dXNP, a Drosophila homolog of XNP/ATRX, induces apoptosis via Jun-N-terminal kinase activation

XNP/ATRX, a causative gene of X-linked alpha-thalassemia/mental retardation syndrome, encodes an SNF2 family ATPase/helicase protein. To better understand the role of XNP/ATRX in development, we isolated and characterized a Drosophila XNP/ATRX homolog, dXNP, which contains highly conserved SNF2 and helicase domains. Ectopically expressed dXNP induced strong apoptosis in the developing eye and wing, but did not affect cell cycle progression or the expression of wingless and engrailed, essential regulators of development. The dXNP-induced apoptosis was strongly suppressed by DJNKK/hemipterous mutation, and dXNP increased JNK activity. Taken together, these results suggest that dXNP regulates apoptosis via JNK activation.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.     

Association of CD38 with nonmuscle myosin heavy chain IIA and Lck is essential for the internalization and activation of CD38

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca(2+) concentration ([Ca(2+)](i)), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca(2+)](i), and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.     

Detection of proteins using a colorimetric bio-barcode assay

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).     

Establishment of a biomimetic device based on tri-layer polymer actuators--propulsion fins

We propose to use bending type tri-layer polymer actuators as propulsion fins for a biomimetic device consisting of a rigid body, like a box fish having a carapace, and paired fins running through the rigid body, like a fish having pectoral fins. The fins or polymer bending actuators can be considered as individually controlled flexible membranes. Each fin is activated with sinusoidal inputs such that there is a phase lag between the movements of successive fins to create enough thrust force for propulsion. Eight fins with 0.125 aspect ratio have been used along both sides of the rigid body to move the device in the direction perpendicular to the longitudinal axis of the body. The designed device with the paired fins was successfully tested, moving in an organic solution consisting of solvent, propylene carbonate (PC), and electrolyte. The design procedure outlined in this study is offered as a guide to making functional devices based on polymer actuators and sensors.     

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

Genetic variation,demographic history and phylogeography of tire track eel,Mastacembelus favus (Synbranchiformes: Mastacembelidae) in Southeast Asia

The complex climatic and geological history of Southeast Asia has been hypothesised to determine the most important aspects of the current phylogeographical structure and distribution of living organisms throughout the region. To test existing hypotheses, the genetic structure of the tire track eel, Mastacembelus favus, was investigated using 823 bp of mitochondrial DNA cytochrome b from 469 individuals from 51 localities encompassing its native range. The results classified all haplotypes into two major lineages, Lineage 1, which was further divided into Lineages 1a (lower Mekong, eastern Gulf of Thailand and Malay—Thai Peninsula), 1b (Banpakong River), 1c (Chao Phraya, Gulf of Thailand and Malay—Thai Peninsula) and 1d (Khlang Yai River), and Lineage 2, the upper reaches of the lower Mekong and the middle Mekong. Strong genetic discontinuities dated approximately 5 MYA were discovered in the Mekong with limited geographical overlap, suggesting a historically dissected drainage between two sections and species colonisation via different routes. The widespread Lineage 1 showed a strong signature of population expansion during the Pleistocene climate oscillation. Haplotype characteristics in the Malay—Thai Peninsula are hypothesised to result from postglacial dispersal from the Mekong and Chao Phraya through an extended Pleistocene drainage network.