Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.

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It is well known that Fas ligand and anti-Fas antibodies can induce apoptosis, although some cancer cells are resistant to their stimuli. On the other hand, phosphatidylinositol 3'-kinase (PI3 K) and Akt mediate the survival signal and allow the cells to escape from apoptosis in various human cancers. Thus, we postulated that LY294002, a PI3 K inhibitor, should inactivate Akt, consequently inhibiting cell proliferation and increase apoptosis in the human gastric carcinoma cell line, MKN-45. Previously, we reported that MKN-45 was resistant against the anti-Fas antibody, CH-11, without interferon-gamma pretreatment in vitro. LY294002 caused a decrease of phosphorylated-Akt and an inhibition of cell proliferation via cell cycle arrest in the G0/G1 phase by P27/Kip1 accumulation, but there was no obvious induction of apoptosis. The simultaneous treatment of LY294002 and CH-11 significantly induced apoptosis confirmed by morphology and DNA ladder formation. Decreased phosphorylated-Akt by LY294002 treatment led to a down-regulation of Mcl-2 and phosphorylated Bad proteins, which are anti-apoptotic factors and belong to the Bcl-2 family. On the other hand, expression levels of the other anti-apoptotic factors, such as FLICE-inhibitory protein (FLIP), Bcl-2 and Bcl-XL, which are associated with the Fas-mediated apoptotic signal pathway, did not change after LY294002 treatment. We concluded that: 1) the PI3K-Akt pathway plays an important role in preventing Fas-mediated apoptosis; and 2) a PI3 K inhibitor, such as LY294002, might be a useful anti-tumoral agent for gastric carcinoma.

These data suggest that RUNX3 may play a physiologic role in chief cells and G cells in gastric mucosa, and that suppression of RUNX3 expression in intestinal metaplasia and carcinoma of human stomach may be implicated in gastric carcinogenesis.

To analyse the characteristics of infections caused by Streptococcus dysgalactiae subsp. equisimilis, clinical isolates (n5145) were collected at 11 medical institutions between September 2003 and October 2005. These isolates belonged to Lancefield group A (n55), group C (n518) or group G (n5122). Among all isolates, 42 strains were isolated from sterile samples such as blood, synovial fluid and tissue specimens from patients who were mostly over 50 years with invasive infections, and included seven cases of streptococcal toxic shock syndrome and necrotizing fasciitis. In contrast, the remaining 103 were isolated mainly from patients of all age groups with non-invasive infections such as pharyngotonsillitis. These isolates were classified into 25 types based on emm genotyping. A significant difference in emm types was observed between isolates from invasive and non-invasive infections (P,0.001): stG485, stG6792 and stG2078 predominated among isolates from invasive infections. A phylogenetic tree of complete open reading frames of emm genes in this organism showed high homology with those of Streptococcus pyogenes, but not with those of other streptococci. The presence of five different clones was estimated based on DNA profiles of isolates from invasive infections obtained by PFGE. Genes for resistance to macrolides [erm(A), three isolates; erm(B), five isolates; mef(A), seven isolates] and levofloxacin (mutations in gyrA and parC, four isolates) were identified in this organism. These results suggest the need for further nationwide surveillance of invasive infections caused by S. dysgalactiae subsp. equisimilis. INTRODUCTIONMost b-haemolytic streptococcal pathogens isolated from humans are identified as Streptococcus pyogenes (group A streptococci; GAS), Streptococcus agalactiae (group B streptococci; GBS), Streptococcus dysgalactiae subsp. equisimilis and species of the anginosus group that belong to Lancefield groups A, B, C, G or F (Facklam, 2002;Ruoff et al., 2003). In contrast to GAS and GBS, which are known to cause serious and systemic invasive infections, streptococci with Lancefield group C or G antigens were long considered to be commensal organisms that only rarely caused invasive infections as opportunistic pathogens.In 1996, S. dysgalactiae subsp. equisimilis was proposed as a new streptococcal taxon (Vandamme et al., 1996). Although rare, S. dysgalactiae subsp. equisimilis strains having group A antigen rather than group C or G antigen have also been reported (Bert & Lambert-Zechovsky, 1997;Brandt et al., 1999;Katsukawa et al., 2002 Notably, S. dysgalactiae subsp. equisimilis possesses many virulence factors shared with GAS, such as M protein (Fischetti, 1989; Schnitzler et al., 1995), streptolysin O (Gerlach et al., 1993Okumura et al., 1994), streptolysin S (Humar et al., 2002 and streptokinase (Walter et al., 1989;Ikebe et al., 2004). It has been suggested that these factors were transmitted from GAS to this species (Kalia et al., 2001).In the present report, we have described S. dysgalactiae ...