Effect of the difference in vehicles on gene expression in the rat liver--analysis of the control data in the Toxicogenomics Project Database

The Toxicogenomics Project is a 5-year collaborative project by the Japanese government and pharmaceutical companies in 2002. Its aim is to construct a large-scale toxicology database of 150 compounds orally administered to rats. The test consists of a single administration test (3, 6, 9 and 24 h) and a repeated administration test (3, 7, 14 and 28 days), and the conventional toxicology data together with the gene expression data in liver as analyzed by using Affymetrix GeneChip are being accumulated. In the project, either methylcellulose or corn oil is employed as vehicle. We examined whether the vehicle itself affects the analysis of gene expression and found that corn oil alone affected the food consumption and biochemical parameters mainly related to lipid metabolism, and this accompanied typical changes in the gene expression. Most of the genes modulated by corn oil were related to cholesterol or fatty acid metabolism (e.g., CYP7A1, CYP8B1, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, squalene epoxidase, angiopoietin-like protein 4, fatty acid synthase, fatty acid binding proteins), suggesting that the response was physiologic to the oil intake. Many of the lipid-related genes showed circadian rhythm within a day, but the expression pattern of general clock genes (e.g., period 2, arylhydrocarbon nuclear receptor translocator-like, D site albumin promoter binding protein) were unaffected by corn oil, suggesting that the effects are specific for lipid metabolism. These results would be useful for usage of the database especially when drugs with different vehicle control are compared.     

Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.     

Current status of genetic disturbances in wild medaka populations (<Emphasis Type="Italic">Oryzias latipes</Emphasis> species complex) in Japan

We revealed the range and current status of genetic disturbances in wild medaka populations (Oryzias latipes species complex) using two DNA markers (cytb gene and b-marker). Genetic disturbances were detected in many wild populations throughout Japan and were primarily caused by artificial introduction of the commercial medaka variety, himedaka. We identified native medaka populations without introgressions, which may be significant conservation targets. To conserve the native genetic diversity of the medaka species complex, further introduction of himedaka should be prevented by educating the public about the current status and risks of introducing non-native medaka varieties into the wild.     

REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

Features of T-cell subset composition in a D-galactose-induced senescence mouse model

Long-term administration of D-galactose induces oxidative stress and accelerates normal age-related changes. Hence, the D-galactose-treated rodent model has been widely used for aging research. In this study, we examined the immunological characteristics, especially CD4⁺ T-cell subset composition, of D-galactose-induced aging model mice to evaluate the model’s utility in immunosenescence studies. The spleens of aging model mice subjected to repeated subcutaneous injections of D-galactose exhibited significant increases in T cells with the memory phenotype (CD62L^low CD44^high) and individual T-cell subsets (Th1, Th2, Th17 and T_reg). Furthermore, cells with the phenotype of T follicular helper (Tfh) cells were spontaneously increased. The features of T-cell subset composition in D-galactose-treated mice were in close agreement with those observed in normal aged mice and appeared to mimic the currently known normal aging processes associated with T-cell homeostasis. Our results suggest that D-galactose-induced aging models would be useful for immunosenescence studies focusing on T-cell homeostasis and give valuable insight into age-related immune system dysregulation.