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Biomedical subjects

Toshio Imai

Publications and source records attributed to Toshio Imai.

At least 37 records · Page 2Linked to original sources
PubMed2004

Inhibition of fractalkine ameliorates murine collagen-induced arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-gamma by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.

Animals↗
PubMed2004

Low expression of human tubulin tyrosine ligase and suppressed tubulin tyrosination/detyrosination cycle are associated with impaired neuronal differentiation in neuroblastomas with poor prognosis.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups. However, the precise mechanism underlying this phenomenon remains largely unknown. To understand the molecular and genetic bases of neuroblastoma, we have generated its cDNA libraries and identified a human ortholog of tubulin tyrosine ligase gene (hTTL/Nbla0660) as a differentially expressed gene at high levels in a favorable subset of the tumor. Tubulin is subjected to several types of evolutionarily conserved posttranslational modification, including tyrosination and detyrosination. Tubulin tyrosine ligase catalyzes ligation of the tyrosine residue to the COOH terminus of the detyrosinated form of alpha-tubulin. The measurement of hTTL mRNA expression in 74 primary neuroblastomas by quantitative real-time reverse transcription-PCR revealed that its high expression was significantly associated with favorable stages (1, 2 and 4s; p = 0.0069), high TrkA expression (p = 0.002), a single copy of MYCN (p < 0.00005), tumors found by mass screening (p = 0.0042), nonadrenal origin (p = 0.0042) and good prognosis (p = 0.023). The log-rank test showed that high expression of hTTL was an indicator of favorable prognosis (p = 0.026). Immunohistochemical analysis using specific antibodies generated by us demonstrated that tyrosinated tubulin (Tyr-tubulin), detyrosinated tubulin (Glu-tubulin) and hTTL as well as Delta2-tubulin were positive in favorable tumors, whereas only Delta2-tubulin was positive in the tumors with MYCN amplification. In an RTBM1 neuroblastoma cell line, hTTL was increased after treating the cells with bone morphogenetic protein 2 (BMP2) or all-trans retinoic acid (RA), which induced neuronal differentiation. These results suggest that the deregulated tubulin tyrosination/detyrosination cycle caused by decreased expression of hTTL is associated with inhibition of neuronal differentiation and enhancement of cell growth in the primary neuroblastomas with poor outcome.

Amino Acid Sequence↗
PubMed2004

Enhancement of serum- and platelet-derived growth factor-induced cell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling.

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.

Animals↗
PubMed2004

Enhancement of hepatocarcinogenesis by kojic acid in rat two-stage models after initiation with N-bis(2-hydroxypropyl)nitrosamine or N-diethylnitrosamine.

Kojic acid (KA) has been used as a food additive for preventing enzymatic browning of crustaceans and as a cosmetic agent for skin whitening. In the present experiments, effects of KA on the induction of hepatic pre-neoplastic lesions in N-bis(2-hydroxypropyl)nitrosamine-initiated (experiment 1) and non-initiated (experiment 2) models, and its promoting influence in a medium-term liver bioassay (experiment 3) were investigated at dietary doses of up to 2% in male F344 rats. In experiment 1, 2% KA feeding induced significant increases in numbers (22.3 +/- 13.0 vs 8.5 +/- 3.4 in the 0%) and areas (0.37 +/- 0.29 vs 0.05 +/- 0.03 in the 0%) of glutathione-S-transferase P form (GST-P)-positive foci and toxic changes such as vacuolation of hepatocytes and microgranulomas. The development of GST-P-positive foci was pronounced in the animals with hepatocellular toxic changes. In experiment 2, numbers (0.65 +/- 0.57 vs 0.17 +/- 0.28 in the 0%) and areas (0.005 +/- 0.005 vs 0.0007 +/- 0.0012 in the 0%) of GST-P-positive foci and hepatocellular proliferating cell nuclear antigen (PCNA) expression (3.8 +/- 2.3 vs 2.6 +/- 0.7 in the 0%) were significantly increased by the 2% treatment. The PCNA-positive hepatocytes were abundantly localized around the vacuolated and granulomatous legions in both experiments 1 and 2. In experiment 3, significant increases in numbers (16.9 +/- 3.2 vs 8.4 +/- 2.7 in the 0%) and areas (1.62 +/- 0.39 vs 0.77 +/- 0.34 in the 0%) of GST-P-positive foci were again observed with 2% KA. These results demonstrate tumor-promoting and possible hepatocarcinogenic activity of KA at 2%, but the carcinogenic potential is likely to be weak. This study also indicated that enhanced replication of hepatocytes related to toxic changes might be involved as an underlying mechanism.

Animals↗
PubMed2004

Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and alpha-actinin in epithelial cells.

Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules that are involved in formation of cadherin-based adherens junctions (AJs). The nectin-based cell-cell adhesion induces activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of AJs through reorganization of the actin cytoskeleton. Although evidence has accumulated that nectins recruit cadherins to the nectin-based cell-cell adhesion sites through their cytoplasm-associated proteins, afadin and catenins, it is not fully understood how nectins are physically associated with cadherins. Here we identified a rat counterpart of the human LIM domain only 7 (LMO7) as an afadin- and alpha-actinin-binding protein. Rat LMO7 has two splice variants, LMO7a and LMO7b, consisting of 1,729 and 1,395 amino acids, respectively. LMO7 has calponin homology, PDZ, and LIM domains. Western blotting revealed that LMO7 was expressed ubiquitously in various rat tissues. Immunofluorescence and immunoelectron microscopy revealed that LMO7 localized at cell-cell AJs, where afadin localized, in epithelial cells of rat gallbladder. In addition, LMO7 localized at the cytoplasmic faces of apical membranes in the same epithelial cells. We furthermore revealed that LMO7 bound alpha-actinin, an actin filament-bundling protein, which bound to alpha-catenin. Immunoprecipitation analysis revealed that LMO7 was associated with both the nectin-afadin and E-cadherin-catenin systems. LMO7 was assembled at the cell-cell adhesion sites after both the nectin-afadin and E-cadherin-catenin systems had been assembled. These results indicate that LMO7 is an afadin- and alpha-actinin-binding protein that connects the nectin-afadin and E-cadherin-catenin systems through alpha-actinin.

Actinin↗
PubMed2004

TNF-alpha and IL-4 regulate expression of fractalkine (CX3CL1) as a membrane-anchored proadhesive protein and soluble chemotactic peptide on human fibroblasts.

The CX(3)C chemokine, fractalkine (FKN, CX(3)CL1), has multiple functions and exists as two distinct forms, a membrane-anchored protein and a soluble chemotactic peptide that cleaves from the cell surface FKN. In this study, we first demonstrated the expression of FKN in tumor necrosis factor (TNF)-alpha- and interleukin (IL)-4-stimulated human fibroblasts. The induction of FKN was observed for both forms. We also demonstrated monocyte chemotactic activity in the culture supernatant from the fibroblasts stimulated with these cytokines. These results suggest that TNF-alpha- and IL-4-stimulated fibroblasts may play an important role in accumulation of monocytes at inflammatory sites.

Cell Adhesion Molecules↗
PubMed2004

Nectin-like molecule-5/Tage4 enhances cell migration in an integrin-dependent, Nectin-3-independent manner.

Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca(2+)-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin alpha(V)beta(3) at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin alpha(V)beta(3) or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin alpha(V)beta(3) in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.

Animals↗
PubMed2004

Identification of novel keratinocyte-secreted peptides dermokine-alpha/-beta and a new stratified epithelium-secreted protein gene complex on human chromosome 19q13.1.

We performed high-throughput in situ hybridization screening of sections of mouse epidermis using an equalized skin cDNA library as probes and identified a novel gene giving rise to two splicing variants, both of which are expressed in the spinous layer. This gene was mapped between two genes encoding keratinocyte-related peptides, suprabasin and keratinocyte differentiation-associated protein (Kdap), on human chromosome 19q13.1. These gene products appeared to carry functional signal sequences. We then designated these two splicing variants as dermokine-alpha and -beta. Northern blotting and quantitative RT-PCR revealed that dermokine-alpha/-beta, suprabasin, and Kdap were highly expressed in stratified epithelia. In mouse embryonic development, dermokine-alpha/-beta began to be expressed during the period of stratification. Also, in differentiating primary cultured human keratinocytes, transcription of dermokine-alpha/-beta, suprabasin, and Kdap was induced. These findings indicated that dermokine-alpha/-beta, suprabasin, and Kdap are secreted from the spinous layer of the stratified epithelia and that these genes form a novel gene complex on the chromosome.

Amino Acid Sequence↗
PubMed2004

Infiltration of CD8+ T cells containing RANTES/CCL5+ cytoplasmic granules in actively inflammatory lesions of human chronic gastritis.

Chronic gastritis is frequently associated with infection of Helicobacter pylori and characterized by tissue infiltration of neutrophils, lymphocytes, and plasma cells. To address the mechanism of lymphocyte infiltration in chronic gastritis, we examined the expression of chemokines and their receptors using frozen sections of chronic gastritis, obtained from 23 patients who underwent gastrectomy for gastric cancer. By immunohistochemistry, lymphocytes in inflamed gastric mucosa expressed CCR5 abundantly, CXCR3 less frequently, and CCR4 sparsely. The numbers of CCR5(+) cells, which were composed of mainly CD8(+) and partly CD4(+) T cells, were positively correlated with the degree of neutrophil infiltration, and decreased in areas with intestinal metaplasia or mucosal atrophy. RANTES/CCL5, one of the ligands of CCR5, was localized mainly in CD8(+) and partly CD4(+) T cells with a characteristic dotted pattern, and such lymphocytes were most densely distributed around the neck region of gastric glands. In situ hybridization confirmed the expression of CCL5 mRNA in these cells, and immunoelectron microscopy revealed localization of CCL5 in the membrane-bound granules, which most probably corresponded to the cytolytic granules of cytotoxic T cells. The numbers of CCL5(+) lymphocytes showed a close correlation with the degree of neutrophil infiltration and markedly decreased in intestinal metaplasia. In conclusion, our data suggest that, together with neutrophils, CCL5(+) T cells, presumably activated cytotoxic T cells, would play important roles in the active inflammatory process of chronic gastritis. Our data also suggest a self-recruiting mechanism involving CCR5 and CCL5 for tissue accumulation of such T cells.

Adult↗
PubMed2004

Sulfation of silk sericin and anticoagulant activity of sulfated sericin.

Silk sericin extracted from a cocoon was sulfated by chlorosulfonic acid. Sulfation of sericin was confirmed by FT-IR and the reaction efficiency was calculated as 44.9%. 1H-NMR suggested that sulfation mainly occurred at serine residues in the sericin molecule. The sulfated sericin was separated in three fractions by gel-filtration chromatography using Sephacryl S-200. The sulfate group content and amino-acid composition of each fraction were almost identical, while the anticoagulant activity differed for each fraction of sulfated sericin. Higher anticoagulant activity was observed for the higher-molecular-mass fraction. The anticoagulant activity of sulfated sericin was estimated at 1/10 to 1/20 of heparin.

Animals↗
PubMed2003

Fractalkine in vascular biology: from basic research to clinical disease.

Fractalkine (now also called CX3CL1) is a unique chemokine that functions not only as a chemoattractant but also as an adhesion molecule and is expressed on endothelial cells activated by proinflammatory cytokines, such as interferon-gamma and tumor necrosis factor-alpha. The fractalkine receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, including natural killer (NK) cells and cytotoxic T lymphocytes, which contain high levels of intracellular perforin and granzyme B, and on macrophages. Soluble fractalkine causes migration of NK cells, cytotoxic T lymphocytes, and macrophages, whereas the membrane-bound form captures and enhances the subsequent migration of these cells in response to secondary stimulation with other chemokines. Furthermore, stimulation through membrane-bound fractalkine activates NK cells, leading to increased cytotoxicity and interferon-gamma production. Recently, accumulating evidence has shown that fractalkine is involved in the pathogenesis of various clinical disease states or processes, such as atherosclerosis, glomerulonephritis, cardiac allograft rejection, and rheumatoid arthritis. In addition, polymorphisms in CX3CR1, which reduce its binding activity to fractalkine, have been reported to increase the risk of HIV disease and to reduce the risk of coronary artery disease. This review will examine new concepts underlying fractalkine-mediated leukocyte migration and tissue damage, focusing primarily on the pathophysiological roles of fractalkine in various clinical conditions, especially in atherosclerosis and vascular injury.

Animals↗
PubMed2003

Rapid induction of colorectal tumors in rats initiated with 1,2-dimethylhydrazine followed by dextran sodium sulfate treatment.

To establish a rapid bioassay system with neoplastic end-points for detection of colorectal carcinogenesis modifiers, we evaluated the effects of dextran sodium sulfate (DSS) treatment on the different stages of carcinogenesis in rats initiated with 1,2-dimethylhydrazine (DMH). F344 male rats were given three subcutaneous injections of DMH (40 mg/kg body weight) in a week, and were administered drinking water containing 1.0% DSS ad libitum either during or after the initiation period for a week, or both during and after initiation periods for 2 weeks. At the 10th week of the experiment, although the numbers of aberrant crypt foci were significantly decreased in all groups treated with DSS and given DMH-initiation as compared with DMH alone, dysplastic foci/adenomas/adenocarcinomas were increased. The incidences and multiplicities of these lesions were highest in rats treated with DSS after DMH-initiation period. At the 26th week, the incidences of adenocarcinomas (100 vs. 20% in DMH alone) and their multiplicities (6.6 +/- 0.8/rat vs. 0.2 +/- 0.4/rat in DMH alone) were also highest in this group. These results indicate that short-term DSS-treatment in the post-initiation period significantly accelerates DMH-induced colorectal tumor development in rats, so that this protocol may effective for establishment of a rapid bioassay system with neoplastic end-points.

1,2-Dimethylhydrazine↗
PubMed2003

Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells.

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that play roles in organization of a variety of cell-cell junctions in cooperation with or independently of cadherins. Four nectins have been identified. Five nectin-like molecules, which have domain structures similar to those of nectins, have been identified, and we characterized here nectin-like molecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1. Necl-2 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and nectin-3. Necl-2 was widely expressed in rat tissues examined. Necl-2 localized at the basolateral plasma membrane in epithelial cells of the mouse gall bladder, but not at specialized cell-cell junctions, such as tight junctions, adherens junctions, and desmosomes. Nectins bind afadin, whereas Necl-2 did not bind afadin but bound Pals2, a membrane-associated guanylate kinase family member known to bind Lin-7, implicated in the proper localization of the Let-23 protein in Caenorhabditis elegans, the homologue of mammalian epidermal growth factor receptor. These results indicate the unique localization of Necl-2 and its possible involvement in localization of a transmembrane protein(s) through Pals2.

Animals↗
PubMed2003

Tage4/Nectin-like molecule-5 heterophilically trans-interacts with cell adhesion molecule Nectin-3 and enhances cell migration.

Malignant transformation of cells causes disruption of cell-cell adhesion, enhancement of cell motility, and invasion into surrounding tissues. Nectins have both homophilic and heterophilic cell-cell adhesion activities and organize adherens junctions in cooperation with cadherins. We examined here whether Tage4, which was originally identified to be a gene overexpressed in colon carcinoma and has a domain structure similar to those of nectins, is involved in cell adhesion and/or migration. Tage4 heterophilically trans-interacted with nectin-3, but not homophilically with Tage4. Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton and cadherins through catenins. Thus, Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its function and domain structure similar to those of nectins.

3T3 Cells↗
PubMed2003

Hepatocellular tumor induction in heterozygous p53-deficient CBA mice by a 26-week dietary administration of kojic acid.

In order to evaluate the tumorigenic potential of kojic acid (KA), used as a food additive for preventing enzymatic browning of crustaceans and a cosmetic agent for the purpose of skin whitening, heterozygous p53-deficient CBA [p53(+/-)] mice, which are recognized as useful for detecting genotoxic carcinogens, and wild-type littermates [p53(+/+) mice] were fed diet containing 0, 1.5, and 3% KA for 26 weeks. KA induced diffuse hypertrophy and hyperplasia of thyroid follicular epithelial cells with decreased serum thyroxine levels in both p53 (+/-) and p53 (+/+) mice, but caused no thyroid tumors. In the liver, the incidence of altered hepatocellular foci was significantly increased at 1.5 and 3% in p 53(+/-) and 1.5% in p53 (+/+) mice, and that of hepatocellular adenomas was increased at 1.5 and 3% in p 53(+/-) and 3% in p53 (+/+) mice. p53 (+/-) mice thus appeared to be more susceptible in terms of the tumorigenic dose of KA with a greater prevalence of hepatic proliferative lesions. The results of the present study indicate tumorigenic potential of KA in the liver, but not thyroid follicular epithelial cells in CBA mice and a contribution of genotoxicity on hepatocellular tumor development cannot be ruled out.

Adenoma, Liver Cell↗
PubMed2003

Membrane-bound form of fractalkine induces IFN-gamma production by NK cells.

Natural killer (NK) cells participate in both innate and adaptive immunity, in part by their prompt secretion of cytokines including IFN-gamma, a pro-inflammatory cytokine with an important role in Th1 polarization. To assess the involvement of fractalkine in inflammatory processes, we examined the effect of fractalkine on IFN-gamma production by NK cells. Although soluble chemokines, including MCP-1 and RANTES as well as fractalkine, had a negligible effect on IFN-gamma production, immobilized fractalkine markedly induced IFN-gamma production by NK cells in a dose-dependent manner. Pretreatment of NK cells with the phosphatidylinositol 3-kinase (PI 3-K) inhibitor, wortmannin, completely inhibited the production of IFN-gamma induced by fractalkine, and pretreatment with the protein tyrosine kinase inhibitor, herbimycin A, partially suppressed the response, suggesting that augmentation of IFN-gamma production in response to fractalkine treatment of NK cells involves signaling through PI 3-K and protein tyrosine kinases. Furthermore, co-culture of NK cells with fractalkine-transfected 293E cells markedly enhanced IFN-gamma production by NK cells compared with co-culture with control 293E cells. These findings may indicate a paracrine feedback loop system in which endothelial cells may be activated to produce more fractalkine, and also suggest a role for fractalkine expressed on endothelial cells in Th1 polarization through the stimulation of IFN-gamma production by NK cells.

Cell Division↗