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Neurocalcin; its un unique expression and a possible role in the Ca2+-signal transduction system in the neural cells
$4-A-1-04 NEUROCALCIN; TRANSDUCTION
ITS UN UNIQUE EXPRESSION AND A POSSIBLE SYSTEM IN THE NEURAL CELLS.
K. Okazaki 1, S, Iino 2, S, Kobayashi 2 and H. Hidaka 1 Department of Pharmacology 1 and Anatomy 2, Nagoya University, School of Nagoya 466, Japan.
R O L E in t h e C a 2 + - S I G N A L
Medicine, Tsurumai 65, Showa-ku,
INTRODUCTION: Neurocalcin is a 3EF-hand calcium-binding protein abundant in the central nervous system(I). Neurocalcin was purified(2), cloned and sequenced(3) from bovine brain. Six isoforms were identified and have a strong homology with recoverin, which is expressed in the photoreceptor cells in retina. Neurocalcin was also expressed in retina, but the distribution is different from that of recoverin(4,5). To elucidate the role of neurocalcin in nervous system, we examined the distribution of this calcium-binding protein in rat various tissues and searched for the target protein of expressed neurocalcin. MATERIAL AND METHODS: Native neurocalcin was purified from bovine brain using W-77 affinity column in a calcium-dependent manner. Expressed neurocalcin was purified from MBP-neurocalcin fusion protein. Neurocalcin-affinity column was prepared by coupling expressed neurocalcin with CN-Br activated sepharose 4B. Immunohistochemistry was perofrmed by using a ABC kit. Expressed neurocalcn was labelled with 125I using a Bolton-Hunter kit. RESULTS: Native neurocalcin immunoreactivity was found in the rat central nervous system and in the sensory organs(6). The ganglion neurons were stained strongly. To the other hand, expressed neurocalcin isoform was found mainly in the glial cells. In neuroealcin-affinity chromatography, several proteins were eluted in a calcium-dependent manner. In a gel over lay analysis using 125I-expressed neurocalcin, two major bands(36kDa and 10kDa) could bind to neurocalcin in a Ca2+-dependent manner. DISCUSSION: This report demonstrated the differential distribution of neurocalcin isoforms in the sensory nerve system. We also found the existence of target proteins. We concluded that Neurocalcin isoforms show different distributions in the rat spinal cord, DRG and other peripheral sensory nerves, hence one may play a distinct role in the Ca2+- signal transduction in sensory systems. We are now investigating the role of neurocalcn isoform, includng the details of their target proteins, in Ca2+ signal tarnsduction system. REFERENCES: 1.Hidaka, H. and Okazaki, K. (1993) Neurosci. Res., 16, 73-77. 2.Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. (1992) J. Biol. Chem., 267, 19596-19599. 3.Okazaki, K., Watanabe, M., Ando, Y., Hagiwara, M., Terasawa, M., and Hidaka, H. (1992) Biochem. Biophys. Res. Commun., 185, 147-153. 4.Nakano, A., Terasawa, M., Watanabe, M., Usuda, N., Morita, T., and Hidaka, H. (1992) Biochem. Biophys. Res. Commun., 186,1207-1211 5.Nakano, A., Terasawa, M., Watanabe, Okazaki, K., Inoue, S., Kato, M., Nirura, Y., Usuda, N., Morita, T., and Hidaka, H. (1993) J. Endocrinol., 138,283-290. 6.Bastianelli, E., Okazaki, K., Hidaka, H. and Pochet, R. Neurosci. Lett.(1993) 161,165-168
S4-BI-I-01
ROLES OF B CELL ACCESSORY MOLECULES, CD40 AND CD23, IN NORMAL AND ABNORMAL IMMUNE RESPONSES. HltOShl Klkutanl Instltute for Molecular and Cellular Blology, Osaka Unlverslty, Osaka, Japan Immune responses are regulated not only by various soluble mediators such as cytokines but also by direct interaction between immune competent cells via adhesion and accessory molecules. For instance, physical interaction between T and B cells are necessary for determination of specificities of humoral immune responses and for providing costimulatory signals to both cells. Some of B cell antigens such as CD40 and CD23 are known to be involved in T and B cell interaction. The CD40 antigen is a glycoprotein expressed on B cells, follicular dendritic cells and some epithelial cells. Its ligand (CD40L) is expressed on activated T cells. An engagement of CD40 with anti-CD40 or CD40L is known to provide a costimulatory signal for proliferation and differentiation of B cells in vitro. Recently, mutations of CD40L have been shown to be responsible for X-linked hyper IgM syndrome, suggesting an importance of the CD40-CD40L system in in vivo immune responses. The CD23 antigen is a low affinity Fc receptor for IgE. A number of functions have been ascribed to CD23, including regulation of IgE production, IgE-mecliatedcytotoxicity and mediator release, IgE-dependent antigen focusing and promotion of B cell growth. To evaluate in vivo functions of CD40 and CD23, CD40- and CD23deficient mice were produced. The CEHO mutant mice failed to mount IgG, IgA and IgE responses to thymus-dependent (TD) antigens, although IgM responses to TD antigens and IgG and IgM responses to thymus independent antigens were normal. Furthermore, the germinal center formation was defective in CD40 mutant mice. The results suggest that CD40 is essential for T-dependent class switching and germinal center formation. The CD23-deficient mice could mount normal antibody responses including IgE responses to TD antigens and Nippostrongyrus brasiliensis infection. However, antigen-specifio IgE mediated enhancement of antibody responses was severely impaired in CD23-deficient mice. These findings demonstrate that beth CD40 and CD23 play important roles in establishment of effective humoral immune responses and that these mice can be animal models suitable for studies of dysfunction of CD40 and CD23 in immunological disorders. It will also be discussed how CD40 and CD23 are involved in viral and bacterial infection, autoimmune diseases and allergic diseases.
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ITS UN UNIQUE EXPRESSION AND A POSSIBLE SYSTEM IN THE NEURAL CELLS.
K. Okazaki 1, S, Iino 2, S, Kobayashi 2 and H. Hidaka 1 Department of Pharmacology 1 and Anatomy 2, Nagoya University, School of Nagoya 466, Japan.
R O L E in t h e C a 2 + - S I G N A L
Medicine, Tsurumai 65, Showa-ku,
INTRODUCTION: Neurocalcin is a 3EF-hand calcium-binding protein abundant in the central nervous system(I). Neurocalcin was purified(2), cloned and sequenced(3) from bovine brain. Six isoforms were identified and have a strong homology with recoverin, which is expressed in the photoreceptor cells in retina. Neurocalcin was also expressed in retina, but the distribution is different from that of recoverin(4,5). To elucidate the role of neurocalcin in nervous system, we examined the distribution of this calcium-binding protein in rat various tissues and searched for the target protein of expressed neurocalcin. MATERIAL AND METHODS: Native neurocalcin was purified from bovine brain using W-77 affinity column in a calcium-dependent manner. Expressed neurocalcin was purified from MBP-neurocalcin fusion protein. Neurocalcin-affinity column was prepared by coupling expressed neurocalcin with CN-Br activated sepharose 4B. Immunohistochemistry was perofrmed by using a ABC kit. Expressed neurocalcn was labelled with 125I using a Bolton-Hunter kit. RESULTS: Native neurocalcin immunoreactivity was found in the rat central nervous system and in the sensory organs(6). The ganglion neurons were stained strongly. To the other hand, expressed neurocalcin isoform was found mainly in the glial cells. In neuroealcin-affinity chromatography, several proteins were eluted in a calcium-dependent manner. In a gel over lay analysis using 125I-expressed neurocalcin, two major bands(36kDa and 10kDa) could bind to neurocalcin in a Ca2+-dependent manner. DISCUSSION: This report demonstrated the differential distribution of neurocalcin isoforms in the sensory nerve system. We also found the existence of target proteins. We concluded that Neurocalcin isoforms show different distributions in the rat spinal cord, DRG and other peripheral sensory nerves, hence one may play a distinct role in the Ca2+- signal transduction in sensory systems. We are now investigating the role of neurocalcn isoform, includng the details of their target proteins, in Ca2+ signal tarnsduction system. REFERENCES: 1.Hidaka, H. and Okazaki, K. (1993) Neurosci. Res., 16, 73-77. 2.Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. (1992) J. Biol. Chem., 267, 19596-19599. 3.Okazaki, K., Watanabe, M., Ando, Y., Hagiwara, M., Terasawa, M., and Hidaka, H. (1992) Biochem. Biophys. Res. Commun., 185, 147-153. 4.Nakano, A., Terasawa, M., Watanabe, M., Usuda, N., Morita, T., and Hidaka, H. (1992) Biochem. Biophys. Res. Commun., 186,1207-1211 5.Nakano, A., Terasawa, M., Watanabe, Okazaki, K., Inoue, S., Kato, M., Nirura, Y., Usuda, N., Morita, T., and Hidaka, H. (1993) J. Endocrinol., 138,283-290. 6.Bastianelli, E., Okazaki, K., Hidaka, H. and Pochet, R. Neurosci. Lett.(1993) 161,165-168
S4-BI-I-01
ROLES OF B CELL ACCESSORY MOLECULES, CD40 AND CD23, IN NORMAL AND ABNORMAL IMMUNE RESPONSES. HltOShl Klkutanl Instltute for Molecular and Cellular Blology, Osaka Unlverslty, Osaka, Japan Immune responses are regulated not only by various soluble mediators such as cytokines but also by direct interaction between immune competent cells via adhesion and accessory molecules. For instance, physical interaction between T and B cells are necessary for determination of specificities of humoral immune responses and for providing costimulatory signals to both cells. Some of B cell antigens such as CD40 and CD23 are known to be involved in T and B cell interaction. The CD40 antigen is a glycoprotein expressed on B cells, follicular dendritic cells and some epithelial cells. Its ligand (CD40L) is expressed on activated T cells. An engagement of CD40 with anti-CD40 or CD40L is known to provide a costimulatory signal for proliferation and differentiation of B cells in vitro. Recently, mutations of CD40L have been shown to be responsible for X-linked hyper IgM syndrome, suggesting an importance of the CD40-CD40L system in in vivo immune responses. The CD23 antigen is a low affinity Fc receptor for IgE. A number of functions have been ascribed to CD23, including regulation of IgE production, IgE-mecliatedcytotoxicity and mediator release, IgE-dependent antigen focusing and promotion of B cell growth. To evaluate in vivo functions of CD40 and CD23, CD40- and CD23deficient mice were produced. The CEHO mutant mice failed to mount IgG, IgA and IgE responses to thymus-dependent (TD) antigens, although IgM responses to TD antigens and IgG and IgM responses to thymus independent antigens were normal. Furthermore, the germinal center formation was defective in CD40 mutant mice. The results suggest that CD40 is essential for T-dependent class switching and germinal center formation. The CD23-deficient mice could mount normal antibody responses including IgE responses to TD antigens and Nippostrongyrus brasiliensis infection. However, antigen-specifio IgE mediated enhancement of antibody responses was severely impaired in CD23-deficient mice. These findings demonstrate that beth CD40 and CD23 play important roles in establishment of effective humoral immune responses and that these mice can be animal models suitable for studies of dysfunction of CD40 and CD23 in immunological disorders. It will also be discussed how CD40 and CD23 are involved in viral and bacterial infection, autoimmune diseases and allergic diseases.
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