Tumor necrosis factor-α enhances hyperbaric oxygen-induced visfatin expression via JNK pathway in human coronary arterial endothelial cells

Background  

Visfatin, a adipocytokine with insulin-mimetic effect, plays a role in endothelial angiogenesis. Hyperbaric oxygen (HBO) has been used in medical practice. However, the molecular mechanism of beneficial effects of HBO is poorly understood. We sought to investigate the cellular and molecular mechanisms of regulation of visfatin by HBO in human coronary arterial endothelial cells (CAECs).     

Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.     

Detection of matrix metalloproteinase (MMP)-2 and MMP-9 in canine seminal plasma

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that play a central role in degradation of protein components of the extracellular matrix and basement membrane. Previous studies have shown that MMP-2 and MMP-9 are present in human seminal plasma, but there is little information available on the presence of MMPs in canine seminal plasma. This study aims to investigate the presence of MMPs in canine seminal plasma and their clinical manifestation at the level of various semen parameters in canine species. Latent and active forms of MMP-2 and MMP-9 were evaluated using gelatin zymography and their association with semen parameters was examined. Results demonstrate that both latent and active forms of MMP-2 and MMP-9 are present in canine seminal plasma and the latent forms are predominant. The latent and active MMP-9 activities were elevated in the semen with unsatisfactory quality traits and proMMP-2 was inversely correlated with semen quality whereas, MMP-2 was positively correlated with semen quality traits. These findings suggest that proMMP-9 and MMP-9 activation contributes to the variation in semen, while the activation of MMP-2 improves the sperm functionality.     

On the use of multifactor dimensionality reduction (MDR) and classification and regression tree (CART) to identify haplotype-haplotype interactions in genetic studies

Haplotype-based approaches may have greater power than single-locus analyses when the SNPs are in strong linkage disequilibrium with the risk locus. To overcome potential complexities owing to large numbers of haplotypes in genetic studies, we evaluated two data mining approaches, multifactor dimensionality reduction (MDR) and classification and regression tree (CART), with the concept of haplotypes considering their haplotype uncertainty to detect haplotype-haplotype (HH) interactions. In evaluation of performance for detecting HH interactions, MDR had higher power than CART, but MDR gave a slightly higher type I error. Additionally, we performed an HH interaction analysis with a publicly available dataset of Parkinson's disease and confirmed previous findings that the RET proto-oncogene is associated with the disease. In this study, we showed that using HH interaction analysis is possible to assist researchers in gaining more insight into identifying genetic risk factors for complex diseases.     

Searching and mining visually observed phenotypes of maize mutants

There are thousands of maize mutants, which are invaluable resources for plant research. Geneticists use them to study underlying mechanisms of biochemistry, cell biology, cell development, and cell physiology. To streamline the understanding of such complex processes, researchers need the most current versions of genetic and physical maps, tools with the ability to recognize novel phenotypes or classify known phenotypes, and an intimate knowledge of the biochemical processes generating physiological and phenotypic effects. They must also know how all of these factors change and differ among species, diverse alleles, germplasms, and environmental conditions. While there are robust databases, such as MaizeGDB, for some of these types of raw data, other crucial components are missing. Moreover, the management of visually observed mutant phenotypes is still in its infant stage, let alone the complex query methods that can draw upon high-level and aggregated information to answer the questions of geneticists. In this paper, we address the scientific challenge and propose to develop a robust framework for managing the knowledge of visually observed phenotypes, mining the correlation of visual characteristics with genetic maps, and discovering the knowledge relating to cross-species conservation of visual and genetic patterns. The ultimate goal of this research is to allow a geneticist to submit phenotypic and genomic information on a mutant to a knowledge base and ask, "What genes or environmental factors cause this visually observed phenotype?".     

Fibrin-Induced Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells as a Mechanism of Peritoneal Fibrosis: Effects of Pentoxifylline

Excessive fibrin deposition in the peritoneum is thought to be involved in the development of encapsulating peritoneal sclerosis (EPS), an important cause of morbidity and mortality in peritoneal dialysis patients. We investigated fibrin-induced epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) as a possible mechanism of fibrin involvement in EPS. In vitro, fibrin overlay of PMCs altered their morphology; increased α-smooth muscle actin, fibronectin, fibroblast specific protein-1, and α_vβ₃ integrin expression; and decreased cytokeratin 18 and E-cadherin expression. Fibrin overlay also increased focal adhesion kinase and Src kinase phosphorylation. Fibrin-induced changes were inhibited by treating the cells with α_vβ₃ integrin antibody or pentoxifylline (PTX). In a rat model, intraperitoneal injection of Staphylococcus aureus and fibrinogen induced severe EPS features, which were attenuated by PTX treatment. PTX-treated rats also showed preserved peritoneal ultrafiltration function and lower concentrations of cytokines than the untreated rats. S. aureus- and fibrinogen-injected rats had higher percentage of cytokeratin-positive cells in the omentum fibrotic tissue than controls; this was also reduced by PTX treatment. Our results suggest that fibrin induces EMT of PMCs by engaging α_vβ₃ integrin and activating associated kinases. Our EPS animal model showed that fibrin-induced EMT was involved in the pathogenesis of peritoneal fibrosis and was inhibited by PTX.     

Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans

Protein interactions are essential components of signal transduction in cells. With the progress in genome-wide yeast two hybrid screens and proteomics analyses, many protein interaction networks have been generated. These analyses have identified hundreds and thousands of interactions in cells and organisms, creating a challenge for further validation under physiological conditions. The bimolecular fluorescence complementation (BiFC) assay is such an assay that meets this need. The BiFC assay is based on the principle of protein fragment complementation, in which two non-fluorescent fragments derived from a fluorescent protein are fused to a pair of interacting partners. When the two partners interact, the two non-fluorescent fragments are brought into proximity and an intact fluorescent protein is reconstituted. Hence, the reconstituted fluorescent signals reflect the interaction of two proteins under study. Over the past six years, the BiFC assay has been used for visualization of protein interactions in living cells and organisms, including our application of the BiFC assay to the transparent nematode Caenorhabditis elegans. We have demonstrated that BiFC analysis in C. elegans provides a direct means to identify and validate protein interactions in living worms and allows visualization of temporal and spatial interactions. Here, we provide a guideline for the implementation of BiFC analysis in living worms and discuss the factors that are critical for BiFC analysis.     

Cloning and functional characterization of the HRASLS2 gene

The HRAS-like suppressor 2 (HRASLS2) gene belongs to the H-REV107 gene family involved in the regulation of cell growth and differentiation. HRASLS2 is expressed at high levels in normal tissues of the small intestine, kidney, and trachea. We cloned HRASLS2 cDNA from human SW480 colon cancer cells. Most wild-type, and some N- and C-terminal truncated HRASLS2 (HRASLS2DeltaNDeltaC) were expressed as a granular pattern located at perinuclear region in HtTA cervical cancer cells, while truncation at the C-terminus only (HRASLS2DeltaC) resulted in a diffuse pattern. Wild-type HRASLS2 significantly suppressed colony formation of HeLa and HCT116 cells. HRASLS2DeltaNDeltaC significantly inhibited colony formation of HCT116 cells, but HRASLS2DeltaC did not affect cell growth. HRASLS2 suppressed the RAS-GTP levels and total RAS protein by 44% and 25%, respectively in HtTA cells; however, the suppression was not observed in truncated HRASLS2 variants. In conclusion, the HRASLS2 protein suppressed growth and RAS activities of cancer cells, and the C-terminal hydrophobic domain appeared to be indispensable for both activities.