Complete genome sequence of Burkholderia gladioli BSR3

We report the complete genome sequence of Burkholderia gladioli BSR3, isolated from a diseased rice sheath in South Korea.     

Expression of truncated Babesia gibsoni thrombospondin-related adhesive proteins in Escherichia coli and evaluation of their diagnostic potential by enzyme-linked immunosorbent assay

Among the previously established enzyme-linked immunosorbent assays (ELISAs), an ELISA using the full length of a recombinant thrombospondin-related adhesive protein of Babesia gibsoni (rBgTRAPf) is considered as the most sensitive diagnostic method for the detection of an antibody to B. gibsoni in dogs. However, the expression of rBgTRAPf in high concentration is poor and, thus, limits its usefulness as a diagnostic antigen. To improve its expression level, we have truncated BgTRAPf into two fragments having either an N- or a C-terminus (BgTRAPn or BgTRAPc, respectively). The expression of BgTRAPc protein in Escherichia coli yielded adequate recombinant protein. The specificity and sensitivity of ELISAs with the truncated proteins were determined using dog sera experimentally infected with B. gibsoni and specific pathogen-free (SPF) dog sera. A total of 254 field dog sera were examined by the ELISA with rBgTRAPn, rBgTRAPc, and rBgTRAPf as well as by an indirect fluorescent antibody test (IFAT). The specificity of rBgTRAPc was the highest (97.15%), and its kappa value was more (0.8003) than rBgTRAPn (0.7083). With a sufficient level of expression as well as higher specificity and reliable sensitivity, rBgTRAPc appears to be a potential candidate antigen for the serodiagnosis of B. gibsoni infection in dogs.     

Glutathione peroxidase 3 of Saccharomyces cerevisiae suppresses non-enzymatic proteolysis of glutamine synthetase in an activity-independent manner

Glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and is important antioxidant enzyme in yeast. It modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathway and protein translocation. Through immunoprecipitation/two-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay, we found glutamine synthetase (GS; EC 6.3.1.2) as a candidate interacting protein with Gpx3. GS is a key enzyme in nitrogen metabolism and ammonium assimilation. It has been known that GS is non-enzymatically cleaved by ROS generated by MFO (thiol/ Fe(3+)/O(2) mixed-function oxidase) system. In this study, it is demonstrated that GS interacts with Gpx3 through its catalytic domain both in vivo and in vitro regardless of redox state. In addition, Gpx3 helps to protect GS from inactivation and degradation via oxidative stress in an activity-independent manner. Based on the results, it is suggested that Gpx3 protects GS from non-enzymatic proteolysis, thereby contributing to cell homeostasis when cell is exposed to oxidative stress.     

Influence of season and parity on the recovery of in vivo canine oocytes by flushing fallopian tubes

Assisted reproductive technology (ART) in dogs largely depends on the in vivo matured oocytes due to lack of a suitable in vitro maturation system. The present study evaluated the technique of flushing fallopian tubes to collect in vivo matured canine oocytes by laparotomy, and determined the effects of seasons, and parity of donor bitches on the success of oocyte retrieval. Oocytes were retrieved from anesthetized bitches by laparotomy. About 7 ml of TCM-199 supplemented with HEPES was used to flush each individual fallopian tube. Oocytes were categorized as good, fair, poor, immature or aged based on the nuclear stage, cumulus cell layers, color and homogeneity of ooplasm. Oocytes categorized as being good or fair were considered usable, while poor, aged or immature oocytes were considered unusable for ART. A significantly higher number of oocytes per bitch were retrieved during the spring (11.2) compared to the winter (7.9). The oocyte recovery rates were 89.4, 92.2, 89.7 and 89.3% for spring, summer, autumn, and winter, respectively. The highest percentage of usable oocytes (74.7%) was retrieved during autumn (P>0.05). The number of oocytes was influenced by the parity of the donor bitch. Significantly more oocytes were collected from the multiparous bitches (10.3) compared to nulliparous bitches (7.7). The percentage of usable oocytes was more in multipara (71.5%) compared to nullipara (64.7%) (P>0.05). Collection of in vivo produced oocytes by laparotomy represents a potential source of matured oocytes for ART in dogs.     

Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance

Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.     

The hsp70 inhibitor VER155008 induces paraptosis requiring de novo protein synthesis in anaplastic thyroid carcinoma cells

In this study, we evaluated the effect of the hsp70 inhibitor VER155008 on survival of anaplastic thyroid carcinoma (ATC) cells. In ATC cells, VER155008 increased the percentages of dead cells and vacuolated cells. VER155008 did not lead to the cleavage of caspase-3 protein regardless of pretreatment with z-VAD-fmk. VER155008 increased LC3-II protein levels but the protein levels were not changed by autophagy inhibitors. VER155008 caused the dilatation of endoplasmic reticulum (ER), and the increased mRNA levels of Bip and CHOP, suggesting paraptosis. VER155008-induced paraptosis was attenuated by pretreatment with cycloheximide. In conclusion, VER155008 induces paraptosis characterized by cytoplasmic vacuolation, independence of caspase, dilatation of ER and induction of ER stress markers in ATC cells. Moreover, VER155008-induced paraptosis requires de novo protein synthesis in ATC cells.     

Interleukin-1β-Induced Wnt5a Enhances Human Corneal Endothelial Cell Migration through Regulation of Cdc42 and RhoA

Wnt5a can activate β-catenin-independent pathways for regulation of various cellular functions, such as migration, that play critical roles in wound repair. Investigation of Wnt5a signaling may help identify therapeutic targets for enhancing corneal endothelial wound healing that could provide an alternative to corneal transplantation in patients with blindness from endothelial dysfunction. However, Wnt5a signaling in corneal endothelial cells (CECs) has not been well characterized. In this study, we show transient induction of Wnt5a by interleukin-1β (IL-1β) stimulation proceeds through NF-κB in human CECs. This leads to binding of Fzd5 to Ror2, resulting in activation of disheveled protein (Dvl) and subsequently disheveled-associated activator of morphogenesis 1 (DAAM1). This leads to activation of Cdc42 and subsequent inhibition of RhoA. Inhibition of RhoA leads to parallel dephosphorylation and inactivation of LIM domain kinase 2 along with dephosphorylation and activation of slingshot 1, resulting in dephosphorylation and activation of cofilin and leading to enhanced cell migration. These findings suggest that Wnt5a enhances cell migration through activation of Cdc42 and inactivation of RhoA in human CECs.     

Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane‐anchored EGFP

Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane‐anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'‐3'‐cyclic nucleotide 3'‐phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt‐Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin‐induced demyelination animal model. Taken together, the membrane‐anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries. genesis 52:341–349, 2014. © 2014 Wiley Periodicals, Inc.