The ryanodine receptor type 3 (RyR-3) functions as a Ca 2؉-induced Ca 2؉ release (CICR) channel and is distributed in a wide variety of cell types including skeletal muscle and smooth muscle cells, neurons, and certain non-excitable cells. However, the physiological roles of RyR-3 are totally unclear. To gain an insight into the function of RyR-3 in vivo, we have generated mice lacking RyR-3 by means of the gene targeting technique. The mutant mice thus obtained showed apparently normal growth and reproduction. Although Ca 2؉ -induced Ca 2؉ release from intracellular Ca 2؉ stores of the mutant skeletal muscle differed in Ca 2؉ sensitivity from that of wild-type muscle, excitation-contraction coupling of the mutant muscle seemed to be normal. Moreover, we could not find any significant disturbance in the smooth muscle and lymphocytes from the mutant mice. On the other hand, the mutant mice showed increased locomotor activity, which was about 2-fold greater than that of the control mice. These results indicate that the loss of RyR-3 causes no gross abnormalities and suggest that the lack of RyR-3-mediated Ca 2؉ signaling results in abnormalities of certain neurons in the central nervous system.

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P ؊/؊ mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.Glycosylation is a major post-translational protein modification, especially for cell surface proteins, which play important roles in a variety of cellular functions including recognition and adhesion. In the last decade, a number of glycosyltransferase genes and related genes have been cloned. Targeted deletion of these genes revealed the roles of cell surface glycans in the modulation of cellular interactions, particularly in the immune system (1, 2). We have been interested in the roles of a neuralspecific carbohydrate, the HNK-1 carbohydrate, which is expressed on glycoproteins as well as on glycolipids and is postulated to be associated with cell adhesion, migration, and neurite outgrowth (3-5). The epitope is a sulfated trisaccharide, HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc (6, 7), and the inner structure, Gal␤1-4GlcNAc, is commonly found on various glycoproteins and glycolipids. To elucidate the roles of the HNK-1 carbohydrate more clearly, we cloned two different glucuronyltransferases (GlcAT-P and GlcAT-S) 1 (8 -11), which are key enzymes in the biosynthesis of the HNK-1 carbohydrate epitope (12,13). In this study, we generated mice with a targeted deletion of the GlcAT-P gene and demonstrated clearly that the HNK-1 carbohydrate is in fact required for higher functions of the brain. EXPERIMENTAL PROCEDURESTargeted Disruption of the GlcAT-P Gene-Cloning of the genomic clones of the 129/Sv mouse GlcAT-P gene was described previously (10). Construction of the targeting vector is schematically represented in Fig. 1A. The neomycin resistance gene cassette in vector pPGKneobpA (14) and diphtheria toxin A (DT-A) gene cassette in vector pMC1DT-A (15) were used as positive and a negative selection markers, respectively. The targeting vector was transfected into E14-1 embryonic stem (ES) cells (16) by electroporation. Two ES clones among 525 tested revealed the desired homologous recombination (13C-2 and 22D-5). To generate chimeric mice, both clones were aggregated with C57BL/6 ϫ BDF1 8-cell-stage embryos, and the embryos were transferred into the uteri of pseudopregnant mice. Both clones gave rise to germline chimeras. Mice heterozygous for the mutation were obtained by cross-breeding of the chimeras with C57BL/6 mice. The heterozygotes were further backcrossed with C57BL/6 mice for more than eight generations, and the resulting heterozygous mutants were interbred ...

In skeletal muscle, excitation-contraction (E-C) coupling requires the conversion of the depolarization signal of the invaginated surface membrane, namely the transverse (T-) tubule, to Ca2+ release from the sarcoplasmic reticulum (SR). Signal transduction occurs at the junctional complex between the T-tubule and SR, designated as the triad junction, which contains two components essential for E-C coupling, namely the dihydropyridine receptor as the T-tubular voltage sensor and the ryanodine receptor as the SR Ca2+-release channel. However, functional expression of the two receptors seemed to constitute neither the signal-transduction system nor the junction between the surface and intracellular membranes in cultured cells, suggesting that some as-yet-unidentified molecules participate in both the machinery. In addition, the molecular basis of the formation of the triad junction is totally unknown. It is therefore important to examine the components localized to the triad junction. Here we report the identification using monoclonal antibody and primary structure by cDNA cloning of mitsugumin29, a novel transmembrane protein from the triad junction in skeletal muscle. This protein is homologous in amino acid sequence and shares characteristic structural features with the members of the synaptophysin family. The subcellular distribution and protein structure suggest that mitsugumin29 is involved in communication between the T-tubular and junctional SR membranes.

We attempted to determine whether cell adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin (endothelial-leukocyte adhesion molecule-1; ELAM-1), are involved in the lymphoid cell infiltration of the salivary and lacrimal glands in Sjogren's syndrome (SS) patients. Both immunohistochemical analysis and the reverse-transcripts polymerase chain reaction (RT-PCR) were used to analyze the expression of VCAM-1, ICAM-1, ELAM-1, very late antigen 4 (VLA-4 [alpha 4,beta 1]), lymphocyte function-associated antigen-1 (LFA-1), interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), and interleukin-1 beta (IL-1 beta). Immunohistochemical analysis of salivary gland biopsies from SS patients showed a marked expression of VCAM-1 and ICAM-1 in the venules surrounded by infiltrated CD4+ CD45RO+ T cells. E-selectin was expressed on vascular endothelium with weak intensity. Increased levels of VCAM-1, ICAM-1, IFN-gamma, and IL-1 beta mRNA were demonstrated by RT-PCR, whereas E-selectin mRNA were weakly expressed in SS lacrimal and salivary gland tissues. This is in contrast with strong expression of ELAM-1 in IL-1 beta-stimulated human umbilical vascular endothelial cells (HUVEC) in vitro. Cytokine-mediated up-regulation of VCAM-1 and ICAM-1 that facilitates the recruitment of VLA-4 and LFA-1 expressing T cells might contribute to lymphoid cell infiltration in the salivary and lacrimal glands in SS.