Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.     

Expression analysis of decapentaplegic gene in the silkworm,Bombyx mori

The decapentaplegic (dpp) gene in Drosophila is involved in multiple developmental processes, and is a highly conserved among various eukaryotic species, including Bombyx mori. Although the gene has well been characterized in Drosophila species the B. mori dpp has not yet been functionally analyzed. In this study, we analyzed the expression pattern of B. mori dpp in 12 different developmental days/stages (7 days for fifth instar larvae, 2 days for spinning stage, 2 days for pupal stages, and 1 day for adults) in both male and female silkworms using quantitative real‐time RT‐PCR (qRT‐PCR). mRNA expression of B. mori dpp was much higher in the female larvae up to the mid‐stage of the fifth instar compared with the corresponding male larvae. Similarly, dpp expression also was much higher in females during the eclosion period than that in the corresponding male pupae. During the embryonic stage, the expression level of the dpp gene was much higher compared to that of adult stage in both male and female silkworms. These results suggest that the B. mori dpp gene plays multiple roles in the developmental of B. mori.     

Syntaxin 16 Binds to Cystic Fibrosis Transmembrane Conductance Regulator and Regulates Its Membrane Trafficking in Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a key membrane protein in the complex network of epithelial ion transporters regulating epithelial permeability. Syntaxins are one of the major determinants in the intracellular trafficking and membrane targeting of secretory proteins. In the present study we demonstrate the biochemical and functional association between CFTR and syntaxin 16 (STX16) that mediates vesicle transport within the early/late endosomes and trans-Golgi network. Immunoprecipitation experiments in rat colon and T84 human colonic epithelial cells indicate that STX16 associates with CFTR. Further analyses using the domain-specific pulldown assay reveal that the helix domain of STX16 directly interacts with the N-terminal region of CFTR. Immunostainings in rat colon and T84 cells show that CFTR and STX16 highly co-localize at the apical and subapical regions of epithelial cells. Interestingly, CFTR-associated chloride current was reduced by the knockdown of STX16 expression in T84 cells. Surface biotinylation and recycling assays indicate that the reduction in CFTR chloride current is due to decreased CFTR expression on the plasma membrane. These results suggest that STX16 mediates recycling of CFTR and constitutes an important component of CFTR trafficking machinery in intestinal epithelial cells.     

Endangered wolves cloned from adult somatic cells

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.     

A Combination of Broadly Neutralizing HIV-1 Monoclonal Antibodies Targeting Distinct Epitopes Effectively Neutralizes Variants Found in Early Infection

Neutralizing antibody protection against HIV-1 may require broad and potent antibodies targeting multiple epitopes. We tested 7 monoclonal antibodies (MAbs) against 45 viruses of diverse subtypes from early infection. The CD4 binding site MAb NIH45-46W was most broad and potent (91% coverage; geometric mean 50% inhibitory concentration [IC(50)], 0.09 μg/ml). Combining NIH45-46W and a V3-specific MAb, PGT128, neutralized 96% of viruses, while PGT121, another V3-specific MAb, neutralized the remainder. Thus, 2 or 3 antibody specificities may prevent infection by most HIV-1 variants.     

Sulfiredoxin protein is critical for redox balance and survival of cells exposed to low steady-state levels of H2O2

Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins (Prxs). Having high affinity toward H2O2, 2-Cys Prxs can efficiently reduce H2O2 at low concentration. We previously showed that Prx I is hyperoxidized at a rate of 0.072% per turnover even in the presence of low steady-state levels of H2O2. Here we examine the novel role of Srx in cells exposed to low steady-state levels of H2O2, which can be achieved by using glucose oxidase. Exposure of low steady-state levels of H2O2 (10-20 μm) to A549 or wild-type mouse embryonic fibroblast (MEF) cells does not lead to any significant change in oxidative injury because of the maintenance of balance between H2O2 production and elimination. In contrast, loss-of-function studies using Srx-depleted A549 and Srx-/- MEF cells demonstrate a dramatic increase in extra- and intracellular H2O2, sulfinic 2-Cys Prxs, and apoptosis. Concomitant with hyperoxidation of mitochondrial Prx III, Srx-depleted cells show an activation of mitochondria-mediated apoptotic pathways including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Furthermore, adenoviral re-expression of Srx in Srx-depleted A549 or Srx-/- MEF cells promotes the reactivation of sulfinic 2-Cys Prxs and results in cellular resistance to apoptosis, with enhanced removal of H2O2. These results indicate that Srx functions as a novel component to maintain the balance between H2O2 production and elimination and then protects cells from apoptosis even in the presence of low steady-state levels of H2O2.     

Crystal structure of phosphoethanolamine methyltransferase from Plasmodium falciparum in complex with amodiaquine

Phosphoethanolamine N-methyltransferase (PMT) is essential for phospholipid biogenesis in the malarial parasite Plasmodium falciparum. PfPMT catalyzes the triple methylation of phosphoethanolamine to produce phosphocholine, which is then used for phosphatidylcholine synthesis. Here we describe the 2.0? resolution X-ray crystal structure of PfPMT in complex with amodiaquine. To better characterize inhibition of PfPMT by amodiaquine, we determined the IC(50) values of a series of aminoquinolines using a direct radiochemical assay. Both structural and functional analyses provide a possible approach for the development of new small molecule inhibitors of PfPMT.