Characterization of Avian Paramyxoviruses Isolated from Feral Ducks in Northern Japan: The Presence of Three Distinct Viruses in Nature

Seventy-eight strains of avian paramyxoviruses (PMV) were isolated from cloacal and/or tracheal swabs taken from 1,342 feral ducks, comprised of spot-bill ducks, mallards, pintails, teals, falcated teals, wigeons and buffie-heads, in Wakuya-cho, Miyagi Prefecture, Japan, between 1976 and 1979. Five and a half percent of the ducks were positive for virus. Serological and structural characterization indicated that three different avian paramyxoviruses arc prevalent in the Japanese feral duck population. The first group of PMV was Newcastle disease virus (NDV), and in vivo pathogenecity tests in embryonated chicken eggs and 1-day-old chicks revealed that all the NDV strains isolated were avirulent. The second and most prevalent strain was closely related to PMV-4, duck/Hong Kong/D3/75 strain. The viruses of the third group were recovered only from pintails. They cross-reacted antigenically with PMV-3 when antisera to the PMV-3 reference strains, turkey/Wisconsin/68 and parakeet/Netherlands/449/75, were employed. However, no cross-reaction was observed when antiserum to pintail/ Wakuya/20/78, the prototype of this group, was used. The viruses of the third group also differed in viral polypeptide profile from the reference strains of PMV-3.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Isoform-specific O-Glycosylation of Osteopontin and Bone Sialoprotein by Polypeptide N-Acetylgalactosaminyltransferase-1

Mucin-type O-glycan biosynthesis is regulated by the family of UDP-GalNAc polypeptide:N-acetylgalactosaminlytransfersases (ppGalNAcTs) that catalyzes the first step in the pathway by transferring GalNAc to Ser or Thr residues in a protein from the sugar donor UDP-GalNAc. Because not all Ser/Thr residues are glycosylated, rules must exist that signal which hydroyxamino acids acquire sugar. To date, no universal consensus signal has emerged. Therefore, strategies to deduce the subset of proteins that will be glycosylated by distinct ppGalNAcTs must be developed. Mucin-type O-glycoproteins are present abundantly in bone, where we found multiple ppGalNAcT isoforms, including ppGalNAcT-1, to be highly expressed. Thus, we compared glycoproteins expressed in wild-type and Galnt1-null mice to identify bone-associated proteins that were glycosylated in a ppGalNAcT-1-dependent manner. A reduction in the apparent molecular masses of two SIBLINGs (small integrin binding ligand N-linked glycoproteins), osteopontin (OPN) and bone sialoprotein (BSP) in the Galnt1-null mice relative to those of the wild-type was observed. Several synthetic peptides derived from OPN and BSP sequences were designed to include either known or predicted (in silico) glycosylation sites. In vitro glycosylation assays of these peptides with recombinant ppGalNAcT-1, ppGalNAcT-2, or ppGalNAcT-3 demonstrated that both SIBLINGs contained Thr/Ser residues that were preferentially glycosylated by ppGalNAcT-1. In addition, lysates prepared from wild-type, but not those from Galnt1-null derived osteoblasts, could glycosylate these peptides efficiently, suggesting that OPN and BSP contain sites that are specific for ppGalNAcT-1. Our study presents a novel and systematic approach for identification of isoform-specific substrates of the ppGalNAcT family and suggests ppGalNAcT-1 to be indispensable for O-glycosylation at specific sites of the bone glycoproteins OPN and BSP.     

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

Background

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.

Methods

Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X₇ receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X₇ receptor.

Results

Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X₇ receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X₇ receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X₇ receptor channel plays a significant role in mediating the ATP release.

General Significance

We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.     

Improving genome assemblies by sequencing PCR products with PacBio

Advances in sequencing technologies have dramatically reduced costs in producing high-quality draft genomes. However, there are still many contigs and possible misassembled regions in those draft genomes. Improving the quality of these genomes requires an efficient and economical means to close gaps and resequence some regions. Sequencing pooled gap region PCR products with Pacific Biosciences (PacBio) provides a significantly less expensive means for this need. We have developed a genome improvement pipeline with this strategy after decreasing a loading bias against larger PCR products in the PacBio process. Compared with Sanger technology, this approach is not only cost-effective but also can close gaps greater than 2.5 kb in a single round of reactions, and sequence through high GC regions as well as difficult secondary structures such as small hairpin loops.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Studies on substantially increased proteins in follicular fluid of bovine ovarian follicular cysts using 2-D PAGE and MALDI-TOF MS

Background  

The objective of this study was to identify substantially increased proteins in bovine cystic follicular fluid (FF) in order to clarify the pathology and etiology of bovine ovarian follicular cysts (BOFC).     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.