear polyhedrosis virus

Abstract Six hybridoma cell lines secreting monoclonal antibodies (MAbs) to the structural polypeptides of occluded virions of Bombyx mori nuclear polyhedrosis virus (BmNPV) were established. These MAbs were characterized by immunoblot analysis and classified into three groups on the basis of their specificity to the viral polypeptides of occluded virions. MAbs classified as group 1 are reactive with occluded virion polypeptides exhibiting a smear pattern ranging in molecular weights (MWs) from 16,000 (16K) to 22K, but show no reactivity with the structural polypeptides of nonoccluded virions. In the BmNPV-infected cells, group 1 MAbs react with a single polypeptide with an apparent MW of 17K, suggesting that group 1 MAbs are directed against the 17K polypeptide or its counterpart present in the occluded virions. Group 2 MAbs react with two occluded virion structural polypeptides with MWs of 66 and 82K that are present in both nonoccluded virions and infected cells, and to a lesser extent, with six additional polypeptides of occluded virions. MAbs in group 3 consisting of four different species recognize the 40K polypeptide and one to five additional polypeptides ranging in MWs from 27 to 39K that are present in the infected cells but not detectable in the nonoccluded virions. In vitro translation of cytoplasmic RNA from infected cells and subsequent immunoprecipitation of translation products showed that polypeptides with MWs similar to those of the polypeptides additionally detected by group 3 MAbs were present among the immunoprecipitates. Immunofluorescence microscopy showed that localization of corresponding antigens in the infected cells differed distinctively among MAbs in the different groups.