Phylogenetic relationships among Phytopythium species,and re-evaluation of Phytopythium fagopyri comb. nov., recovered from damped-off buckwheat seedlings in Japan

We examined the phylogenetic relationships among Phytopythium species using the rDNA ITS region, the LSU rDNA region, and the mitochondrial coxI and coxII genes. The genus was resolved into three monophyletic clades (1–3). Clade 1 was the largest clade, composed of 12 known species. Clades 2 contained two known and one new species candidate and clade 3 contained two known species. Three isolates in clade 2 (FP1, HonMa, and a strain designated as P. helicoides CBS293.35) formed a monophyletic group with high bootstrap support. This monophyletic group was distinct from P. helicoides sensu stricto. All three isolates came from damped-off buckwheat seedlings. The isolates were morphologically identical with one another and were characterized by globose, sub-globose, or pyriform sporangia with apical papillae; internally or internally nested proliferating sporangia; simple sympodia; coiling antheridial stalks; and wavy, sessile, or clavate antheridial cells. The isolates grew at temperatures between 15 °C and 40 °C, and the optimum temperature was 30 °C, with a radial growth rate of 20 mm/24 h. The phylogenetic and morphological analyses indicated that these isolates belong to a distinct species, which was previously under the genus Pythium, named here Phytopythium fagopyri comb. nov.     

Derivation of Transgene-Free Human Induced Pluripotent Stem Cells from Human Peripheral T Cells in Defined Culture Conditions

Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.     

Endothelins specifically recognize lysophosphatidylcholine micelles

Lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low‐density lipoprotein (ox‐LDL), is implicated in numerous inflammatory diseases, including atherosclerosis. Here, to clarify the relationship between bioactive endothelins (ETs) (which are considered to be potent proinflammatory mediators) and LPC/ox‐LDL, we investigated the interaction between ETs and LPC/ox‐LDL by fluorescence spectroscopy and western blotting. Tryptophan fluorescence measurements revealed ETs specifically interacted with LPC at concentrations that exceeded the critical micelle concentration (CMC). The tryptophan residue in ETs was not likely to be involved directly in the interaction between ETs and LPC micelles. Tryptophan fluorescence quenching revealed tryptophan residue in ETs where LPC concentrations were below the CMC may be buried deeply in the peptide or may interact with other amino acid residues, whereas tryptophan residue in ETs in the presence of LPC at concentrations exceeding the CMC was exposed outside of the peptide. Furthermore, ETs bind to ox‐LDL in a concentration‐dependent manner. These results strongly suggest that ox‐LDL contains micelle‐rich LPCs and that ETs specifically interact with the bioactive LPC micelles. Further study of the interaction between ETs and LPC micelles contained in ox‐LDL will provide important information on the development and progression of many inflammatory diseases, including atherosclerosis. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Omeprazole suppresses endothelial calcium response and eNOS Ser1177 phosphorylation in porcine aortic endothelial cells

Molecular Biology Reports - Although high doses of proton pump inhibitors can elicit an anticancer effect, this strategy may impair vascular biology. In particular, their effects on endothelial...     

Intracellular delivery of serum-derived hepatitis C virus

A robust and reliable cell culture system for serum-derived HCV (HCVser) has not been established yet because of the presence of neutralizing antibody and tropism for infection. To overcome this obstacle, we employed a lipid-mediated protein intracellular delivery reagent (PIDR) that permits internalization of proteins into cells. Although entry of HCVcc was not enhanced by the treatment with PIDR, entry of HCVser into hepatoma cell lines (Huh7 and HepG2) and immortalized primary hepatocytes (Hc and HuS/E2) was significantly enhanced by the PIDR treatment. The entry of HCVser into Huh7 cells in the presence of PIDR was resistant to the neutralization by an anti-hCD81 antibody, suggesting that PIDR is capable of internalizing HCVser in a receptor-independent manner. Interestingly, the PIDR-mediated entry of HCVser and HCVcc was enhanced by the addition of sera from chronic hepatitis C patients but not from healthy donors. In addition, neutralization of HCVcc infection by anti-E2 antibody was canceled by the treatment with PIDR. In conclusion, the PIDR is a valuable tool to get over the obstacle of neutralizing antibodies to internalize HCV into cells and might be useful for the establishment of in vitro propagation HCVser.     

Protrudin serves as an adaptor molecule that connects KIF5 and its cargoes in vesicular transport during process formation

Neurons are highly polarized cells with long neurites. Vesicular transport is required for neurite extension. We recently identified protrudin as a key regulator of vesicular transport during neurite extension. Expression of protrudin in nonneuronal cells thus induces formation of neurite-like membrane protrusions. We adopted a proteomics approach to identify proteins that associate with protrudin. Among the protrudin-associated proteins, including many with a function related to intracellular trafficking, we focused on KIF5, a motor protein that mediates anterograde vesicular transport in neurons. A coimmunoprecipitation assay confirmed that endogenous protrudin and KIF5 interact in mouse brain. Overexpression of KIF5 induced the formation of membrane protrusions in HeLa cells, reminiscent of the effect of protrudin overexpression. Forced expression of both protrudin and KIF5 promoted protrusion extension in a synergistic manner, whereas depletion of either protein attenuated protrusion formation. Protrudin facilitated the interaction of KIF5 with Rab11, VAP-A and -B, Surf4, and RTN3, suggesting that protrudin serves as an adaptor protein and that the protrudin-KIF5 complex contributes to the transport of these proteins in neurons. Given that mutation of protrudin or KIF5 is a cause of human hereditary spastic paraplegia, the protrudin-KIF5 axis appears to be integral to neuronal function.     

Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges

This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing β-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 μm in diameter and elongated pores 250-450 μm in length and 100-150 μm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 μm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.