The small nuclear ribonucleoprotein U1A interacts with NS5 from yellow fever virus

The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.

Utp25p, a nucleolar Saccharomyces cerevisiae protein, interacts with U3 snoRNP subunits and affects processing of the 35S pre-rRNA

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. Two intermediate complexes, pre-40S and pre-60S, are formed at the early stages of 35S pre-rRNA processing and give rise to the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, some ribosomal proteins and processing factors. The novel yeast protein Utp25p has previously been identified in the nucleolus, an indication that this protein could be involved in ribosome biogenesis. Here we show that Utp25p interacts with the SSU processome proteins Sas10p and Mpp10p, and affects 18S rRNA maturation. Depletion of Utp25p leads to accumulation of the pre-rRNA 35S and the aberrant rRNA 23S, and to a severe reduction in 40S ribosomal subunit levels. Our results indicate that Utp25p is a novel SSU processome subunit involved in pre-40S maturation.

Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. Acute hyperglycemia increased synthesis of intrarenal Ang I and Ang II and resulted in activation of both Ang II receptors, AT1 and AT2, in the kidney. Losartan (specific AT1 antagonist) or PD123319 (specific AT2 antagonist) did not affect hyperglycemia but prevented activation of renal AT1 and AT2, respectively. In murine renal cortex, acute hyperglycemia increased VEGF protein but not mRNA content after 24 h, which suggested translational regulation. Blockade of AT2, but not AT1, prevented increase in VEGF synthesis by inhibiting translation of VEGF mRNA in renal cortex. Acute hyperglycemia increased VEGF expression in wild type but not in AT2 knockout mice. Binding of heterogeneous nuclear ribonucleoprotein K to VEGF mRNA, which stimulates its translation, was prevented by blockade of AT2, but not AT1. The Akt-mTOR-p70(S6K) signaling pathway, involved in the activation of mRNA translation, was activated in hyperglycemic kidneys and was blocked by the AT2 antagonist. Elongation phase is an important step of mRNA translation that is controlled by elongation factor 1A (eEF1A) and 2 (eEF2). Expression of eEF1A and activity of eEF2 was higher in kidney cortex from hyperglycemic mice and only the AT2 antagonist prevented these changes. To assess selectivity of translational control of VEGF expression, we measured expression of fibronectin (FN) and laminin beta 1 (lam beta 1): acute hyperglycemia increased FN expression at both protein and mRNA levels, indicating transcriptional control, and did not affect the expression of lam beta 1. To confirm results obtained with PD123319, we induced hyperglycemia in AT2 knockout mice and found that in the absence of AT2, translational control of VEGF expression by hyperglycemia was abolished. Our data show that acute hyperglycemia stimulates Ang II synthesis in murine kidney cortex, this leads to AT2 activation and stimulation of VEGF mRNA translation, via the Akt-mTOR-p70(S6K) signaling pathway. Our data show that exclusive translational control of protein expression in the kidney by acute hyperglycemia is not a general phenomenon, but do not prove that it is restricted to VEGF. (C) 2010 Elsevier Inc. All rights reserved.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Phylogeography of the common vampire bat (Desmodus rotundus): Marked population structure, Neotropical Pleistocene vicariance and incongruence between nuclear and mtDNA markers

Background: The common vampire bat Desmodus rotundus is an excellent model organism for studying ecological vicariance in the Neotropics due to its broad geographic range and its preference for forested areas as roosting sites. With the objective of testing for Pleistocene ecological vicariance, we sequenced a mitocondrial DNA (mtDNA) marker and two nuclear markers (RAG2 and DRB) to try to understand how Pleistocene glaciations affected the distribution of intraspecific lineages in this bat. Results: Five reciprocally monophyletic clades were evident in the mitochondrial gene tree, and in most cases with high bootstrap support: Central America (CA), Amazon and Cerrado (AMC), Pantanal (PAN), Northern Atlantic Forest (NAF) and Southern Atlantic Forest (SAF). The Atlantic forest clades formed a monophyletic clade with high bootstrap support, creating an east/west division for this species in South America. On the one hand, all coalescent and non-coalescent estimates point to a Pleistocene time of divergence between the clades. On the other hand, the nuclear markers showed extensive sharing of haplotypes between distant localities, a result compatible with male-biased gene flow. In order to test if the disparity between the mitochondrial and nuclear markers was due to the difference in mutation rate and effective size, we performed a coalescent simulation to examine the feasibility that, given the time of separation between the observed lineages, even with a gene flow rate close to zero, there would not be reciprocal monophyly for a neutral nuclear marker. We used the observed values of theta and an estimated mutation rate for the nuclear marker gene to perform 1000 iterations of the simulation. The results of this simulation were inconclusive: the number of iterations with and without reciprocal monophyly of one or more clades are similar. Conclusions: We therefore conclude that the pattern exhibited by the common vampire bat, with marked geographical structure for a mitochondrial marker and no phylogeographic structure for nuclear markers is compatible with a historical scenario of complete isolation of refuge-like populations during the Pleistocene. The results on demographic history on this species is compatible with the Carnaval-Moritz model of Pleistocene vicariance, with demographic expansions in the southern Atlantic forest.

Long range rapidity correlations and jet production in high energy nuclear collisions

The STAR Collaboration at the Relativistic Heavy Ion Collider presents a systematic study of high-transverse-momentum charged-di-hadron correlations at small azimuthal pair separation Delta phi in d+Au and central Au+Au collisions at s(NN)=200 GeV. Significant correlated yield for pairs with large longitudinal separation Delta eta is observed in central Au+Au collisions, in contrast to d+Au collisions. The associated yield distribution in Delta eta x Delta phi can be decomposed into a narrow jet-like peak at small angular separation which has a similar shape to that found in d+Au collisions, and a component that is narrow in Delta phi and depends only weakly on Delta eta, the ""ridge."" Using two systematically independent determinations of the background normalization and shape, finite ridge yield is found to persist for trigger p(t)>6 GeV/c, indicating that it is correlated with jet production. The transverse-momentum spectrum of hadrons comprising the ridge is found to be similar to that of bulk particle production in the measured range (2 < p(t)< 4 GeV/c).

Haplotype distribution of five nuclear genes based on network genealogies and Bayesian inference indicates that Trypanosoma cruzi hybrid strains are polyphyletic

Chagas disease is still a major public health problem in Latin America. Its causative agent, Trypanosoma cruzi, can be typed into three major groups, T. cruzi I, T. cruzi II and hybrids. These groups each have specific genetic characteristics and epidemiological distributions. Several highly virulent strains are found in the hybrid group; their origin is still a matter of debate. The null hypothesis is that the hybrids are of polyphyletic origin, evolving independently from various hybridization events. The alternative hypothesis is that all extant hybrid strains originated from a single hybridization event. We sequenced both alleles of genes encoding EF-1 alpha, actin and SSU rDNA of 26 T. cruzi strains and DHFR-TS and TR of 12 strains. This information was used for network genealogy analysis and Bayesian phylogenies. We found T. cruzi I and T. cruzi II to be monophyletic and that all hybrids had different combinations of T. cruzi I and T. cruzi II haplotypes plus hybrid-specific haplotypes. Bootstrap values (networks) and posterior probabilities (Bayesian phylogenies) of clades supporting the monophyly of hybrids were far below the 95% confidence interval, indicating that the hybrid group is polyphyletic. We hypothesize that T. cruzi I and T. cruzi II are two different species and that the hybrids are extant representatives of independent events of genome hybridization, which sporadically have sufficient fitness to impact on the epidemiology of Chagas disease.

Clinicopathological evaluation of in vivo epidermal nuclear fluorescence

Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear. To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples. A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF. The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus (n = 9), mixed CTD (n = 3), overlap syndrome (n = 3), Sjogren`s syndrome (n = 1), and CREST (calcinosis, Raynaud`s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome (n = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%. The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.

Phylogeny of the Neotropical genus Acestrorhynchus (Ostariophysi: Characiformes) based on nuclear and mitochondrial gene sequences and morphology: A total evidence approach

Acestrorhynchus is the sole genus of the family Acestrorhynchidae which includes 14 species currently recognized as valid. Species of Acestrorhynchus comprise small-to-medium sized piscivorous fishes and have been traditionally grouped on the basis of well-defined color patterns. A recent phylogeny, based on morphological characters, could not resolve the phylogenetic affinities of A. heterolepis and the relationships among the species of the clade formed by A. abbreviatus, A. altus, A. falcatus, A. lacustris, and A. pantaneiro. The simultaneous analysis of two mitochondrial genes (16S and ATP synthase subunits 6 and 8) and one nuclear intron (S7) was able to resolve the latter clade, but the position of A. heterolepis remained unresolved. The combination of the molecular and morphological data sets in a total evidence analysis resulted in a well-resolved hypothesis regarding the phylogenetic relationships of Acestrorhynchus species. (C) 2009 Elsevier Inc. All rights reserved.

Phosphoproteomics Profiling Suggests a Role for Nuclear beta IPKC in Transcription Processes of Undifferentiated Murine Embryonic Stem Cells

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal and differentiation However, the function of specific PKC Isoenzymes have yet to be determined Of the PKCs expressed in undifferentiated ESCs, beta IPKC was the only isoenzyme abundantly expressed in the nuclei To investigate the role of beta IPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one beta IPKC-specific inhibitor peptide We identified 13 nuclear proteins that are direct or indirect beta IPKC substrates in undifferentiated ESCs These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation Inhibiting beta IPKC had no effect on DNA synthesis in undifferentiated ESCs However, upon differentiation many cells seized to express beta IPKC and beta IPKC was frequently found in the cytoplasm Taken together, our results suggest that beta IPKC takes part in the processes that maintain ESCs in their undifferentiated state

Results of Joint Experiments and other IAEA activities on research using small tokamaks

This paper presents an overview of the results obtained during the Joint Experiments organized in the framework of the IAEA Coordinated Research Project on `Joint Research Using Small Tokamaks` that have been carried out on the tokamaks CASTOR at IPP Prague, Czech Republic (2005), T-10 at RRC `Kurchatov Institute`, Moscow, Russia (2006), and the most recent one at ISTTOK at IST, Lisbon, Portugal, in 2007. Experimental programmes were aimed at diagnosing and characterizing the core and the edge plasma turbulence in a tokamak in order to investigate correlations between the occurrence of transport barriers, improved confinement, electric fields and electrostatic turbulence using advanced diagnostics with high spatial and temporal resolution. On CASTOR and ISTTOK, electric fields were generated by biasing an electrode inserted into the edge plasma and an improvement of the global particle confinement induced by the electrode positive biasing has been observed. Geodesic acoustic modes were studied using heavy ion beam diagnostics on T-10 and ISTTOK and correlation reflectometry on T-10. ISTTOK is equipped with a gallium jet injector and the technical feasibility of gallium jets interacting with plasmas has been investigated in pulsed and ac operation. The first Joint Experiments have clearly demonstrated that small tokamaks are suitable for broad international cooperation to conduct dedicated joint research programmes. Other activities within the IAEA Coordinated Research Project on Joint Research Using Small Tokamaks are also overviewed.

Constraining the nuclear gluon distribution in eA processes at RHIC

A systematic determination of the gluon distribution is of fundamental interest in understanding the parton structure Of nuclei and the QCD dynamics. Currently, the behavior of this distribution at small x (high energy) is completely undefined. In this Letter we analyze the possibility of constraining the nuclear effects present in Xg(A) using the inclusive observables which would be measured in the future electron-nucleus collider at RHIC. We demonstrate that the Study of nuclear longitudinal and charm structure functions allows to estimate the magnitude of shadowing and antishadowing effects in the nuclear gluon distribution. (C) 2008 Elsevier B.V. All rights reserved.

Cwc24p, a novel Saccharomyces cerevisiae nuclear ring finger protein, affects pre-snoRNA U3 splicing

U3 snoRNA is transcribed from two intron-containing genes in yeast, snR17A and snR17B. Although the assembly of the U3 snoRNP has not been precisely determined, at least some of the core box C/D proteins are known to bind pre-U3 co-transcriptionally, thereby affecting splicing and 3 `-end processing of this snoRNA. We identified the interaction between the box C/D assembly factor Nop17p and Cwc24p, a novel yeast RING finger protein that had been previously isolated in a complex with the splicing factor Cef1p. Here we show that, consistent with the protein interaction data, Cwc24p localizes to the cell nucleus, and its depletion leads to the accumulation of both U3 pre-snoRNAs. U3 snoRNA is involved in the early cleavages of 35 S pre-rRNA, and the defective splicing of pre-U3 detected in cells depleted of Cwc24p causes the accumulation of the 35 S precursor rRNA. These results led us to the conclusion that Cwc 24p is involved in pre-U3 snoRNA splicing, indirectly affecting pre-rRNA processing.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.