Biomedical subjects
Publications and source records attributed to Kenichi Shiiba.
Twenty lactobacilli isolated from human feces were studied for binding to the human blood type B-antigen [Galalpha1-3 (Fucalpha1-2) Gal-] and H-antigen (Fucalpha1-2Gal-] expressed sugar chains in human intestinal mucosa. We found two strains, L. gasseri OLL2755 and L. gasseri OLL2877 that firmly adhere to human B-antigen, and we found L. gasseri OLL2827 bound to the H-antigen.
Adherent lactic acid bacteria (LAB) in the human intestine were investigated using the surface plasmon resonance technique with the biosensor BIACORE-1000. Ninety-three LAB strains were isolated from human feces and evaluated for binding to human blood type-A antigen [GalNAcalpha1-3 (Fucalpha1-2) Gal-: A-trisaccharide] expressed in the intestinal mucosa. Eleven strains showed strong adherence to an A-trisaccharide biotinyl polymer (BP) probe, and slightly or no adherence to a B-trisaccharide BP probe. Four strains with high adherence (high A/B ratio) were selected and their surface layer proteins (SLPs) were evaluated for A-antigen ligand binding using BIACORE. The SLP from L. brevis strain OLL2772 showed a single band at ca. 48 kDa by SDS-PAGE analysis and it had a very strong adherence to the human A-antigen, as shown using an anti-A lectin blocking technique. A partial N-terminal sequence of the band showed strong homology to an S-layer protein of L. brevis ATCC8287T. The probiotic LAB binds to human blood type-A antigen expressed in the intestinal mucosa which may aid in colonization of the gut.
Specific efflux transporters, such as P-glycoprotein, have been shown to confer drug resistance by decreasing the intracellular accumulation of anticancer drugs. Understanding influx transporters, as well as efflux transporters, is essential to overcome this resistance. We report the expression profile and pharmacological characterization of an organic cation transporter, SLC22A16. The results of our experiments indicate that SLC22A16 is a mediator of doxorubicin uptake in cancer cells. Quantitative real-time RT-PCR analyses show that SLC22A16 is expressed in primary samples taken from patients with acute leukemia. Xenopus oocytes injected with SLC22A16 cRNA import doxorubicin, a widely used anticancer drug for hematological malignancies, in a saturable and dose-dependent manner. The apparent Km value for doxorubicin import was 5.2+/-0.4 microM. In cytotoxic assays, stable transfectants of leukemic Jurkat cells overexpressing SLC22A16 cells became significantly more sensitive to doxorubicin (2 microM) treatment. Characterization of SLC22A16 will help in designing novel therapies targeting hematological malignancies.
Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). Here, we confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 was blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was further shown by immunohistochemical analysis. We therefore examined whether DSCR1 played any roles in angiogenesis. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibited VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibited the spreading of ECs on vitronectin, as well as on the immobilized anti-alphavbeta3 mAb, but not on anti-alphavbeta5 mAb. Moreover, AS-ODN inhibited tyrosine phosphorylation of focal adhesion kinase when ECs were plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin alphavbeta3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin alphavbeta3.
Angiopoietin-2 (Ang-2) plays an important role in destabilizing vessels for angiogenesis, however its transcriptional regulation has not been determined. Here we isolated about 3.2kb of the 5' upstream of the human Ang-2 gene and further characterized the transcriptional regulation of Ang-2. We found 10 Ets binding sites (EBSs) in this region. Ets-1 and Ets-2 but not Erg-1 augmented its promoter activity. Sequential deletion of EBSs and mutation analysis revealed that EBS8 located at -416/-406 from the translation initiation site was critical for Ets-1- or Ets-2-stimulated promoter activity. VEGF induced the expression of Ets-1 and Ang-2 in human umbilical vein endothelial cells (HUVECs). Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Ets-1 bound to EBS8 in HUVECs. Moreover, synthetic double-strand oligonucleotides containing corresponding EBS8 abrogated the induction of Ang-2 by VEGF in HUVECs. These results indicate that Ets-1 and the corresponding EBS are critical for the induction of Ang-2.
A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcalpha1-3(Fucalpha1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.
A 73-year-old male was referred to our hospital for abdominal pain, diarrhea and general fatigue lasting for 3 weeks. Physical examination of the abdomen revealed a firm mass in the left abdominal region. Computed tomography revealed a mass around the descending colon. Colonoscopy and barium enema revealed poor extensibility of the lumen with edematous mucosa, and narrowing of the descending colon with rugged mucosal surface. Because of the clinical symptoms and findings, the patient was diagnosed clinically as suffering from panniculitis of the descending colon. He underwent the left hemi-colectomy with side-to-side colo-colostomy after making of a loop ileostomy. Histological analysis of the resected colon showed an infiltration of inflammatory cells, predominantly lymphocytes, into veins and venules of the submucosa, muscularis propria and fat tissue of the colonic mesentery, with an involvement of all layers of the vessel wall. Arteries were escaped from inflammatory changes. The histopathological diagnosis of enterocolic phlebitis and venulitis was made because of these findings.
Anti-tumor immune responses are considered to be one of the key host reactions in human colorectal cancer, with T cells as important effector cells. For the induction of tumor-specific immunity, processing of cancer cells and priming of T cells by antigen-presenting cells are important. The present study was designed to clarify the relationship between Fas ligand (FasL; CD95L) expression and apoptotic cancer cells. Immunohistochemistry using frozen sections taken from 58 patients with colorectal cancer revealed that stromal cells composed mainly of tumor-associated macrophages expressed FasL, leaving cancer cells negative for FasL. These macrophages were abundantly distributed along the invasive margin. In situ hybridization revealed that these macrophages as well as cancer cells expressed FasL mRNA, whereas macrophages in the normal colon mucosa rarely expressed FasL. Apoptotic cancer cells recognized by monoclonal antibody M30 CytoDEATH were localized not only in cancer cell nests, but also in the stroma along the invasive margin showing a dissociated pattern, which was particularly evident in the areas of FasL+ macrophages. Furthermore, these macrophages, phenotypically similar to dendritic cells, occasionally contained M30+ apoptotic cancer cells in the cytoplasm. Clinicopathologic analyses in 123 cases revealed 1) a positive correlation between the degree of dissociated M30+ apoptotic cancer cells and the number of macrophages along the invasive margin and 2) an inverse association between the degree of dissociated M30+ apoptotic cancer cells and the occurrence of hematogenous metastasis after surgical resection of the primary tumor. In conclusion, the present study shows the importance of FasL+ activated macrophages as one of the host defense mechanisms against cancer cell spread in human colorectal cancer.