Biomedical subjects

K Toyoshima

Publications and source records attributed to K Toyoshima.

At least 37 records · Page 2Linked to original sources

PubMed2000

Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.

3T3 Cells↗

PubMed2000

Down-regulation of beta-catenin by the colorectal tumor suppressor APC requires association with Axin and beta-catenin.

The tumor suppressor adenomatous polyposis coli (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. APC forms a complex with beta-catenin, Axin, and glycogen synthase kinase-3beta and induces the degradation of beta-catenin. In the present study, we examined whether APC association with Axin is required for degradation of beta-catenin. We found that a fragment of APC that induces beta-catenin degradation was rendered inactive by disruption of its Axin-binding sites. Also, overexpression of an Axin fragment spanning the regulator of the G-protein signaling domain inhibited APC-mediated beta-catenin degradation. An APC fragment with mutated beta-catenin-binding sites but intact Axin-binding sites also failed to induce degradation of beta-catenin. These results suggest that APC requires interaction with Axin and beta-catenin to down-regulate beta-catenin.

Adenomatous Polyposis Coli Protein↗

PubMed2000

The APC-hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase.

The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in many sporadic colorectal tumors. The carboxyl-terminal S/TXV motif of the APC gene product interacts with the PDZ domain of hDLG, the human homolog of the Drosophila lethal (1) discs larige-1 (dlg) tumor suppressor. In the present study, we found that overexpression of hDLG suppresses cell proliferation by blocking cell cycle progression from the G0/G1 to S phase. This inhibition of cell cycle progression was abolished when the PDZ, SH3 or guanylate kinase-like domain of hDLG was mutated. Moreover, overexpression of these mutant hDLGs partially interfered with the cell cycle blocking activity of APC. Consistent with this result, mutant APC lacking the S/TXV motif exhibited weaker cell cycle blocking activity than the intact APC. These results suggest that APC-hDLG complex formation plays an important role in transducing the APC cell cycle blocking signal.

3T3 Cells↗

PubMed2000

Urine levels of CD46 (membrane cofactor protein) are increased in patients with glomerular diseases.

Soluble membrane cofactor protein (MCP, CD46) has not been detected by conventional ELISA in human urine. Here, we established a highly sensitive assay method for determination of urinary MCP (uMCP) using monoclonal antibody-coated paramagnetic beads. This method enabled us to detect less than 0.05 ng/ml of purified membrane and recombinant soluble MCP, a sensitivity 10-fold higher than that of conventional ELISA. In normal subjects, the levels of uMCP were <0. 05 ng/ml. The levels of uMCP were elevated in patients with IgA nephropathy and more prominently in patients with rapidly progressive glomerulonephritis. The levels of uMCP were correlated significantly with those of serum MCP (sMCP) and N-acetyl-beta-glucosaminidase and nonsignificantly with those of beta(2)-microglobulin, total urine protein, or serum creatinine. The properties of uMCP were inconsistent with those of the reported sMCP, since uMCP showed three bands on SDS-PAGE/immunoblotting with molecular mass profiles different from those of sMCP. uMCP exhibited factor I cofactor activity for cleavage of C3b comparable to that of sMCP. The origin of uMCP, however, remains to be determined. These results, taken together with the parameter correlation profiles, suggested that uMCP is secreted or produced secondary to tubular or glomerular damage. The physiological role and clinical significance of uMCP are now within the scope of our investigation by establishment of this assay.

Acetylglucosaminidase↗

PubMed2000

Innate immune therapy for cancer. Screen for molecules capable of activating the innate immune system.

Tumor cells usually express antigens which are distinguishable from normal "self" antigens and are thereby recognized by the host immune system. However, the host immune system barely responds to tumors in patients. Supplementation with adjuvant (such as BCG-CWS) in patients with cancer contributes to regression of intrinsically growing cancer. The adjuvant targets antigen-presenting cells, i.e. innate immunity, but not lymphocytes, and promotes up-regulation of MHC, co-stimulators and initial cytokines in antigen-presenting cells. We hypothesized that the role of the adjuvant is to provide conditions suitable for antigen-presentation where antigens are available and the lack of adjuvant-induced priming of antigen-presenting cells results in unresponsiveness to tumor antigens. Here, we report innate immune therapy applicable to cancer patients by supplementation with adjuvants for induction of potent immune responses against tumors.

Humans↗

PubMed2000

A comparative analysis of the antigenic, structural, and functional properties of three different preparations of recombinant human interleukin-18.

We compared the structural and functional properties of three recombinant human interleukin-18 (rIL-18) preparations, commercially available (Pep rIL-18) and prepared in our laboratory (active and inactive, according to their ability to potentiate IL-12-mediated interferon-gamma [IFN-gamma] induction in lymphocytes). All three preparations showed multimer formation on SDS-PAGE/immunoblotting using monoclonal antibodies (mAb) against the inactive form of rIL-18. In contrast, only the 18-kDa bands were recognized in each sample by mAb against the active form of rIL-18. The amounts of multimers and the 18-kDa moiety of Pep rIL-18 resembled those of the inactive rather than the active form. Likewise, the reaction profile of Pep rIL-18 toward mAb was very similar to that of inactive but not active rIL-18 on sandwich ELISA. Pep rIL-18 potentiated IFN-gamma-inducing activity together with IL-12, but its potency was 100-fold less than that of the active rIL-18, and excess doses were required for its activity. The inactive rIL-18 showed virtually no IFN-gamma-inducing ability, but when reduced and reconstituted, it inhibited the IFN-gamma-inducing activity of active rIL-18. These results suggest that there are two categories of recombinant IL-18 that are structurally, functionally, and antigenically different, and the mAb 125-2H and 21 can discriminate these two IL-18 populations by recognizing the epitopes specifically expressed on active and inactive IL-18, respectively.

Animals↗

PubMed2000

Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors.

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.

Antigen Presentation↗

PubMed2000

Role of serotype-specific polysaccharide in the resistance of Streptococcus mutans to phagocytosis by human polymorphonuclear leukocytes.

To clarify the role of cell surface components of Streptococcus mutans in resistance to phagocytosis by human polymorphonuclear leukocytes (PMNs), several isogenic mutants of S. mutans defective in cell surface components were studied with a luminol-enhanced chemiluminescence (CL) assay, a killing assay, and a transmission electron microscope. The CL responses of human PMNs to mutant Xc11 defective in a major cell surface antigen, PAc, and mutant Xc16 defective in two surface glucosyltransferases (GTF-I and GTF-SI) were the same as the response to the wild-type strain, Xc. In contrast, mutant Xc24R, which was defective in serotype c-specific polysaccharide, induced a markedly higher CL response than the other strains. The killing assay showed that human PMNs killed more Xc24R than the parent strain and the other mutants. The transmission electron microscopic observation indicated that Xc24R cells were more internalized by human PMNs than the parental strain Xc. These results may be reflected by the fact that strain Xc24R was more phagocytosed than strain Xc. The CL response of human PMNs to a mutant defective in polysaccharide serotype e or f was similar to the response to Xc24R. Furthermore, mutants defective in serotype-specific polysaccharide were markedly more hydrophobic than the wild-type strains and the other mutants, suggesting that the hydrophilic nature of polysaccharides may protect the bacterium from phagocytosis. We conclude that the serotype-specific polysaccharide, but not the cell surface proteins on the cell surface of S. mutans, may play an important role in the resistance to phagocytosis.

Antigens, Bacterial↗

PubMed2000

Complement activation in Mycoplasma fermentans-induced mycoplasma clearance from infected cells: probing of the organism with monoclonal antibodies against M161Ag.

Mycoplasma fermentans, a cell wall-less prokaryote, is capable of infecting humans and has been suggested to serve as a cofactor in AIDS development. Recently, we discovered a novel lipoprotein with a molecular mass of 43 kDa originating from M. fermentans. This protein, named M161Ag, activated human complement via the alternative pathway and efficiently induced the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor alpha, IL-6, IL-10, and IL-12 in human peripheral blood monocytes. It is likely that M161Ag of M. fermentans affects the host immune system upon mycoplasma infection. In this study, we developed monoclonal antibodies (MAbs) against M161Ag and examined the direct role of complement in M. fermentans infection using these MAbs as probes. M. fermentans was rapidly cleared from the surfaces of infected cells by human complement, but a low-grade infection persisted in human tumor cell lines. Mycoplasma particles remaining alive in host cells may cause recurrent infection, and liberated M161Ag may serve as a biological response modifier affecting both innate and acquired immunity.

Animals↗

PubMed2000

Participation of a MEK-independent pathway in MAP kinase activation and modulation of cell growth in mouse hepatoma cell lines.

The mechanism of cell growth was investigated in GIT medium-supplemented in vitro assay using high and low metastatic mouse hepatoma cell sublines, G-5 and G-1, respectively. G-5 cells exhibited high growth rate compared to G-1 cells. The PI3-kinase inhibitor LY294002 and P70 S6 kinase inhibitor rapamycin partially blocked both G-1 and G-5 cell growth, suggesting that these two kinases are involved in hepatoma cell growth. In contrast, the MEK1 inhibitor PD98059 partially blocked G-5 cell growth but not G-1 cell growth. MAP kinases (MAPK) in both G-1 and G-5 cells were indistinguishably phosphorylated, yet MEK-dependent MAPK activation was observed only in G-5 cells. In G-1 cells, MAPK was phosphorylated in a manner not connected to MEK activation. Thus, the low degree of cell growth in G-1 cells was attributable to disruption of the MEK-dependent MAPK cascade. However, the molecular mechanism whereby MAPK phosphorylation does not parallel MAPK activation in G-1 cells remains unknown. Here, we suggest that there may be an as yet unidentified MAPK phosphorylation pathway in malignantly transformed cells, which may affect in vivo cell growth and metastatic capacities of cancers.

Animals↗

PubMed2000

[Effect of beclomethasone increment on airflow limitation in asthmatic children treated with high dose beclomethasone].

In order to evaluate the effect of higher dose BDP therapy (1200-1660 micrograms/day), we studied 12 asthmatic children (mean age 9.7 years-old) with airflow limitation on respiratory function test who were asymptomatic with high-dose BDP therapy (800 micrograms/day). After 4 weeks of higher dose BDP therapy, FVC, FEV1 and V50 were significantly improved, but those improvement were insufficient compared with those after salbutamol inhalation. The personal best values after salbutamol inhalation were not different in every parameter of respiratory function test between BDP 800 micrograms/day and 1200 micrograms/day. We conclude that less than 800 micrograms of daily BDP is generally adequate for prevention in most asthmatic children, because higher dose BDP therapy is no more effective on respiratory function in those treated with 800 micrograms of daily BDP, and that the best value of respiratory function after salbutamol inhalation is not always a goal of high dose BDP therapy.

Anti-Asthmatic Agents↗

PubMed2000

[Reevaluation of immunopotentiation therapy for cancer].

Various cancer immunotherapies are thought to be insufficient to suppress cancers by themselves. Thus, they are usually applied in cancer therapy as an immunopotentiator in combination with chemotherapy. However, chemotherapy itself seems to be hazardous for activated immune cells. On the other hand, immunotherapy after surgical treatment for cancer patients of stages I through III significantly reduced cancer deaths compared to historical control data for the same surgeon team. Some lymph node metastases are cured by immunotherapy after resection of the main tumors. The potential of immunocancer therapy and problems in the future development of new cancer therapies are discussed.

Adjuvants, Immunologic↗

PubMed1999

Merkel cells are responsible for the initiation of taste organ morphogenesis in the frog.

Taste organs in the frog have a distinctive cell type located exclusively in the basal portion. In the same fashion as type III cells in mammalian taste buds, these basal cells show immunoreactivity for serotonin antibody. Further, these cells are morphologically similar to epidermal Merkel cells. To determine the significance of these serotonergic basal cells, we examined the early development of taste organs during metamorphosis of the frog by focusing on the origin and possible roles of serotonergic basal cells. For convenience of description, five stages of development (metamorphic stage to climax stages A-D) are defined. In the metamorphic stage, a few noninnervated Merkel cells appear at the upper layer of the lingual epithelium. No neuronal elements are seen in the epithelium at this stage. At climax stages A-B, immature fungiform papillae become discernible in the dorsal surface of the tongue, where the Merkel cells are located. Merkel cells then move downward and extend their cytoplasmic processes toward the basal lamina. These cells are identified by their intense immunoreactivity for serotonin. During the later stages, many nerve fibers in the subepithelial connective tissue approach the epithelium containing Merkel cells. At climax stages C-D, Merkel cells extend cytoplasmic processes along the basal lamina toward the center of the newly forming fungiform papillae. The morphology of these Merkel cells exactly coincides with that of serotonergic basal cells in adult taste organs. Profuse exocytotic release of dense-cored granules of Merkel cells toward the nerve fibers through the basal lamina is frequently seen in these stages. The present study indicates that serotonergic basal cells are derived from intraepithelial Merkel cells, which act as target sites for growing nerves and may be responsible for the initiation of taste organ morphogenesis.

Animals↗

PubMed1999

Human MD-1 homologue is a BCG-regulated gene product in monocytes: its identification by differential display.

BCG-CWS is a therapeutically potent immune activator which improves the prognosis of cancer patients. However, the targeting effector cells and molecules for BCG-CWS in the human immune system have not been determined. Here, we found that BCG-CWS activates human monocytes and concomitantly down-regulates expression of a human homologue of chicken MD-1 in the activated monocytes by differential display. According to a previous study, MD-1 forms a complex with the Toll family protein RP-105 on murine B cell lines to facilitate its stable expression. Thus, MD-1 may participate in regulation of innate immune activation on human monocytes. Our results, taken together with these recent findings regarding Toll family proteins, suggest that BCG-CWS acts on monocytes to modulate the human innate immune system via regulation of Toll family proteins.

Amino Acid Sequence↗

PubMed1999

Negative regulation of Wingless signaling by D-axin, a Drosophila homolog of axin.

Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the amount of beta-catenin/Armadillo, which in turn activates gene transcription. Here the Drosophila protein D-Axin was shown to interact with Armadillo and D-APC. Mutation of d-axin resulted in the accumulation of cytoplasmic Armadillo and one of the Wingless target gene products, Distal-less. Ectopic expression of d-axin inhibited Wingless signaling. Hence, D-Axin negatively regulates Wingless signaling by down-regulating the level of Armadillo. These results establish the importance of the Axin family of proteins in Wnt/Wingless signaling in Drosophila.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Expression of Mash1 in basal cells of rat circumvallate taste buds is dependent upon gustatory innervation.

Mash1, a mammalian homologue of the Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. In this study, using RT-PCR and in situ RT-PCR, we investigated if Mash1 gene expression occurs in rat taste buds. Further, we examined dynamics of Mash1 expression in the process of degeneration and regeneration in denervated rat taste buds. In rat tongue epithelium, Mash1 gene expression is confined to circumvallate, foliate, and fungiform papilla epithelia that include taste buds. In taste buds, Mash1-expressing cells are round cells in the basal compartment. In contrast, the mature taste bud cells do not express the Mash1 gene. Denervation and regeneration experiments show that the expression of Mash1 requires gustatory innervation. We conclude that Mash1 is expressed in cells of the taste bud lineage, and that the expression of Mash1 in rat taste buds is dependent upon gustatory innervation.

Animals↗

PubMed1999

The 3'-UT of the ubiquitous mRNA of human CD46 confers selective suppression of protein production in murine cells.

Mice express CD46 protein and its approximately 1.5-kb mRNA only in the testicular germ cells, unlike primates and pigs which ubiquitously express CD46 and its approximately 4 kb mRNA. Human CD46 is not well expressed in transgenic mice carrying human CD46 cDNA. To analyze the mechanism of regulation of human CD46 expression in mouse cells, we cloned the long (ubiquitous approximately 4 kb, L-form) and short ( approximately 1.5 kb, S-form) forms of human CD46 cDNA whose size difference is due to a stretch of the 3'-UT. Transfection of either cDNA resulted in marked S-form-dependent protein generation in all mouse cell lines tested. In contrast, there were virtually no differences in protein synthesis between S- and L-form cDNA in the simian and swine cell lines. Quantitative mRNA analyses and luciferase reporter gene assays suggested that one major cause of this interspecies discrepancy is transcriptional regulation, i. e. selective suppression of the 4-kb mRNA leading to low levels of protein synthesis. Although other mechanisms such as mRNA stability and translational regulation may lead to the low expression levels of L-form-derived CD46 in mice, the silencer activity in the L-form 3'-UT appears to function in human CD46 transcriptional regulation in mice.

3' Untranslated Regions↗

PubMed1999

Membrane and secretory forms of mouse membrane cofactor protein (CD46) generated from a single gene through alternative splicing.

A cDNA encoding a new secretory form of mouse membrane cofactor protein (MCP, CD46) was identified additionally to the membrane form cDNA. The secretory MCP, predicted from the cDNA sequence, consisted of the conserved four short consensus repeats (SCRs) plus a four amino acid-stretch. Unlike human MCP which comprises many isoforms, mouse MCP cDNA predicted a single isoform of membrane MCP with cytoplasmic tail 1 (CYT1) and serine/ threonine-rich domain C (ST(C)). To clarify the genomic origin and monomorphic alteration of these cDNAs, we cloned and analyzed a mouse genomic DNA harboring the full coding sequence of MCP from a 129/SV mouse genomic library. The mouse Mcp was a single gene approximately 50 kilobases long. Eleven of the 14 coding exons of the human MCP gene and intron-exon boundary sequences were found to be conserved in the mouse gene. The STC homologue but not the STA or STB homologue in the mouse exons was functional: the latter being due to deletions and lack of consensus sequences for splicing. The sequence equivalent to cytoplasmic tail 2 (CYT2) has not been identified in the Mcp genome. Thus, the three exons (ST(A), ST(B), and probably CYT2) responsible for the polymorphism of human MCP by differential splicing were missing in the mouse Mcp gene. Unlike the case in humans, no Mcp-related genes or pseudogenes were observed in the mouse genome. The single mouse Mcp gene was mapped to the R-positive H5 band of mouse Chromosome 1 by FISH. Strikingly, one alternative exon with 73 base pairs (encoding the four new amino acids and a TGA stop codon) was discovered between the SCRIV and the STC exons; alternative splicing causes the generation of the secretory form of mouse MCP. These results on mouse MCP, together with the information concerning other mouse SCR proteins, infer that the regulator of complement activation (RCA) gene cluster is genetically diverged between humans and mice.

Alternative Splicing↗