A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix

Mesenchymal stem cells (MSC) show a very short proliferative life span and readily lose the differentiation potential in culture. However, the growth rate and the proliferative life span of the stem cells markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix, which was produced by PYS-2 cells or primary endothelial cells. Furthermore, the stem cells expanded on the extracellular matrix, but not those on plastic tissue culture dishes, retained the osteogenic, chondrogenic, and adipogenic potential throughout many mitotic divisions. The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor. Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies, which require a large number of stem or progenitor cells.     

Plasma interleukin (IL)-18 (interferon-gamma-inducing factor) and other inflammatory cytokines in patients with gouty arthritis and monosodium urate monohydrate crystal-induced secretion of IL-18

To determine whether levels of interleukin (IL)-18, together with those of IL-1beta, tumor necrosis factor-alpha, IL-6, and IL-8, are elevated in the plasma of patients with gouty arthritis, the plasma concentrations of those cytokines were measured in 31 males with gouty arthritis. Further, CD14+ cells were obtained from human blood and thioglycolate medium-induced peritoneal cells obtained from caspase 1-deficient mice, and then separately cultured in the presence of monosodium urate monohydrate (MSU) crystals. In addition, in an animal in vivo experiment, MSU crystals were injected into subcutaneous air pouches of IL-18-deficient mice. The plasma concentrations of IL-18, IL-6, and IL-8 were elevated in the presence of gouty arthritis in the gout patients. In the in vitro study, the presence of MSU crystals stimulated CD14+ cells (monocytes) to secrete IL-18 and increased the activity of caspase 1 in CD14+ cells, whereas there was no significant effect on IL-18 messenger RNA in CD14+ cells and only a slight induction of IL-18 secretion from thioglycolate medium-induced caspase 1-deficient peritoneal cells. In the in vivo experiment, MSU crystals injected into the air pouch promoted neutrophil accumulation along with an increase in concentrations of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-1alpha in air-pouch fluids in both IL-18-deficient and wild-type mice. However, there was no increase in the concentration of IL-18 in air-pouch fluids in either mouse strain. Our results suggest that plasma IL-18, IL-6, IL-8, and C-reactive protein (CRP) levels reflect local inflammation associated with gouty arthritis, though IL-18 does not play an important role in neutrophil accumulation. Further, they suggest that MSU crystals accelerate the processing of IL-18 from an inactive to active form via the activation of caspase 1.     

Calcium ion propagation in cultured keratinocytes and other cells in skin in response to hydraulic pressure stimulation

We have previously suggested that a variety of environmental factors might be first sensed by epidermal keratinocytes, which represent the frontier of the body. To further examine this idea, in the present study, we examined the intracellular calcium responses of cultured keratinocytes to external hydraulic pressure. First, we compared the responses of undifferentiated and differentiated keratinocytes with those of fibroblasts, vascular endothelial cells (VEC), and lymphatic endothelial cells. Elevation of intracellular calcium was observed after application of pressure to keratinocytes, fibroblasts, and VEC. The calcium propagation extended over a larger area and continued for a longer period of time in differentiated keratinocytes, as compared with the other cells. The response of the keratinocytes was dramatically reduced when the cells were incubated in medium without calcium. Application of a non‐selective transient receptor potential (TRP) channel blocker also attenuated the calcium response. These results suggest that differentiated keratinocytes are sensitive to external pressure and that TRP might be involved in the mechanism of their response. J. Cell. Physiol. 224:229–233, 2010 © 2010 Wiley‐Liss, Inc.     

Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells

We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. Root extract from Gentiana triflora triggered cell death of human Daudi cells in culture. In addition, daily administration of the extract to mice inhibited growth of implanted solid tumors. Extract treatment of cultured cells resulted in the appearance of shranken, fragmented, or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. But, the extract-treated cells did not show DNA fragmentation, which exhibits a nucleosome ladder, suggesting that extract-triggered cell death is not mediated through a typical apoptotic pathway.     

Effects of dietary myo-inositol, D-chiro-inositol and L-chiro-inositol on hepatic lipids with reference to the hepatic myo-inositol status in rats fed on 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane

The effects of dietary 0.2% inositol stereoisomers on the hepatic lipids and myo-inositol (MI) status in rats fed with 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) were investigated. Dietary MI reduced the hepatic lipids in the rats fed with DDT. Dietary D-chiro-inositol (DCI) and L-chiro-inositol (LCI) both had a promoting effect on the increase in hepatic lipids due to DDT feeding. Dietary MI enhanced the hepatic free MI level and the phosphatidylinositol/phosphatidylcholine ratio, but dietary DCI reduced the level and ratio.     

Binding of AlF-C, an Orc1-binding transcriptional regulator, enhances replicator activity of the rat aldolase B origin

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Lipid polarity is maintained in absence of tight junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.     

Annexin A4 is a possible biomarker for cisplatin susceptibility of malignant mesothelioma cells

Mesothelioma is a highly malignant tumor with a poor prognosis and limited treatment options. Although cisplatin (CDDP) is an effective anticancer drug, its response rate is only 20%. Therefore, discovery of biomarkers is desirable to distinguish the CDDP-susceptible versus resistant cases. To this end, differential proteome analysis was performed to distinguish between mesothelioma cells of different CDDP susceptibilities, and this revealed that expression of annexin A4 (ANXA4) protein was higher in CDDP-resistant cells than in CDDP-susceptible cells. Furthermore, ANXA4 expression levels were higher in human clinical malignant mesothelioma tissues than in benign mesothelioma and normal mesothelial tissues. Finally, increased susceptibility was observed following gene knockdown of ANXA4 in mesothelioma cells, whereas the opposite effect was observed following transfection of an ANXA4 plasmid. These results suggest that ANXA4 has a regulatory function related to the cisplatin susceptibility of mesothelioma cells and that it could be a biomarker for CDDP susceptibility in pathological diagnoses.