| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1992: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1990: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Research Abstract |
Followings were obtained mainly by means of immunohisto- & cytochemistry at the light and electron microscopic level.
1)Chick embryos have two different kinds of beta-galactoside binding lectins, the 14- and 16-kDa lectins. In the present study, localization of endogenous beta-galactoside binding lectins and their gene expression patterns were investigated during differentiation of chick embryonic skin in vivo and in vitro. By light microscopy, immunostaining of the lectins was weak in the undifferentiated epidermis, while it became intense in the keratinized epidermis, particularly in the intermediate cells. The gene expression pattern as revealed by in situ hybridization technique was consistent with these immunohistochemical observations. In the Vitamin A-pretreated cultured skin, mucous metaplasia of the epidermis was induced and marked changes in localization of these two isolectins were observed. In the metaplastic epidermis, the 16-kDa lectin expression was increased especially i
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n the superficial cells, and the gene expression was detected in all layers of the epidermis, while distribution of 14-kDa lectin and its gene expression was markedly reduced. In the keratinized epidermis, detailed localization of two isolectins under the electron microscope was almost the same. Both of them were located primarily along the plasma membrane, in the intercellular space, and in the desmosomes. In the mucous metaplastic epidermis, however, the localization of the 16-kDa lectin was completely different from that of the 14-kDa lectin; e.g., the former was detected in the while the later was scarcely observed in the epidermis. To characterize the complementary sugar residue recognized by these endogenous lectins, conventional plant lectins such as PNA, RCA and ECA were applied and found that binding patterns of these exogenous lectins were coincided with the expression of the 16-kDa lectin, but not with that of 14-kDa lectin in the mucous metaplastic skin. The present results indicated that 1)the two isolectins are expressed during the epidermal differentiation, 2)much 16-kDa lectin is produced and secreted from the Vitamin A-pretreated culture epidermis, while 14-kDa lectin disappears, and 3)the gene expression of 14- and 16-kDa lectins is regulated independently.
2)The production of extracellular matrix components such as laminin, Type IV collagen, fibronectin, and tenascin during the formation of basement membrane in cultured epidermis-dermis recombinant skin of 13-day-old chick embryo was analyzed immunohistochemically. The epidermis and dermis were separated from each other by treatment with EDTA and/or dispase. The basal lamina of the basement membrane was thus removed from both epidermis and dermis. The isolated epidermis was overlaid onto the isolated dermis, i.e., recombined, and then cultured for 1-7 days in a chemically defined medium (BGJb) on a Millipore filter. Immunofluorescence labeling was used for light microscopy and HRP or colloidal gold labeling for electron microscopy. In specimens from 2-day cultures, positive sites of anti-laminin and anti-fibronectin reaction were observed light microscopically as patches which, at the electron microscopic level, corresponded to fragments of the basal lamina located immediately beneath and in the vicinity of the attachment plaques of the hemidesmosomes. The staining pattern became continuous 7 days after recombination. Fluorescence labeling of laminin and fibronectin appeared somewhat earlier than that of Type IV collagen and tenascin. All of the four components were found localized primarily in the basal lamina. Furthermore, fibronectin and tenascin were also distributed in the extracellular matrix of the dermis. The expression of tenascin, which does not exist in the basement membrane of 13-day-old intact embryonic skin, was induced in vitro. These results suggest that hemidesmosomes may play an important role in the reconstruction of the basement membrane appeared at different times during the reconstruction.
3)Recently, it has been revealed that changes in carbohydrate structures of glycolipids and glycoproteins constituting the plasma membrane occur in association with canceration of the cell. GAT (galactosyltransferase associated with tumor) is one of the glycosyltransferases which are regarded to be responsible for malignant changes. Differences were detected in ultrastructural location of GAT by light and electron microscopy in the normal and malignant endometrium.
4)Glucose transporters are integral membrane proteins that transport glucose across the plasma membrane. At least five isoforms of facilitated diffusion glucose transporter (GLUT1-5) have been identified in mammalian cells. Using isoform-specific antibodies, we determined their localization using immunofluorescence and immunogold staining of frozen sections. GLUT1 was found at the plasma membrane in various cells, while GLUT4, an isoform expressed in insulin sensitive cells, was found intracellularly in skeletal muscle cells in the absence of insulin. Ultracytochemical examination revealed that GLUT4 was localized in the trans side of the Golgi apparatus and vesicles and tubules near the plasma membrane. Insulin treatment induced the translocation of GLUT4 to the plasma membrane. To see whether their different intracellular localization is dependent on the cell types that express these glucose transporter isoforms, or on the isoform molecules themselves, GLUT1 and GLUT4 were stably expressed in Chinese hamster ovary cells by the transfection of their cDNAs. GLUT1 was localized at the plasma membrane, while GLUT4 remained intracellularly in the trans-Golgiarea as well as vesicles and tubules, suggesting that each isoform has a signal to determine its cellular localization. To elucidate these differential intracellular targeting mechanisms, several chimeric glucose transporters in which portions of GLUT4 are replaced with corresponding portions of GLUT1, are expressed in Chinese hamster ovary cells. Two domains of GLUT4 seems to determine its targeting to the cytoplasmic vesicles. One is the second transmembrane domain, which contains the consensus sequence of leucine zipper structure. The other is the portion including the eighth transmembrane domain. These domains would be specifically recognized by the intracellular targeting mechanism, resulting the differential localization of the isoforms. Less
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