Search for gravity-responsive genes by PCR-based mRNA differential display in human cells

Mechanisms of molecular responses of human cells to gravity change and/or space radiation are one of the most important physiological problems in space science. We have previously reported that expression levels of several genes are changed in cultured human cells after UVC irradiation, and a few of those genes are responsible for UVC sensitivity. In this study, to find candidates for genes that play roles in susceptibility of human cells to gravity stressors, including those responsible for genetic stability in humans, we analyzed genes expressed differentially after gravity stress in human cells, using a PCR-based mRNA differential display (D.D.) method. Cells used were RSa and its variant cell lines, with discrepant sensitivity to radiation cell-killing and mutagenicity [correction of mutagenecity].     

Pharmacophore Modeling for Anti-Chagas Drug Design Using the Fragment Molecular Orbital Method

BackgroundChagas disease, caused by the parasite Trypanosoma cruzi, is a neglected tropical disease that causes severe human health problems. To develop a new chemotherapeutic agent for the treatment of Chagas disease, we predicted a pharmacophore model for T. cruzi dihydroorotate dehydrogenase (TcDHODH) by fragment molecular orbital (FMO) calculation for orotate, oxonate, and 43 orotate derivatives.Conclusions/SignificanceFMO-based interaction energy analyses revealed a pharmacophore model for TcDHODH inhibitor. Hydrogen bond acceptor pharmacophores correspond to Lys43 and Lys214, hydrogen bond donor and acceptor pharmacophores correspond to Asn67 and Asn194, and the aromatic ring pharmacophore corresponds to FMN, which shows important characteristics of compounds that inhibit TcDHODH. In addition, the Lys214 residue is not conserved between TcDHODH and human DHODH. Our analysis suggests that these orotate derivatives should preferentially bind to TcDHODH, increasing their selectivity. Our results obtained by pharmacophore modeling provides insight into the structural requirements for the design of TcDHODH inhibitors and their development as new anti-Chagas drugs.     

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity

Background

Osteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.

Aims

To investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.

Methods

We compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.

Results

OPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.

Conclusions

OPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.     

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH?/?) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494–504, 2015     

Assessing Depression Related Severity and Functional Impairment: The Overall Depression Severity and Impairment Scale (ODSIS)

Background

The Overall Depression Severity and Impairment Scale (ODSIS) is a brief, five-item measure for assessing the frequency and intensity of depressive symptoms, as well as functional impairments in pleasurable activities, work or school, and interpersonal relationships due to depression. Although this scale is expected to be useful in various psychiatric and mental health settings, the reliability, validity, and interpretability have not yet been fully examined. This study was designed to examine the reliability, factorial, convergent, and discriminant validity of a Japanese version of the ODSIS, as well as its ability to distinguish between individuals with and without a major depressive disorder diagnosis.

Methods

From a pool of registrants at an internet survey company, 2830 non-clinical and clinical participants were selected randomly (619 with major depressive disorder, 619 with panic disorder, 576 with social anxiety disorder, 645 with obsessive–compulsive disorder, and 371 non-clinical panelists). Participants were asked to respond to the ODSIS and conventional measures of depression, functional impairment, anxiety, neuroticism, satisfaction with life, and emotion regulation.

Results

Exploratory and confirmatory factor analysis of three split subsamples indicated the unidimensional factor structure of ODSIS. Multi-group confirmatory factor analysis showed invariance of factor loadings between non-clinical and clinical subsamples. The ODSIS also showed excellent internal consistency and test–retest intraclass correlation coefficients. Convergence and discriminance of the ODSIS with various measures were in line with our expectations. Receiver operating characteristic curve analyses showed that the ODSIS was able to detect a major depressive syndrome accurately.

Conclusions

This study supports the reliability and validity of ODSIS in a non-western population, which can be interpreted as demonstrating cross-cultural validity.     

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

Translation elongation factor G (EF‐G) in bacteria plays two distinct roles in different phases of the translation system. EF‐G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post‐termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF‐G are distributed over two different EF‐G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF‐G is also found in bacteria. Two EF‐G paralogues are found in the spirochaete Borrelia burgdorferi, EF‐G1 and EF‐G2. We demonstrate that EF‐G1 is a translocase, while EF‐G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF‐G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF‐3) is present in the reaction. These results indicate that two B. burgdorferi EF‐G paralogues are close relatives to mitochondrial EF‐G paralogues rather than the conventional bacterial EF‐G, in both their phylogenetic and biochemical features.     

Identification of genes related to heart failure using global gene expression profiling of human failing myocardium

Although various management methods have been developed for heart failure, it is necessary to investigate the diagnostic or therapeutic targets of heart failure. Accordingly, we have developed different approaches for managing heart failure by using conventional microarray analyses. We analyzed gene expression profiles of myocardial samples from 12 patients with heart failure and constructed datasets of heart failure-associated genes using clinical parameters such as pulmonary artery pressure (PAP) and ejection fraction (EF). From these 12 genes, we selected four genes with high expression levels in the heart, and examined their novelty by performing a literature-based search. In addition, we included four G-protein-coupled receptor (GPCR)-encoding genes, three enzyme-encoding genes, and one ion-channel protein-encoding gene to identify a drug target for heart failure using in silico microarray database. After the in vitro functional screening using adenovirus transfections of 12 genes into rat cardiomyocytes, we generated gene-targeting mice of five candidate genes, namely, MYLK3, GPR37L1, GPR35, MMP23, and NBC1. The results revealed that systolic blood pressure differed significantly between GPR35-KO and GPR35-WT mice as well as between GPR37L1-Tg and GPR37L1-KO mice. Further, the heart weight/body weight ratio between MYLK3-Tg and MYLK3-WT mice and between GPR37L1-Tg and GPR37L1-KO mice differed significantly. Hence, microarray analysis combined with clinical parameters can be an effective method to identify novel therapeutic targets for the prevention or management of heart failure.