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Expression of alpha-hemoglobin stabilizing protein and cellular prion protein in a subclone of murine erythroleukemia cell line MEL
2008
Otsuka, Y.(Hokkaido Univ., Sapporo (Japan)) | Ito, D. | Katsuoka, K. | Arashiki, N. | Komatsu, T. | Inaba, M.
alpha-Hemoglobin stabilizing protein (AHSP) functions as the erythroid-specific molecular chaperon for alpha-globin. AHSP gene expression has been reported to be downregulated in hematopoietic tissues of animals suffering from prion diseases though the mechanism remains to be clarified. Herein, we demonstrate that MELhipod8 cells, a subclone of murine erythroleukemia (MEL) cells, have prion protein (PrPsup(C)) on the cell surface and have highly inducible expression of the AHSP and alpha- and beta-globin genes, resembling the expression pattern of the PrP and AHSP genes in bipotential erythroid- and megakaryocyte-lineage cells followed by erythroid differentiation in normal erythropoiesis. Moreover, MELhipod8 cells exhibit greater effective erythroid differentiation with a population of hemoglobinized normoblast-like cells than that observed for the parental MEL cells. These findings suggest that MELhipod8 cells could provide a mechanism for downregulation of the AHSP gene in prion diseases.
Mostrar más [+] Menos [-]Coculture of equine mesenchymal stem cells and mature equine articular chondrocytes results in improved chondrogenic differentiation of the stem cells
2010
Lettry, V., Hokkaido Univ., Sapporo (Japan) | Hosoya, K. | Takagi, S. | Okumura, M.
Bone marrow derived mesenchymal stem cells (MSCs) can be used to repair articular cartilage defects, these cells should be properly stimulated so that they could differentiate morphologically and hold cellular synthetic features closer to maturely differentiated chondrocytes. It is well known that tissue specific environment plays an important role in cell fate determination. Once improved isolation, proliferation and differentiation protocols have been developed, the likelihood of spontaneous differentiation of MSCs into divergent lineages will be reduced, thus increasing their value for cartilage repair. The purpose of this study was to improve chondrogenic differentiation of equine MSCs using coculture with mature equine articular chondrocytes (ACs), along with the determination of the effect of adding transforming growth factor (TGF) beta1 in the pellet culture system. Following confirmation of multilineage (adipogenic, osteogenic and chondrogenic) differentiation, isolated MSCs, ACs and coculture of both cell types were transferred into pellet culture system in a DMEM-based medium supplemented with or without TGFbetal. Chondrogenic differentiation was evaluated histologically and the relative mRNA expressions of collagen type 1 alpha1 (COL1A1), collagen type 2 alpha1 (COL2A1), aggrecan (ACAN) and SRY-box 9 (SOX9) were estimated by quantitative RT-PCR. Cocultured cells showed diffuse distribution of extracellular matrix (ECM), whereas in chondrocyte pellets it was more localized to central regions. Expression of COL2A1, ACAN and SOX9 genes were higher in cocultured pellets when compared to MSCs and ACs-composed pellets. Addition of TGFbeta1 in chondrogenic differentiating medium did not consistently amplify expression of the above mentioned genes. Differentiation of equine MSCs was enhanced by coculturing in association with mature ACs, improving expression of cartilage-specific genes and producing a more homogeneous production of ECM within the newly formed cocultured cartilage. The use of the coculture system could possibly enhance the capacity of MSC-derived chondrocytes to build up stable articular cartilage-like constructs, which could play an important role in articular cartilage repair and regeneration.
Mostrar más [+] Menos [-]Genetic and antigenic analyses of a Puumala virus isolate as a potential vaccine strain
2008
Daud, N.H.A.(Hokkaido Univ., Sapporo (Japan)) | Kariwa, H. | Tkachenko, E. | Dzagurnova. T. | Medvedkina, O. | Tkachenko, P. | Ishizuka, M. | Seto, T. | Miyashita, D. | Sanada, T. | Nakauchi, M. | Yoshii, K. | Maeda, A. | Yoshimatsu, K. | Arikawa, J. | Takashima, I.
Puumala virus (PUUV), a causative agent of hemorrhagic fever with renal syndrome (HFRS), is prevalent in Europe and European Russia. No vaccine has been developed for PUUV-associated HFRS, primarily because of the low viral yield in cultured cells. A PUUV strain known as DTK/Ufa-97 was isolated in Russia and adapted for growth in Vero E6 cells maintained in serum-free medium. The DTK/Ufa-97 strain produced a higher viral titer in serum-free medium, suggesting that it may prove useful in the development of an HFRS vaccine. When PUUV-infected Vero E6 cells were grown in serum-free medium, the DTK/Ufa-97 strain yielded more copies of intracellular viral RNA and a higher viral titer in the culture fluid than did the Sotkamo strain. Phylogenetic analysis revealed that PUUVs can be classified into multiple lineages according to geographical origin, and that the DTK/Ufa-97 strain is a member of the Bashkiria-Saratov lineage. The deduced amino acid sequences of the small, medium, and large segments of the DTK/Ufa-97 strain were 99.2% to 100%, 99.3% to 99.8%, and 99.8% identical, respectively, to those of the Bashkirian PUUV strains and 96.9%, 92.6%, and 97.4% identical, respectively, to those of the Sotkamo strain, indicating that the PUUVs are genetically diverse. However, DTK/Ufa-97 and other strains of PUUV exhibited similar patterns of binding to a panel of monoclonal antibodies against Hantaan virus. In addition, diluted antisera (i.e., ranging from 1:160 to 1:640) specific to three strains of PUUV neutralized both homologous and heterologous viruses. These results suggest that the DTK/Ufa-97 strain is capable of extensive growth and is antigenically similar to genetically distant strains of PUUV.
Mostrar más [+] Menos [-]Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic defferentiation in pellet culture system
2006
Bosnakovski, D.(Hokkaido Univ., Sapporo (Japan)) | Mizuno, M. | Kim, G. | Takagi, S. | Okumura, M. | Fujinaga, T.
Bovine bone marrow mesenchymal stem cells (MSCs) cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative 'Real Time' reverse transcriptase polymerase chain reaction (qRT-PCR). Results showed that bovine MSCs underwent complete chondrogenesis; the initial stage was characterized by expression of sox 9 messenger ribonucleic acid (mRNA), followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1 and beta2, basic fibroblast growth factor (FGF 2), bone morphogenic protein 6 (BMP 6), insulin-like growth factors 1, parathyroid hormone related peptide and indian hedgehog (Ihh) were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day 21 of the culture, FGF 2, BMP 6 and Ihh were highly expressed, compared to cells cultured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells. Furthermore, this study contributes to our in understanding of known chondrogenic-growth factors in autocrine and/or paracrine manner playing a role in the spontaneous differentiation.
Mostrar más [+] Menos [-]Analysis of the cell cycle of fibroblasts derived from the LEC rat after X-irradiation
2006
Masuda, K.(Nagoya City Univ. (Japan)) | Miyamoto, T. | Cho, A.R. | Agui, T.
The LEC rat is reported to exhibit hypersensitivity to X-irradiation, deficiency in DNA double-strand break repair, and radio-resistant DNA synthesis. This character of the LEC rat has been thought to be due to abnormal G1 arrest in cells after X-irradiation. In this report, we re-investigated the effect of X-irradiation on the cell cycle in primary-cultured fibroblasts. Primary-cultured fibroblasts derived from LEC and BN rats were exposed to 4 Gy of X-ray and their cell cycle analysis was performed with a flow cytometer. Fibroblasts derived from both rats showed normal response of the cell cycle, indicating the arrest at both G1- and G2/M-phase and no difference in the cell cycle population between fibroblasts derived from both rats. In contrast, when the same analysis was performed using the cell line, L7 and W8, which had been established from the lung fibroblasts of LEC and control WKAH rats, respectively, by immortalizing with SV40 T-antigen, L7 cells but not W8 cells showed impaired G1 arrest and abnormal cell cycle. These results suggest that fibroblasts derived from LEC rats possess the normal cell cycle response after X-irradiation, if they are kept naive as not immortalized with SV40 T-antigen.
Mostrar más [+] Menos [-]Nonspecific cell-mediated cytotoxicity of peripheral blood lymphocytes derived from suckling piglets
1987
Onizuka, N. (Hokkaido Univ., Sapporo (Japan). Faculty of Veterinary Medicine) | Maede, Y. | Ohsugi, T. | Namioka, S.