In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver fibrosis in mice.

INTRODUCTION: Liver transplantation is a gold standard treatment for intractable liver diseases. Because of the shortage of donor organs, alternative therapies have been required. Due to their potential to differentiate into a variety of mature cells, stem cells are considered feasible cell sources for liver regeneration. Stem cells from human exfoliated deciduous teeth (SHED) exhibit hepatogenic capability in vitro. In this study, we investigated their in vivo capabilities of homing and hepatocyte differentiation and therapeutic efficacy for liver disorders in carbon tetrachloride (CCl4)-induced liver fibrosis model mice. METHODS: We transplanted SHED into CCl4-induced liver fibrosis model mice through the spleen, and analyzed the in vivo homing and therapeutic effects by optical, biochemical, histological, immunological and molecular biological assays. We then sorted human leukocyte antigen-ABC (HLA-ABC)-positive cells from primary CCl4-damaged recipient livers, and analyzed their fusogenicity and hepatic characteristics by flow cytometric, genomic DNA, hepatocyte-specific gene assays. Furthermore, we examined the treatment effects of HLA-positive cells to a hepatic dysfunction by a secondary transplantation into CCl4-treated mice. RESULTS: Transplanted SHED homed to recipient livers, and expressed HLA-ABC, human hepatocyte specific antigen hepatocyte paraffin 1 and human albumin. SHED transplantation markedly recovered liver dysfunction and led to anti-fibrotic and anti-inflammatory effects in the recipient livers. SHED-derived HLA-ABC-positive cells that were sorted from the primary recipient liver tissues with CCl4 damage did not fuse with the host mouse liver cells. Sorted HLA-positive cells not only expressed human hepatocyte-specific genes including albumin, cytochrome P450 1A1, fumarylacetoacetase, tyrosine aminotransferase, uridine 5'-diphospho-glucuronosyltransferase, transferrin and transthyretin, but also secreted human albumin, urea and blood urea nitrogen. Furthermore, SHED-derived HLA-ABC-positive cells were secondary transplanted into CCl4-treated mice. The donor cells homed into secondary recipient livers, and expressed hepatocyte paraffin 1 and human albumin, as well as HLA-ABC. The secondary transplantation recovered a liver dysfunction in secondary recipients. CONCLUSIONS: This study indicates that transplanted SHED improve hepatic dysfunction and directly transform into hepatocytes without cell fusion in CCl4-treated mice, suggesting that SHED may provide a feasible cell source for liver regeneration.

In vitro bactericidal effects of near-ultraviolet light from light-emitting diodes on Helicobacter pylori.

We investigated whether near-ultraviolet light emitted from light-emitting diodes (LEDs) effects Helicobacter pylori viability and whether this new method can potentially apply to eradication therapy. Three H. pylori strains were used for near-ultraviolet (UV) LED irradiation experiments. Viability of isolates exposed to near-UV light was compared with controls by counting colony forming units. A time-dependent bactericidal effect of near-UV light was definitely observed. LED irradiation with near-UV light showed effective bactericidal activity against H. pylori strains. Eradication therapy with LED might provide a new avenue of treatment in patients refractory to eradication due to antibiotic resistance and/or adverse effects of antibiotics.

Impact of negative frequency-dependent selection on mating pattern and genetic structure: a comparative analysis of the S-locus and nuclear SSR loci in Prunus lannesiana var. speciosa.

Mating processes of local demes and spatial genetic structure of island populations at the self-incompatibility (S-) locus under negative frequency-dependent selection (NFDS) were evaluated in Prunus lannesiana var. speciosa in comparison with nuclear simple sequence repeat (SSR) loci that seemed to be evolutionarily neutral. Our observations of local mating patterns indicated that male-female pair fecundity was influenced by not only self-incompatibility, but also various factors, such as kinship, pollen production and flowering synchrony. In spite of the mating bias caused by these factors, the NFDS effect on changes in allele frequencies from potential mates to mating pollen was detected at the S-locus but not at the SSR loci, although the changes from adult to juvenile cohorts were not apparent at any loci. Genetic differentiation and isolation-by-distance over various spatial scales were smaller at the S-locus than at the SSR loci, as expected under the NFDS. Allele-sharing distributions among the populations also had a unimodal pattern at the S-locus, indicating the NFDS effect except for alleles unique to individual populations probably due to isolation among islands, although this pattern was not exhibited by the SSR loci. Our results suggest that the NFDS at the S-locus has an impact on both the mating patterns and the genetic structure in the P. lannesiana populations studied.

Fragmented CagA protein is highly immunoreactive in Japanese patients.

BACKGROUND: High-molecular-weight cell-associated proteins (HM-CAP) assay is the most popular serological immunoassay worldwide and has been developed from US isolates as the antigens. The accuracy is reduced when the sera are from adults and children in East Asia including Japan. To overcome the reduced accuracy, an enzyme immunoassay using Japanese strain-derived HM-CAP (JHM-CAP) was developed, in which the antigens were prepared by exactly the same procedure as HM-CAP. The performance of JHM-CAP was better than that of HM-CAP in Japanese adults as well as in children. The higher sensitivity was because of the presence of 100-kDa protein that was absent in the preparation of HM-CAP antigen. MATERIALS AND METHODS: Immunoblot analysis and peptide mass fingerprinting methods were used to identify the distinctive 100-kDa protein present in JHM-CAP antigens. The peptide sequence and identification were analyzed by Mascot Search on the database of Helicobacter pylori. The identified protein was confirmed by immunoblot with a specific antibody and inhibition assay by the sera. RESULTS: The distinctive 100-kDa protein was a fragment of CagA derived from Japanese clinical isolates, and the sera of Japanese patients had strongly reacted to the protein, probably to the exposed epitope on the fragmented CagA. The fragmentation of CagA had occurred in the process of antigen preparation in Japanese isolates, not in US isolates even under the same preparation. CONCLUSION: The distinctive 100-kDa protein was a fragment of CagA protein of H. pylori derived from Japanese clinical isolates, and Japanese patients including children are likely to react strongly to the exposed epitopes on fragmented CagA.

Salmonella enterotoxin (Stn) regulates membrane composition and integrity.

The mechanism of action of Salmonella enterotoxin (Stn) as a virulence factor in disease is controversial. Studies of Stn have indicated both positive and negative effects on Salmonella virulence. In this study, we attempted to evaluate Stn function and its effects on Salmonella virulence. To investigate Stn function, we first performed in vitro and in vivo analysis using mammalian cells and a murine ileal loop model. In these systems, we did not observe differences in virulence phenotypes between wild-type Salmonella and an stn gene-deleted mutant. We next characterized the phenotypes and molecular properties of the mutant strain under various in vitro conditions. The proteomic profiles of the total cell membrane protein fraction differed between wild type and mutant in that there was an absence of a protein in the mutant strain, which was identified as OmpA. By far-western blotting, OmpA was found to interact directly with Stn. To verify this result, the morphology of Salmonella was examined by transmission electron microscopy, with OmpA localization being analyzed by immunogold labeling. Compared with wild-type Salmonella, the mutant strain had a different pole structure and a thin periplasmic space; OmpA was not seen in the mutant. These results indicate that Stn, via regulation of OmpA membrane localization, functions in the maintenance of membrane composition and integrity.

Environmental polarity estimation in living cells by use of quinoxaline-based full-colored solvatochromic fluorophore PQX and its derivatives.

Fluorosolvatochromic probes have recently attracted interest for live cell imaging. We recently tested our quinoxaline-based fluorosolvatochromic probes, PQX and MPQX, for in vivo imaging applications. PQX and MPQX are characterized by donor (pyrrole site)-acceptor (quinoxaline site) structure, and the peak emission wavelength of these compounds varies over the visible wavelength range depending on the polarity of the solvent used, for a variety of solvents from hexane to water. A linear relationship was obtained between peak emission wavenumber and E(T)(N) (normalized solvent polarity). When tested on cultured HEp-2 cells, the results showed that the PQX and MPQX were not harmful at the applied concentrations (10(-5) M), and site-dependent fluorescence spectra in living cells were observed with PQX treatment, which indicates that PQX penetrates into the plasma membrane, followed by delocalization throughout the cells. MPQX was also able to penetrate the cell membrane, distribute throughout the cells and emit fluorescence, but did not show site-dependent intracellular fluorescence spectra. These results suggest that PQX is a remarkable tool for investigating the local polarity environment in cells after appropriate conjugation to biomolecules.

Up-regulation of 42 kDa tubulin alpha-6 chain fragment in well-differentiated hepatocellular carcinoma tissues from patients infected with hepatitis C virus.

We performed proteomic differential display analysis of hepatitis C virus-associated 21 human hepatocellular carcinoma (HCV-HCC) tissues by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). One of the numerous spots which was located next to three spots of glutamine synthetase showed stronger intensity in well-differentiated HCC tissues compared to non-cancerous tissues. Samples from 6 out of 21 patients showed up-regulation of this spot compared to non-cancerous tissues. After in-gel digestion, MALDITOF/MS identified the spot as tubulin alpha-6 chain. Two-dimensional immunoblot analysis confirmed that this spot was indeed tubulin alpha, and this spot was stronger in cancerous tissues than in noncancerous tissues. These results suggest that tubulin alpha-6 chain is one of the candidates for biomarkers for well-differentiated HCV-associated HCC.

Helicobacter pylori CagA inhibits endocytosis of cytotoxin VacA in host cells.

Helicobacter pylori, a common pathogen that causes chronic gastritis and cancer, has evolved to establish persistent infections in the human stomach. Epidemiological evidence suggests that H. pylori with both highly active vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), the major virulence factors, has an advantage in adapting to the host environment. However, the mechanistic relationship between VacA and CagA remains obscure. Here, we report that CagA interferes with eukaryotic endocytosis, as revealed by genome-wide screening in yeast. Moreover, CagA suppresses pinocytic endocytosis and the cytotoxicity of VacA in gastric epithelial cells without affecting clathrin-dependent endocytosis. Our data suggest that H. pylori secretes VacA to attack distant host cells while injecting CagA into the gastric epithelial cells to which the bacteria are directly attached, thereby protecting these attached host cells from the cytotoxicity of VacA and creating a local ecological niche. This mechanism might allow H. pylori to balance damage to one population of host cells with the preservation of another, allowing for persistent infection.

A novel approach of protein immobilization for protein chips using an oligo-cysteine tag.

Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamond-like, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.

Helicobacter pylori tissue tropism: mouse-colonizing strains can target different gastric niches.

Studies with the mouse-adapted Helicobacter pylori strain SS1 had supported an idea that infections by this pathogen start in the gastric antrum and spread to the corpus after extensive mucosal damage. This paper shows that the unrelated strain X47 colonizes the corpus preferentially. Differences between strains in preferred gastric region were detected by co-inoculating mice with a mixture of SS1 and X47, and genotyping H. pylori recovered after 2-8 weeks of infection by vacA s allele PCR and RAPD fingerprinting. Mixed infections were found in each of 59 co-inoculated young C57BL/6J mice. On average, however, SS1 was fourfold more abundant than X47 in the antrum and X47 was threefold more abundant than SS1 in the corpus. Similar results were obtained in mice inoculated first with one strain and then the other strain 2 weeks later. SS1 was even more abundant in the antrum of elderly (>1 year old) mice (97 % of isolates). Qualitatively similar SS1 and X47 tissue distributions were seen using unrelated mouse lines (AKR/J, A/J, DBA/2J, BALB/cJ, LG/J, SM/J), but with significantly different SS1 : X47 ratios in some cases. These results suggest the existence of at least two distinct gastric niches whose characteristics may be affected by host genotype and age (physiology), and indicate that strains differ in how effectively they colonize each niche. Differences among gastric regions and the mixed infections that these allow may contribute to H. pylori diversity and genome evolution.

Tob proteins enhance inhibitory Smad-receptor interactions to repress BMP signaling.

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.

Mice lacking a transcriptional corepressor Tob are predisposed to cancer.

tob is a member of antiproliferative family genes. Mice lacking tob are prone to spontaneous formation of tumors. The occurrence rate of diethylnitrosamine-induced liver tumors is higher in tob(-/-) mice than in wild-type mice. tob(-/-)p53(-/-) mice show accelerated tumor formation in comparison with single null mice. Expression of cyclin D1 mRNA is increased in the absence of Tob and is reduced by Tob. Tob acts as a transcriptional corepressor and suppresses the cyclin D1 promoter activity through an interaction with histone deacetylase. Levels of tob mRNA are often decreased in human cancers, implicating tob in cancer development.

Synthesis and Herbicidal Activity of Opened Hydantoin-ring Derivatives of Hydantocidin.

We synthesized three hydantocidin derivatives and evaluated their herbicidal activity in order to elucidate the role of the spirohydantoin system at the anomeric center of hydantocidin. With application to foliage at 1000 ppm, only α-ureidoamide 14 demonstrated activity, the remaining compounds being found to be inactive.