E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.     

Phylogenetic relationships among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

Bacillus thuringiensis Berliner has previously been classified via the serological identification of flagellar antigens. However, the phylogenetic relationships among strains of B. thuringiensis cannot be investigated by serotyping. Furthermore, high levels of homology have been found in gene sequences among various strains, complicating the determination of their evolutionary relationships. In order to elucidate the phylogenetic relationships within B. thuringiensis, we analyzed 40 strains belonging to typical serotypes using two approaches: an analysis of small subunit (SSU) rRNA sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The SSU rRNA analysis resulted in all 40 strains forming a single cluster with Bacillus cereus Frankland & Frankland. The distances among the subclusters were too small to further classify the strains. On the other hand, the phylogenetic analysis based on GP resulted in three clusters of B. thuringiensis strains. These results suggest that GP is a better method for the determination of phylogenetic relationships within B. thuringiensis.     

Detection of ER stress in vivo by Raman spectroscopy

The endoplasmic reticulum (ER) is an organelle in which most membrane and secretory proteins are synthesized. If these proteins are not folded correctly, unfolded proteins accumulate in the ER lumen, causing a cellular situation known as ER stress. Recently, many studies on the relationship between ER stress and diseases have been reported. Thus, studies of ER stress in vivo should yield information that is useful in pathology. Model mice have been developed as a powerful tool to visualize ER stress in vivo, but this approach depends on transgenic technology. Here, we report on a method of detecting ER stress in vivo by Raman spectroscopy. Our experiments revealed that two specific Raman bands were reduced in both cultured cells and animal tissues in an ER stress dependent manner. This suggests that Raman spectroscopy could be a useful tool to detect ER stress in vivo without transgenic technology.     

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.     

Artificial production and natural breeding of the endangered frog species Odorrana ishikawae, with special reference to fauna conservation in the laboratory

Odorrana ishikawae is listed as a class IB endangered species in the IUCN Red List and is protected by law in both Okinawa and Kagoshima Prefectures, Japan. Here, in an effort to help effectively preserve the genetic diversity of this endangered species in the laboratory, we tested a farming technique involving the artificial breeding of frogs, and also promoted natural breeding in the laboratory. Field-caught male/female pairs of the Amami and Okinawa Island populations were artificially bred using an artificial insemination method in the 2004, 2006, and 2008 breeding seasons (March to April). Although fewer than 50% of the inseminated eggs achieved metamorphosis, approximately 500, 300, and 250 offspring from the three respective trials are currently being raised in the laboratory. During the 2009 and 2010 breeding seasons, second-generation offspring were produced by the natural mating activities of the first offspring derived from the two artificial matings in 2004. The findings and the methods presented here appear to be applicable to the temporary protection of genetic diversity of local populations in which the number of individuals has decreased or the environmental conditions have worsened to levels that frogs are unable to survive by themselves.     

AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS1

Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat‐based dimorphism of chloroplast DNA (cpDNA). Here, we examined the “shallow” and “deep” subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima’s D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both “shallow” and “deep” haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat‐based dimorphism of cpDNA from field‐collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes.     

Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase

The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro. Further modification of its C-2 substituent has led to the discovery of RO-09-4879, which exhibits antifungal activity in vivo. The drug design is based on X-ray crystal analysis of a CaNmt complex with benzofuran derivative 4a. The optimization incorporates various biological investigations including a quasi in vivo assay and pharmacokinetic study. The computer aided drug design, synthesis, structure-activity relationships, and biological properties of RO-09-4879 are described in detail.     

Accumulation of 8-oxoguanine in the cellular DNA and the alteration of the OGG1 expression during ischemia-reperfusion injury in the rat kidney

During ischemia-reperfusion (I/R) injury in the rat kidney, apoptosis was observed in the distal tubules of the cortico-medullary region and outer medulla (OM) while severe necrosis was seen in the proximal straight tubules of the OM. The majority of these changes disappeared within 2 weeks. We examined the contents of 8-oxo-2'-deoxyguanosine (8-oxo-dG), which is a major type of oxidative damage in DNA, in the rat kidney during I/R injury, and also investigated the expression level of the OGG1 gene encoding the 8-oxoguanine DNA glycosylase. High-performance liquid chromatography with an MS/MS analysis of the nuclear DNA revealed an immediate accumulation of 8-oxo-dG in the nuclear DNA prepared from the cortex and OM of the kidney 1h after I/R, and an immunohistochemical analysis demonstrated the immediate accumulation of 8-oxo-dG in the nuclei of renal tubular cells both in the cortex and OM. A delayed increase of cytoplasmic staining with anti-8-oxo-dG was observed only in the cortico-medulla and OM, where the cytoplasmic staining in the proximal tubular cells is higher than in the distal tubular cells. The level of cytoplasmic staining representing 8-oxo-dG in mitochondrial DNA, peaked at 6h after I/R and preceded the necrosis of proximal tubular cells in the OM. An RNase protection assay showed a high level of OGG1 mRNA in the normal kidney, and the level decreased within 3h only in the OM, and increased thereafter 1-7 days of I/R both in the cortex and OM. In situ hybridization showed higher levels of OGG1 mRNA expression in the renal tubules in the OM than in the cortex of the normal kidney, which decreased rapidly within 3h of I/R. Thus, the accumulation of 8-oxo-dG in the mitochondrial DNA rather than in nuclear DNA is likely to be involved in the pathogenic responses such as necrosis of renal tubular cells during I/R injury of the kidney, together with an altered level of OGG1 expression.     

Decreased Amyloid-β Pathologies by Intracerebral Loading of Glycosphingolipid-enriched Exosomes in Alzheimer Model Mice

Elevated levels of amyloid-β peptide (Aβ) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular Aβ can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in Aβ metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped Aβ and with the associated Aβ were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-β precursor protein transgenic mice resulted in marked reductions in Aβ levels, amyloid depositions, and Aβ-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for Aβ binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for Aβ by carrying it on the exosome surface GSLs and suggest a role of exosomes in Aβ clearance in the central nervous system. Improving Aβ clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.