Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.     

Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method

Active nuclear import of proteins requires the recognition of a nuclear localization sequence (NLS) by members of the importin (IMP) family of proteins. We have developed a modified AlphaScreen-based assay able to estimate the solution binding affinities of such interactions using biotinylated IMPs and His6-tagged NLS-containing proteins. We describe this assay in detail as well as its application in documenting the phenomenon of intramolecular masking of NLSs using recombinant green fluorescent protein (GFP) fusion proteins containing sequences from the SV40 large tumor T antigen (T-ag). We also use it to examine, for the first time, IMP binding to the cancer cell-specific proapoptotic factor viral protein 3 (VP3) from the chicken anemia virus (CAV). High-affinity binding of the IMPalpha/beta heterodimer to the T-ag NLS was observed when the GFP tag was fused to its N terminus but not to its C terminus. Effects of flanking residues were also observed in GFP-T-ag fusion derivatives containing the Thr128 NLS-inactivating mutation, whereby the absence of flanking sequences N terminal to the T-ag NLS appeared to decrease the specificity of the mutation in terms of oblating IMPalpha/beta binding. IMPbeta, but not IMPalpha or the IMPalpha/beta heterodimer, was found to bind to CAV VP3 with high affinity. Interestingly, GFP-VP3(74-121) bound to IMPbeta with threefold higher affinity than the full-length protein, GFP-VP3(1-121), implying that the NLS is masked to a significant extent in the context of full-length protein. This may represent a regulatory mechanism to control nuclear import in a tumor cell-specific fashion.     

A novel multiplex PCR/RFLP assay for the identification of Streptococcus bovis/Streptococcus equinus complex members from dairy microbial communities based on the 16S rRNA gene

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises pathogenic species associated with different degrees with human infections but also spontaneously fermented dairy products. We aimed therefore at developing a specific identification assay for the SBSEC targeting the 16S rRNA gene comprising a multiplex PCR followed by a differentiating restriction fragment length polymorphisms (RFLP). The multiplex PCR assay was positively applied on 200 SBSEC isolates including reference strains. The assay did not yield false-positive amplifications with strains of closely related bacteria and isolates of non-SBSEC streptococci, lactococci, enterococci, and other genera of dairy origin. The downstream RFLP using MseI and XbaI enabled further discrimination of Streptococcus infantarius/S.?bovis (biotype II.1) from Streptococcus gallolyticus (biotype I and II.2)/Streptococcus alactolyticus and S.?equinus. Furthermore, the newly developed primers can be used directly for Sanger sequencing. Conclusively, this novel PCR/RFLP assay is applicable in the complex dairy microbial communities and provides an important tool to assess the prevalence of members of the SBSEC in dairy products.     

Characterization of new microsatellite loci isolated from Santiria trimera (Burseraceae)

? Premise of the study: To study the genetic structure among three morphotypes of an African rainforest tree species, Santiria trimera, nuclear microsatellite markers were isolated and characterized. ? Methods and Results: Seven polymorphic loci were isolated using a pyrosequencing-based protocol and successfully amplified on three different morphotypes of S. trimera. For six of the seven loci, there is at least one private allele for one of the three morphotypes. The mean effective number of alleles is about four for each of the three morphotypes. ? Conclusions: These microsatellite markers are promising to explore the genetic delimitation among sympatric morphotypes found in Gabonese forests and to study the spatial genetic structure within each gene pool.     

A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, p protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.     

A biophysical model of Sugarcane growth

Scientists predict that global agricultural lands will expand over the next few decades due to increasing demands for food production and an exponential increase in crop‐based biofuel production. These changes in land use will greatly impact biogeochemical and biogeophysical cycles across the globe. It is therefore important to develop models that can accurately simulate the interactions between the atmosphere and important crops. In this study, we develop and validate a new process‐based sugarcane model (included as a module within the Agro‐IBIS dynamic agro‐ecosystem model) which can be applied at multiple spatial scales. At site level, the model systematically under/overestimated the daily sensible/latent heat flux (by ?10.5% and 14.8%, H and λE, respectively) when compared against the micrometeorological observations from southeast Brazil. The model underestimated ET (relative bias between ?10.1% and –12.5%) when compared against an agro‐meteorological field experiment from northeast Australia. At the regional level, the model accurately simulated average yield for the four largest mesoregions (clusters of municipalities) in the state of São Paulo, Brazil, over a period of 16 years, with a yield relative bias of ?0.68% to 1.08%. Finally, the simulated annual average sugarcane yield over 31 years for the state of Louisiana (US) had a low relative bias (?2.67%), but exhibited a lower interannual variability than the observed yields.     

产超广谱β- 内酰胺酶肺炎克雷伯菌耐药性及其基因分型分析

目的:了解新疆独山子地区肺炎克雷伯菌超广谱β-内酰胺酶(ESBLs)的发生率、作为指示剂的五种抗生素的检出情况及ESBLs主要基因型。方法:收集临床分离的148株肺炎克雷伯菌,采用双纸片协同筛选法、NCCLS推荐的表型筛选和确证试验对细菌进行ESBLs产酶株的识别;耐药基因的质粒重组、转化,聚合酶链反应(PCR)扩增阳性产物测序,通过GenBank对序确定基因型。结果:本地区肺炎克雷伯菌ESBLs的分离率达31.1%,头孢曲松(CRO)安曲南(ATM)和头孢噻肟(CTX)头孢泊肟(CPD)、头孢他定(CAZ)作为指示剂检出率分别为97.8%、95.6%、93.4%、76.0%、65.2%;本地区产ESBLs菌株耐药基因型CTX-M-22占54.3%,CTX-M-18占41.3%,TEM61.8%,52.1%的产ESBLs肺炎克雷伯菌SHV耐药基因阳性。结论:CRO、ATM和CTX对检测ESBLs阳性率较高;CTX-M-22、CTX-M-18是本地区产ESBLs菌株的主要基因型。     

A new species of Eugenia (Myrtaceae) from south‐eastern Brazil

A new species, Eugenia marambaiensis M.C.Souza & M.P.Morim, is described and illustrated. It is characterized by having large translucent gland dots densely distributed on the leaves, with a caudate apex and slightly wavy margins and a glabrous raceme, with only two flowers. Eugenia marambaiensis was only found in the locality of Restinga da Marambaia, Rio de Janeiro, Brazil. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 306–308.     

Species limits and hybridization zones in Icterus cayanensis–chrysocephalus group (Aves: Icteridae)

One of the best examples of differentiation and hybridization among South American passerine birds is exhibited by Icterus cayanensis (Epaulet Oriole) and Icterus chrysocephalus (Moriche Oriole). Icterus chrysocephalus is a monotypic species restricted to northern South America. Icterus cayanensis is a polytypc species that ranges from Suriname and French Guyana to northern Argentina. Five subspecies are recognized to I. cayanensis. Hybrid zones are known between I. cayanensis and I. chrysocephalus as well as between subspecies of I. cayanenis, even though character variation has never been adequately assessed and mapped. Although molecular data support the hypothesis that I. cayanensis and I. chrysocephalus form a monophyletic group, they do not support the species limits currently recognized within this group. We analysed the geographic variation of plumage characters along the range of this group to map the geographic variation of individual plumage characters and identify the populations that have uniform phenotypic character expression and therefore represent genuine phylogenetic species. We also used molecular data to investigate the phylogenetic relationships among these species. Geographic variation of plumage characters, habitat preferences and molecular data identified four species within I. cayanensis–chrysocephalus clade: an Amazonian species group, formed by I. cayanensis and I. chrysocephalus and a Southern species group composed of I. pyrrhopterus and I. tibialis. The Amazonian species are separated by a relatively narrow hybrid zone along the Amazon valley, whereas the Southern species are separated by a hybrid zone that is larger than the ranges of the two species individually. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 583–597.     

博尔纳病病毒对BALB/c小鼠感染性检测

目的分析博尔纳病病毒（Borna disease virus,BDV）H1766株对BALB/c小鼠的感染性。方法选择病毒滴度为2.0×107FFU/ml的BDV病毒液分别对新生和成年BALB/c小鼠进行脑内接种,并用相同病毒液对原代培养的新生BALB/c小鼠脑细胞进行接种。经过一定时间的病毒作用后分别提取总RNA,采用巢式RT-PCR方法检测BDV-p40基因,并通过免疫组化方法检测脑内接种脑组织中BDV-P40蛋白。结果脑内接种病毒的小鼠脑组织中可以检测到BDV-p40基因和BDV-P40蛋白,培养的小鼠脑细胞中可以检测到BDV-p40基因。结论BDVH1766株可以感染新生和成年的BALB/c小鼠。