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High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet‐derived growth factor B‐driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma‐bearing mice and low‐grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S‐deficient gliomas. Re‐expression of GD3S restored expression of MMP9 in primary‐cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α‐MMP9 axis. This is the first report to describe the tumor‐enhancing functions of GD3S in vivo., Genetically engineered mice were used for evaluation of ganglioside GD3 synthase in murine gliomas. Deficiency of GD3 synthase resulted in prolonged life‐span of glioma‐bearing mice and low grade pathology in generated gliomas. Reduced activation of signaling molecules and MMP proteins in the KO mice has been described.

Loading is indispensable for the growth, development, and maintenance of joint tissues, including mandibular condylar cartilage, but excessive loading or reduced host adaptive capacity can considerably damage the temporomandibular joint (TMJ), leading to temporomandibular joint osteoarthritis (TMJ-OA). TMJ-OA, associated with other pathological conditions and aging processes, is a highly degenerative disease affecting the articular cartilage. Many treatment modalities for TMJ-OA have been developed. Traditional clinical treatment includes mainly nonsurgical options, such as occlusal splints. However, non-invasive therapy does not achieve joint tissue repair and regeneration. Growing evidence suggests that low-intensity pulsed ultrasound (LIPUS) accelerates bone fracture healing and regeneration, as well as having extraordinary effects in terms of soft tissue repair and regeneration. The latter have received much attention, and various studies have been performed to evaluate the potential role of LIPUS in tissue regeneration including that applied to articular cartilage. The present article provides an overview of the status of LIPUS stimulation used to prevent the onset and progression of TMJ-OA and enhance the tissue regeneration of mandibular condylar cartilage. The etiology and management of TMJ-OA are explained briefly, animal models of TMJ-OA are described, and the effectiveness of LIPUS on cell metabolism and tissue regeneration in the TMJ is discussed.

Summary Objective Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease characterized by progressive cartilage degeneration, abnormal bone remodeling, and chronic pain. In this study, we aimed to investigate effective therapies to reverse or suppress TMJOA progression. Design To this end, we performed intravenous administration of serum free conditioned media from human exfoliated deciduous teeth stem cells (SHED-CM) into a mechanical-stress induced murine TMJOA model. Results SHED-CM administration markedly suppressed temporal muscle inflammation, and improved bone integrity and surface smoothness of the destroyed condylar cartilage. Moreover, SHED-CM treatment decreased the number of IL-1β, iNOS, and MMP-13 expressing chondrocytes, whereas it specifically increased PCNA-positive cells in the multipotent polymorphic cell layer. Notably, the numbers of TdT-mediated dUTP nick end labeling (TUNEL)-positive apoptotic chondrocytes in the SHED-CM treated condyles were significantly lower than in those treated with DMEM, whereas the proteoglycan positive area was restored to a level similar to that of the sham treated group, demonstrating that SHED-CM treatment regenerated the mechanical-stress injured condylar cartilage and subchondral bone. Secretome analysis revealed that SHED-CM contained multiple therapeutic factors that act in osteochondral regeneration. Conclusions Our data demonstrated that SHED-CM treatment promoted the regeneration and repair of mechanical-stress induced mouse TMJOA. Our observations suggest that SHED-CM has potential to be a potent tissue-regenerating therapeutic agent for patients with severe TMJOA.

Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be "autologous typing" performed by Old's group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient's sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.

To analyze the binding specificity of a sialic acid–recognizing lectin, sialic acid-binding Ig-like lectin 7 (SIGLEC7), to disialyl gangliosides (GD3s), here we established GD3-expressing cells by introducing GD3 synthase (GD3S or ST8SIA1) cDNA into a colon cancer cell line, DLD-1, that expresses no ligands for the recombinant protein SIGLEC7-Fc. SIGLEC7-Fc did not recognize newly-expressed GD3 on DLD-1 cells, even though GD3 was highly expressed, as detected by an anti-GD3 antibody. Because milk-derived GD3 could be recognized by this fusion protein when incorporated onto the surface of DLD-1 cells, we compared the ceramides in DLD-1–generated and milk-derived GD3s to identify the SIGLEC7-specific GD3 structures on the cell membrane, revealing that SIGLEC7 recognizes only GD3-containing regular ceramides but not phytoceramides. This was confirmed by knockdown/knockout of the sphingolipid delta(4)-desaturase/C4-monooxygenase (DES2) gene, involved in phytoceramide synthesis, disclosing that DES2 inhibition confers SIGLEC7 binding. Furthermore, knocking out fatty acid 2-hydroxylase also resulted in the emergence of SIGLEC7 binding to the cell surface. To analyze the effects of binding between SIGLEC7 and various GD3 species on natural killer function, we investigated cytotoxicity of peripheral blood mononuclear cells from healthy donors toward GD3S-transfected DLD-1 (DLD-1–GD3S) cells and DLD-1–GD3S cells with modified ceramides. We found that cytotoxicity is suppressed in DLD-1–GD3S cells with dehydroxylated GD3s. These results indicate that the ceramide structures in glycosphingolipids affect SIGLEC7 binding and distribution on the cell surface and influence cell sensitivity to killing by SIGLEC7-expressing effector cells.

Cancer-associated glycosphingolipids have been used as markers for diagnosis and targets for immunotherapy of malignant tumors. Recent progress in the analysis of their implications in the malignant properties of cancer cells revealed that cancer-associated glycosphingolipids are not only tumor markers, but also functional molecules regulating various signals introduced by membrane microdomains, lipid rafts. In particular, a novel approach, enzyme-mediated activation of radical sources combined with mass spectrometry, has enabled us to clarify the mechanisms by which cancer-associated glycosphingolipids regulate cell signals based on the interaction with membrane molecules and formation of molecular complexes on the cell surface. Novel findings obtained from these approaches are now providing us with insights into the development of new anticancer therapies targeting membrane molecular complexes consisting of cancer-associated glycolipids and their associated membrane molecules. Thus, a new era of cancer-associated glycosphingolipids has now begun.

BackgroundAlthough recent studies have revealed the powerful antinociceptive effect of human dental pulp stem cells in an animal model for diabetes and osteoarthritis, its analgesic mechanisms are still largely elusive. We have previously reported that conditioned medium (CM) from dental pulp stem cells of deciduous teeth (SHED-CM) or its components, monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (sSiglec-9), directly induces anti-inflammatory M2 macrophages, however the antinociceptive activity of induced M2 is unknown. In this study, we investigated the antinociceptive effect of SHED-CM, MCP-1, and sSiglec-9 or secretome from M2-induced by SHED-CM (M2-CM) against neuropathic pain (NP) using a partial sciatic nerve ligation (PSL) mouse model and analyzed the mechanical bases of their antinociceptive effects. MethodsPSL mice were treated using SHED-CM with or without mannosylated-Clodrosome, specifically depleting M2 macrophages, recombinant MCP-1 and sSiglec-9 protein, M2-CM, or fibroblast-CM. Human Schwann cells activated by TNF-α in vitro were treated with M2-CM. The expression of pro-inflammatory mediators, neuroprotective factors, the nociceptive receptor, and markers for M1, M2, and activated glial cells in injured sciatic nerve (SCN), dorsal root ganglion, or spinal cord were evaluated by RT-PCR and immunohistochemistry. Mechanical allodynia of PSL mice was analyzed via von Frey test. ResultsIn the behavioral test, intravenous administration of SHED-CM greatly improved the PSL-induced hypersensitivity. SHED-CM treatment recruited M2 macrophages in the injured SCN and ipsilateral L4/L5 dorsal root ganglion and suppressed microglial activation in the spinal cord. Specific depletion of the M2 by mannosylated-Clodrosome markedly reduced the antinociceptive effect of SHED-CM. Intravenous administration of both MCP-1/sSiglec-9 and M2-CM ameliorated the PSL-induced hypersensitivity. We found that M2-CM directly suppressed the expression of nociceptive receptors as well as proinflammatory mediators in Schwann cells. ConclusionTaken together, our data suggest that SHED-CM ameliorates NP through the induction of the analgesic anti-inflammatory M2 macrophages. Thus, SHED-CM may present as a potential novel therapeutic candidate for NP.

Ganglioside GD2 is specifically expressed in small-cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme-mediated activation of radical sources combined with mass spectrometry in GD2+ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2+ SCLC cells were enhanced by glutamine uptake, and were suppressed by L-γ-glutamyl-p-nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.

Gene knockout mice of glycosyltransferases have clearly showed roles of their products in the bodies, while there are examples where phenotype of knockout was much less severe than expected probably due to functional redundancy. The most striking novel finding obtained from ganglioside-deficient mice was that progressive inflammatory reaction took place, leading to neurodegeneration. In particular, dysfunction of complement-regulatory proteins due to deteriorated architecture of lipid rafts seemed to be essential mechanisms for the inflammation. Furthermore, roles of gangliosides in neurons were demonstrated by neuron-specific transgenic of B4galnt1 with genetic background of B4galnt1 deficiency. From study of gene knockout mice of St8sia1, new roles of b-series gangliosides in leptin secretion from adipocytes, and roles of a-series gangliosides in leptin receptor, ObR in hypothalamus were demonstrated, leading to apparent intact balance of energy. Essential roles of b-series gangliosides in malignant properties of gliomas were also shown, suggesting their roles in the regulation of inflammation and proliferation in nervous tissues. How to apply these findings for the control of newly discovered patients with ganglioside deficiency remains to be investigated. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Acidic glycosphingolipids, i.e., gangliosides, are predominantly and consistently expressed in nervous tissues of vertebrates at high levels. Therefore, they are considered to be involved in the development and function of nervous systems. Recent studies involving genetic engineering of glycosyltransferase genes have revealed novel aspects of the roles of gangliosides in the regulation of nervous tissues. In this review, novel findings regarding ganglioside functions and their modes of action elucidated mainly by studies of gene knockout mice are summarized. In particular, the roles of gangliosides in the regulation of lipid rafts to maintain the integrity of nervous systems are reported with a focus on the roles in the regulation of neuro-inflammation and neurodegeneration via complement systems. In addition, recent advances in studies of congenital neurological disorders due to genetic mutations of ganglioside synthase genes and also in the techniques for the analysis of ganglioside functions are introduced.

Some gangliosides, sialic acid-containing glycosphingolipids, have been considered as tumor-associated antigens. GD1α or a GD1α synthase gene ST6GalNAc5 was reported to be involved in the metastasis of murine lymphomas or human breast cancers, respectively. But expression patterns of 0-series gangliosides GD1α and its precursor GM1b in human cancers have not yet been investigated mainly due to lack of specific antibodies. We established specific monoclonal antibodies (mAbs) reactive with GD1α or GM1b using gangliosides from brain tissues of GM3 synthase (St3gal5)-deficient mice as immunogens. We used GM2/GD2 synthase (B4galnt1)-deficient mice to immunize by liposomes embedded with GD1α or acidic glycolipid fractions from brain of St3gal5-deficient mice. Specificities of established mAbs as analyzed by enzyme-linked immunosorbent assay and thin-layer chromatography-immunostaining were very high among various gangliosides. Increased expression of GD1α and reduced GM1b in the St6galnac5 cDNA-transfected RAW117 cell line also substantiated the specificities of two mAbs. Then, we analyzed expression of GD1α and GM1b, and of relevant glycosyltransferase genes in various human cancer cell lines using generated anti-GD1α mAb 122 or anti-GM1b mAb MR155A-7. A few human cancer cell lines showed significant expression of these gangliosides with reasonable expression of relevant glycosyltransferase genes.

B4GALNT1 is an enzyme essential for the synthesis of complex gangliosides, whose absence leads to progressive neurodegeneration with aging in mice. Recently, eleven cases of hereditary spastic paraplegia with mutation in the coding region of B4GALNT1 were reported. However, changes in the enzymatic activity of their products have never been studied. We have constructed expression vectors for individual mutant cDNAs, and examined their activities by cell-free in vitro enzyme assays, and flow cytometry of cells transfected with their expression vectors. Among them, almost all mutant genes showed the complete loss of B4GALNT1 activity in both the in vitro enzyme assays and flow cytometry. Two mutants exceptionally showed weak activity. One of them, M4, had a mutation at amino acid 228 with a premature termination codon. Interestingly, the intensity of fluorescence of GM2 measured by flow cytometry was equivalent between the WT and M4 mutant, although the positive cell population was relatively small in M4. Western immunoblotting of cell lysates from transfectants with cDNA plasmids revealed 67-kDa bands except those containing premature termination codons or frame-shift mutation. Taken together with the clinical findings of patients, loss of enzyme activity may be responsible for the clinical features of hereditary spastic paraplegia, whereas the intensity of neurological disorders was relatively milder than expected. These clinical features of patients including those with male hypogonadism are very similar to the abnormal phenotypes detected in B4galnt1-deficient mice.

Recently, the combined use of multiple drugs for coexisting multiple diseases in elderly patients has become a problem. In facilities for elderly persons, pharmacists contribute to the intervention and optimization of prescriptions. However, the procedures have been conducted smoothly in only a few facilities. We established five procedures for prescription intervention by pharmacists and implemented these interventions in 80 institutionalized individuals. The total results over 2 years(January 1, 2015 to December 31, 2016)revealed 118 cases of prescription proposals from pharmacists and a reduction of 2,411,937 yen in medical expense. The purpose of prescription proposals from pharmacists is to reduce burden on patients by optimizing prescriptions, but not to reduce the number or dose of drug. In doing so, it was important to hear patients' opinions on medical care. Patient-centered prescription proposals from pharmacists are considered to have improved the quality of medical care and may enable the promotion of prescription intervention.

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.

There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.

Gangliosides play roles in the regulation of cell signaling that are mediated via membrane microdomains, lipid rafts. In this review, functions of gangliosides in the maintenance of nervous systems with a focus on regulation of inflammation and neurodegeneration are addressed. During analyses of various ganglioside-lacking mutant mice, we demonstrated that nervous tissues exhibited inflammatory reactions and subsequent neurodegeneration. Among inflammation-related genes, factors of the complement system showed up-regulation with aging. Analyses of architectures and compositions of lipid rafts in nervous tissues from these mutant mice revealed that dysfunctions of complement regulatory proteins based on disrupted lipid rafts were main factors to induce the inflammatory reactions resulting in neurodegeneration. Ganglioside changes in development and senescence, and implication of them in the integrity of cell membranes and cellular phenotypes in physiological and pathological conditions including Alzheimer disease have been summarized. Novel directions to further analyze mechanisms for ganglioside functions in membrane microdomains have been also addressed.

Although regulatory mechanisms for immune cells with inhibitory signals via immunoreceptor tyrosine-based inhibitory motifs are well known, signals transduced via interaction between Siglecs and sialyl compounds on their counterreceptors into target cells have not been reported to date. In this study, we found that an astrocytoma cell line, AS, showed detachment from culture plates when co-cultured with Siglec-9-expressing cells and/or soluble Siglec-9. Moreover, detached AS cells regrew as co-cultured cells with Siglec-9-deficient cells. They also showed increased motility and invasiveness upon Siglec-9 binding. In immunoblotting, rapid degradation of focal adhesion kinase (FAK) and related signaling molecules such as Akt, paxillin, and p130Cas was observed immediately after the co-culture. Despite degradation of these molecules, increased p-Akt was found at the front region of the cytoplasm, probably reflecting increased cell motility. Calpain was considered to be a responsible protease for the protein degradation by the inhibition experiments. These results suggest that protein degradation of FAK and related molecules was induced by Siglec-9 binding to its counterreceptors via sialylglycoconjugates, leading to the modulation of adhesion kinetics of cancer cells. Thus, this might be a mechanism by which cancer cells utilize Siglec-9-derived signals to escape from immunosurveillance.

Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, β1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of β4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that β4GalT6 plays a role as a LacCer synthase, whereas β4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.

PCBは有害物質であり，国内では2016年までの処理が法律で義務づけられている。本研究では，高濃度PCBに汚染されたコンデンサおよび高圧トランスからのPCB溶出実験と，マイクロ波・触媒反応による脱塩素実験を同時に行った。一方，内部部材内でのPCBの拡散方程式と，溶出したPCBの脱塩素反応についての反応速度式を同時に解き，この手法が実験で計測されたPCB濃度の経時変化を定量的に説明することができるか検討した。室内で行ったコンデンサ無害化試験においてはPCB濃度の計算値は実測値とよく一致したことから，今回のシミュレーションがPCB濃度の予測に有効と考えられた。一方，外気温に左右される倉庫で行われた高圧トランス無害化試験においては，反応速度定数を反応時間帯において変化させないと，計算値は実測値と一致せず，その原因の一つとして溶液温度が下がった場合にKOHが析出し不足することによる反応速度の低下などが考えられた。

Inositol 1,4,5-trisphosphate (IP(3)) is an intracellular messenger that elicits a wide range of spatial and temporal Ca(2+) signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP(3) biosensors that exhibit strong pH stability and varying affinities for IP(3), as well as a method for the quantitative measurement of cytosolic concentrations of IP(3) ([IP(3)](i)) in single living cells. We applied this method to elucidate IP(3) dynamics during agonist-induced Ca(2+) oscillations, and we demonstrated cell type-dependent differences in IP(3) dynamics, a nonfluctuating rise in [IP(3)](i) and repetitive IP(3) spikes during Ca(2+) oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP(3) spikes in HSY-EA1 cells varied from 10 to 100 nm, and the [IP(3)](i) spike peak was preceded by a Ca(2+) spike peak. These results suggest that repetitive IP(3) spikes in HSY-EA1 cells are passive reflections of Ca(2+) oscillations, and are unlikely to be essential for driving Ca(2+) oscillations. In addition, the interspike periods of Ca(2+) oscillations that occurred during the slow rise in [IP(3)](i) were not shortened by the rise in [IP(3)](i), indicating that IP(3)-dependent and -independent mechanisms may regulate the frequency of Ca(2+) oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP(3) and Ca(2+).

SUMMARYThe ciliated protozoan Paramecium spontaneously changes its swimming direction in the absence of external stimuli. Such behavior is based on resting potential fluctuations, the amplitudes of which reach a few mV. When the resting potential fluctuation is positive and large, a spike-like depolarization is frequently elicited that reverses the beating of the cilia associated with directional changes during swimming. We aimed to study how the resting potential fluctuation is amplified. Simultaneous measurements of the resting potential and intracellular Ca2+([Ca2+]i) from a deciliated cell showed that positive potential fluctuations were frequently accompanied by a small increase in[Ca2+]i. This result suggests that Ca2+influx through the somatic membrane occurs during the resting state. The mean amplitude of the resting potential fluctuation was largely decreased by either an intracellular injection of a calcium chelater (BAPTA) or by an extracellular addition of Ba2+. Hence, a small increase in[Ca2+]i amplifies the resting potential fluctuation. Simulation analysis of the potential fluctuation was made by assuming that Ca2+ and K+ channels of surface membrane are fluctuating between open and closed states. The simulated fluctuation increased to exhibit almost the same amplitude as the measured fluctuation using the assumption that a small Ca2+ influx activates Ca2+ channels in a positive feedback manner.

Using mouse and human monoclonal antibodies, a number of cancer-associated antigens were defined, and many of their epitopes have turned out to be carbohydrates. Besides as a tumor marker, some of them really play roles as regulatory molecules on the cell membrane. Generally, disialyl glycosphingolipids (disialyl gangliosides) such as GD3 and GD2 transduce activating signals to enhance malignant properties of cancer cells, while monosialyl gangliosides such as GM1 transduce suppressive signals on cancer properties. In particular, roles of GD3 in melanomas and GD2 in small cell lung cancers, osteosarcomas, and breast cancers have been well investigated. Recently, roles of GM1 in the suppression of cancer metastasis have been demonstrated using a murine lung cancer system. These regulatory functions of cancer-associated glycolipids are performed at membrane microdomains named glycolipid-enriched microdomain (GEM)/rafts. As mechanisms for these regulatory functions, molecular interactions between glycolipids and various membrane proteins existing in the vicinity of them at GEM/rafts are of importance and now under vigorous investigation.

To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.

Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophagesin vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.

Gamma nailing is in principle performed for fractures proximal to the femur at our hospital. We conducted intraoperative and postoperative investigations in 97 cases who had undergone gamma nailing, and studied 86 cases for which follow-up data were available. This paper is a report of our findings.Complications were found in 2.3% intraoperatively and in 5.8% postoperatively.Studies of indications for surgery with consideration given to the medullary space diameter, complete reduction of the fractured region, skill in surgical techniques and regular postoperative follow-up are considered important as measures to deal with complications.

Of 213 patients who were treated for femoral neck fracture at our hospital 153 patients could be followed up. We investigated their condition and walking ability after discharge, and studied the ideal liaison medical service for this fracture. Twenty percent of these patients were sent home after discharg while the remaining 80 percent changed to another hospital. This shows the importance of liaison medical service, including welfare, for this fracture. Our department has prepared a critical path and is using it to dissipate anxiety and dissatisfaction of patients' families over the liaison medical service, and also to maintain the quality of medical care at hospitals receiving these patients. We plan to prepare a critical path incorporating the opinions of liaison hospitals for the overall improvement of the liaison medical service. In this paper, we report on our methods for carrying out the liaison medical service efficiently.

Moderately loaded CWM started in the USSR in the 1950's as a means of transporting coal, and there is one operating long distance coal-slurry pipeline at present in the United States. Highly loaded CWM based on bituminous coal was developed in Asia. Europe and the United States after the second oil shock in 1978 and it is now ready for commercial use. Because of the stabilization of oil prices at low levels after the reverse oil shock in 1986, the CWM market has been retarded. High concentration slurries of other types of coal, such as sub-bituminous coal, brown coal and petroleum coke are still under development and in the demonstration stage in Australia, Japan and the United States.

Orthognathic surgery was performed on 362 patients from 1987 to 1997 at the Second Department of Oral and Maxillofacial Surgery, Osaka Dental University.The results were as follows: 1. The male-female ratio was 1: 2.4.2. The mean age was 22.4 years old.3. The diagnosis of mandibular prognathism was made for most of the patients (89.2%).4. Sagittal splitting ramus osteotomy (SSRO) was performed on 274 cases (75.7%).5. The mean time of operation for SSRO was 3hr. 7 min±57min, and for mandibular body osteotomy was 3hr. 15min±60.7min.6. The mean blood loss during operation for SSRO was 275.8±221.4ml and for mandibular body osteotomy was 171.8±147.5ml.7. Paresthesia of the lip and mental area was observed in 404 sides after opration. 80.7% of patients treated for SSRO recovered within a year.8. TMJ were examined in 202 cases for 1 year after SSRO. The symptom of TMJ was recognized in 5 cases.

Titanium oxide powder and thin films were prepared from an (NH4)2TiF6 aqueous solution upon addition of boric acid in a process denoted as liquid phase deposition (LPD). In this process, titanium oxide powder and films were formed by the chemical equilibrium reaction between titanium fluoro-complex ions and metal oxide in the aqueous solution. The prepared powder and deposited films showed some features which differed from titanium oxide prepared from titanium alkoxide. The prepared powder and the deposited films included anatase (TiO2) and showed photocatalytic activity without prior calcination. The decomposition rate of CH3CHO reached a maximum when the powder or films were calcined at 300 °C. The LPD process enabled a uniform layer of TiO2 film to form homogeneously on the entire surface of glass wool. The prepared TiO2 contained fluorine and nitrogen. The amount of these impurities was reduced upon calcination.

The activity indices of the masticatory and temporal muscles and the maxillofacial morphologies in patients with skeletal mandibular prognathism at the initial examination were compared with those in individuals with normal occlusion. The results were as follows:1. The muscle activity index was significantly lower in the prognathic patients than in the normal individuals.2. The items of maxillofacial morphometry that correlated with the muscle activity index were fewer and weaker in the prognathic patients than in the normal individuals.These findings suggested that patients with skeletal mandibular prognathism have functional and morphological disturbances.

The observed rapidity distribution of particles from diffractive state X in \=pp→\=pX is asymmetric with respect to the kinematic central value \(\bar{y}=\ln(\sqrt{s}/M)\) of the system X, where \(\sqrt{s}\) and M are c.m.s. energy and invariant mass of X, respectively. In the framework of the geometric quark jet model, this asymmetry suggests existence of a leading particle in fragmentation of diffractively excited states.

The purpose of this study was to clarify the effect of long-term exercise on the maintenance of concentration, as an indicator of the activity level of the cerebral cortex, and on the Profile of Mood States (POMS) in healthy elderly persons. Forty-six subjects were divided into three groups: a long-term exercise group (LTE-G) of 21 subjects (mean age: 67 years); a health exercise group (HE-G) of 12 subjects (mean age: 69 years); and a sedentary control group (SC-G) of 13 subjects (mean age: 70 years). We assessed the target aiming function (TAF), the POMS, and the presence in the urine of the following compounds: 8-hydroxydeoxyguanosine (8-OHdG) and acrolein (ACR) as indicators of oxidative stress; adrenaline, noradrenaline, and dopamine as indicators of sympathetic nervous activity. The mean TAF-L values, which represent the level of mental concentration, were statistically lower in the LTE-G and HE-G than in the SC-G. The mean TAF-D values, indicating deviation levels, were significantly lower in the HE-G than in the SC-G. Total Mood Disturbance (TMD) scores, which are the scored totals of POMS tests, and the scores on the "Confusion" factor were clearly lower in the LTE-G and HE-G than in the SC-G. On the other hand, there were no significant differences in 8-OHdG, ACR, adrenaline, noradrenaline or dopamine values among the three groups. These results suggest that the continuation of exercise improves the cerebral cortical activity, the maintenance of concentration, and the POMS scores of healthy elderly persons.

Determining the oxygenation level of skeletal muscle is an effective non-invasive method of evaluating its physiological changes. Nevertheless, the relationship between muscle fiber composition and intramuscular oxygenation is still unclear. In the present study we examined the differences in muscle fiber composition and their relationship to muscle oxygenation levels by comparing power lifters (Training group), who regularly engaged in muscular resistance training, with ordinary healthy people (Control group). The vastus lateralis muscle oxygenation level was measured via near infrared continuous wave spectroscopy, and muscle tissues were collected and used for histochemical analyses in order to calculate muscle fiber compositions. When the rate of decrease in muscle oxygenation after performing a single squat at 50 and 80% of the participants’ maximal lifting weight (MLW) was examined, the rate of decrease was significantly higher at 80% of MLW than at 50% of MLW in Training group, and was significantly higher in the Training group than in the Control group. Moreover, after performing multiple squat movements at 80% of MLW, compared with the Control group, the Training group showed a significant delay in the time it took for their muscle oxygenation level to recover to 50% of its original level at resting time (T1/2). A significant correlation between the occupancy of Type IIa fiber and T1/2, or between an average cross-sectional area of muscle fibers and T1/2 was noted. The present study demonstrated that in the Training group, in a cross-sectional area of Type IIa fiber that had increased, the decreased muscle oxygenation level due to performing squat exercises exhibited a delay in recovery at the resting time, suggesting that the amount of oxygen consumption was increased in the Training group because the crosssectional area of Type IIa fiber was larger, compared with the Control group.

We study the multiplicity distributions both in non-single diffractive and whole inelastic pp(\=pp) interactions in the framework of the geometric quark jet model. It is shown that the model reproduces successfully the approximate KNO scaling of multiplicity distributions at ISR and scaling violation at SPS collider energies, if the average internal multiplicity in the impact parameter space is suitably chosen. For multiplicity distributions in the rapidity interval | y

A clinical statistical study was carried out on surgical correction of jaw deformation in patients who visited Osaka Dental University.1) The annual number of the new patients with jaw deformation slightly increased. A marked increase was observed after 1990.2) The male/female ratio in the new patients was about 1: 2.3) The mean age at the time of initial consultation was 20.4 years for males and 20.3 years for females.4) According to age groups, the number of patients aged 16-20 years accounted for 47.6% and that of patients aged 21-25 years for 30% of all patients.5) Concerning area distribution in the new patients, Osaka prefecture accounted for the highest percentage (71%), followed in order by Hyogo, Nara, and Oyoto prefectures.6) According to the type of jaw deformation, skeletal mandibular protrusion accounted for the highest percentage (84%).

Expression and implication of carbohydrate antigens in squamous cell carcinomas (SCCs) in oral cavity was examined. In the cell lines, type 2H and Lewis y antigens were markedly expressed. In the tissues from SCC patients and benign disorders, type 2H was highly expressed in hyperplasia (96.4 %), displasia (92.9 %) and SCC (100 %). Lewis y was, in turn, expressed mainly in cancer tissues (91.3 %), suggesting that Lewis y is a cancer-associated antigen. Normal oral mucosa showed no expression of these blood group antigens. Surprisingly, Lewis y antigen disappeared in the invasion sites where Ki-67 was definitely stained. Over-expression of Lewis y with manipulation of a fucosyltransferase cDNA resulted in suppression of cell growth and invasion, and knockdown of Lewis y also brought about increased cell growth and invasion. In either situations, no changes in the expression of sialyl-Lewis x could be found. Lowered tumor growth and invasion into surrounding tissues were also shown in Lewis y-positive SCC grafts in nu/nu mice. All these results together with alternative staining between Lewis y and Ki-67 in cancer tissues and FUT1 transfectants suggested that loss of Lewis y is a crucial event for the late stage of SCCs.

In order to identify membrane proteins whose expression is induced by X-ray irradiation, we developed an antibody (Ab)-directed strategy using a phage Ab library. X-Ray-irradiated cells were screened with a phage Ab library in the presence of a large excess of polyclonal Abs prepared against membrane proteins that are commonly present at the surface of both X-ray-irradiated and nonirradiated cells. After isolation of Ab that bound only to X-ray-irradiated cells, the antigen was identified using MS. Using this approach, we found that expression of LY6D is induced in MCF10A cells by X-ray irradiation. The induction of LY6D expression is triggered through a pathway regulated by ATM, CHK2 and p53. This method is a new Ab-directed proteomic strategy for analysis of membrane proteins, and is applicable to various biological phenomena in situations in which both target molecule-expressing cells and nonexpressing cells are available.