Kazukiyo Onodera

Isolation and characterization of a yeast gene, MPD1, the overexpression of which suppresses inviability caused by protein disulfide isomerase depletion

MPD1, a yeast gene the overexpression of which suppresses the inviability caused by the loss of protein disulfide isomerase (PDI) was isolated and characterized. The MPD1 gene product retained a single disulfide isomerase active site sequence (APWCGHCK), an N‐terminal putative signal sequence, and a C‐terminal endoplasmic reticulum (ER) retention signal, and was a novel member of the PDI family. The gene product, identified in yeast extract, contained core size carbohydrates. MPD1 was not essential for growth, but overexpression of the gene suppressed the maturation defect of carboxypeptidase Y caused by PDI1 deletion, indicative of the related function to PDI in the yeast ER.

Cell-cycle dependent biosynthesis and localization of p53 protein in untransformed human cells

Summary— Localization of p53 in human cultured lymphocytes and in cultured skin fibroblasts was studied by immuno‐fluorescent microscopy and post‐embedded immunoelectron microscopy using Lowicryl K4M. In quiescent lymphocytes, p53 was found in small amounts in both the cytoplasm and the nucleus. p53 in the nucleus was found associated with the non‐chromatin structure. At 24 h or 72 h of PHA stimulation, p53 increased markedly just beneath the plasma membrane and in the nucleus, which stained diffusely with anti‐p53. In resting fibroblasts, small amounts of p53 were present in both the cytoplasm and the nucleus. After 16 h of stimulation of confluent‐resting fibroblasts by trypsinization and replating, a phase just prior to the initiation of DNA synthesis, p53 slightly increased in both the cytoplasm and the nucleus. Afterwards, p53 was present predominantly in the cytoplasm, closely associated with the cytoskeletal actin filaments. In mitotic cells, p53 was distributed throughout the cytoplasm. When fibroblasts were extracted with saponin, p53 was still associated with the actin filaments, as well as mitochondrial membranes and granular structures of the nuclear matrix. Our data suggest that the initial increase of p53 in cells that enter the cell cycle through G1 first bind to the actin cytoskeleton, and that some of the p53 then move into the nucleus to initiate gene activation and DNA synthesis for cell proliferation. This implies that there is some functionally significant interaction between p53 and actin in the cells.

Altered molecular structure of HLA-DR antigens synthesized in the presence of tunicamycin

Summary Effects of tunicamycin on the synthesis of proteins and glycoproteins, especially the surface antigens of a human lymphoblastoid cell line (Raji), were investigated. Electrophoretic profiles of [3H]leucine labeled membrane components were similar in the tunicamycin treated cells and untreated cells. However, the respective electrophoretic profiles of [3H]glucosamine labeled membrane components were remarkably different. In the presence of tunicamycin, the relative amount of HLA-DR antigens in the newly synthesized membrane was remarkably reduced, and both subunits of the antigens showed reduced apparent molecular weights. Moreover, one of the subunits completely lost its [3H]glucosamine labeled portion(s), and the other subunit still contained [3H]glucosamine labeled portion(s). These results imply that HLA-DR antigens possess either lipid carrier dependent oligosaccharides or other type(s) of oligosaccharide(s).

Immunohistochemistry of superoxide dismutase-1 in developing human brain

The developmental changes in superoxide dismutase (SOD)-1 were studied in brains ranging in age from human fetuses to adults by immunohistochemistry. SOD-positive neurons and glial cells appeared with maturation in each region, and increased progressively with gestational and postnatal age. This phenomenon implies a relationship between SOD-1 gene expression and the anti-oxidant defence mechanism in developing neurons and glia.

The orientation of primary cilia during the wound response in 3Y1 cells

Summary— The behavior of the primary cilia of 3Y1 cells in the interphase was investigated by indirect immunofluorescence microscopy and transmission electron microscopy, using an antibody for tubulin. At 4.5 h after scraping a part of a confluent cell sheet, the primary cilia of cells facing the wound were located predominantly forward of the nucleus on the wounded side, and were oriented in the direction of the leading lamellae. Cytoplasmic microtubules (MTs), emanating from around the base of the cilia, were well developed in the leading lamellae on the wounded side. On the other hand, in the cells of an unperturbed area away from the wounded edge, the primary cilia remained randomly distributed near the nucleus. The position and a certain well‐defined orientation of a pair of centrioles seem to play an important role for the development of cytoplasmic MTs, and consequently the orientation of the centrioles is controlled by the primary cilia.

Characterization of lung squamous cell carcinoma-derived T-cell suppressive factor.

Background. The immunosuppressive state of a tumor‐bearing patient is possibly mediated by tumor‐derived factor. In this study, the authors characterized lung squamous cell carcinoma‐derived immunosuppressive factor (LSCF).

Chemically defined medium for the production of biologically active substances of CHO cells

A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.

The spatial distribution of spindle microtubules in anaphase 3Y1 cells.

Summary— The spatial distribution of the microtubules (MT) in the rat 3Y1 cells in mitosis was investigated by immunoelectron microscopy and computer‐graphic reconstruction of serial thin sections. In anaphase the interzone‐MT increased in number gradually with advancing phase, while the kinetochore‐MT in half‐spindles decreased. The interzone‐MT overlapped with each other at the equatorial region of the cell, and they formed a specific structure called the ‘stem bodies’. The ends of the interzone‐MT opposite to the stem bodies often attached to chromosomes but not to the poles. The stem bodies were not labeled with immunogold particles of anti‐α tubulin. Some of the stem bodies or MT which originate from stem bodies were found just beneath the plasma membrane in the equatorial region where abundant actin filaments appear showing the formation of the contractile ring and subsequently the cleavage furrow begins. On the basis of these observations it is assumed that the interzone‐MT is involved both in the separation of chromosomes in anaphase and in the formation of the cleavage furrow in telophase.

Production of the Human Growth Hormone in Serum-Free UC203 Medium

We developed a serum-free medium from UC202 medium [1] that could support cell growth and enhance the production of the human growth hormone (hGH) coded by the introduced human gene. This developed UC203 medium was better than alpha-MEM [21 with 5% dialyzed fetal calf serum (DFCS) in terms of supporting cell growth and enhancing the production of hGH.

Distribution of Glial Fibrillary Acidic Protein (GFAP) in the Intermediate Filaments of the Cultured Cells from a Patient with Tuberous Sclerosis

We established a cell line (TS) from adenoma sebaceum of a patient with tuberous sclerosis. Through our previous studies, the abnormal cell division and dysfunction of TS cells were indicated. Glial fibrillary acidic protein (GFAP) and 55kd protein had been found to be major cytoskeletal proteins in these cells. This time we have examined the structure and distribution of cytoskeletons in TS cells with immunoelectron microscopy. TS cells were found to coexpress GFAP and vimentin‐like substance; both structures seem to be closely related. The coexistence of a few kinds of proteins integrated in the cytoskeletons might lead to the abnormal behavior of the nucleus during the process of cell division.