High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.     

Mitochondrial genome and a nuclear gene indicate a novel phylogenetic position of deep-sea tube-eye fish (Stylephoridae)

The rare, monotypic deep-sea fish family Stylephoridae has long been considered a member of the order Lampridiformes (opahs, velifers, ribbonfishes), and no systematic ichthyologist has questioned its placement within the order for over 80 years. Recently three individuals of Stylephorus chordatus were collected from different oceans, and we sequenced the whole mitochondrial genome and a partial nuclear recombination activating gene 1 (RAG1) gene sequences for each specimen. We aligned these sequences with those available from higher teleosts, including representative lampridiforms, and constructed two separate datasets from the sequences. The resulting trees derived from partitioned Bayesian analyses strongly indicated that S. chordatus is not a lampridiform but is closely related to the order Gadiformes (cod and their relatives). Lampridiformes is diagnosed on the basis of four synapomorphies, three of which are correlated with the rare and possibly unique ability to extend both the maxilla and premaxilla as a unit during feeding. Stylephorus also possesses such unique ability, but lacks two and possibly three of the four synapomorphies, suggesting that further morphological analysis is needed. Considering its unique morphologies with no indication of affinities within Gadiformes (or any other presently recognized order), the present results warrant a recognition of the new order for S. chordatus in fish systematics.     

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.     

Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences

During a survey fungal diversity of xylariaceous fungi in Thailand, a new Nemania species, N. plumbea, was identified. Nemania plumbea is characterized by soft-textured grey stromata on a persistent mat of white hyphae, pale brown ascospores with a short germ-slit on the more convex side. It also produces a Geniculosporium-like anamorph in culture. In order to evaluate its phylogenetic relationships among related species and genera, ITS-5.8S rDNA and RPB2 were analysed separately and simultaneously. Results from the phylogenetic analyses indicate that there is close phylogenetic association between N. plumbea and N. aenea. A preliminary account into the natural grouping of Xylariaceae based on ITS-5.8S rDNA and RPB2 sequences is also discussed.     

Romellia is congeneric with Togninia, and description of Conidiotheca gen. nov. for one species of this genus with polysporous asci

During a continued survey of fungi accommodated in the Calosphaeriales, we observed that the genus Romellia is morphologically heterogeneous. Historically, four species were placed in the genus. Romellia was introduced for taxa with globose, dark, superficial, papillate perithecia, a paraphysate centrum, and octosporous asci containing eight allantoid, hyaline ascospores. The type species, R. vibratilis, was revised in our study, and its freshly collected material was cultured. The anamorph obtained in vitro matches the generic concept of the hyphomycete genus Phaeoacremonium, the anamorph of Togninia. The phylogenies inferred from newly obtained sequences of LSU rDNA, β-tubulin, and actin of R. vibratilis revealed that the species is congeneric with Togninia (Togniniaceae) and represents a good species. Revision of type and herbarium material of other Romellia species confirmed they are not congeneric with Togninia. Romellia tympanoides possesses immersed, subglobose perithecia with sessile asci containing eight ascospores, which during maturation produce ascoconidia filling the entire ascus. This maturation process represents a new kind of apparent polyspory known in the ascomycetes. We introduce a new genus, Conidiotheca, and a new combination for R. tympanoides. The species is compared with the genera Barrina and Tympanis. The relationships of two other Romellia species, R. ambigua and R. cornina, lie with Wegelina and Jattaea of the Calosphaeriales.     

Phylogenetic systematics of leaffishes (Teleostei: Polycentridae,Nandidae)

The Asian (nandid) and Afro‐Neotropical (polycentrid) leaffishes represent two superficially similar, but historically poorly diagnosed families – a situation resulting in a convoluted systematic history. Here, and including for the first time in a molecular study all leaffish genera, we generate a hypothesis of the phylogenetic history of both groups. We analyse a multilocus molecular data set encompassing 257 acanthomorph taxa, carry out a survey and assessment of selected osteological characters for the polycentrid leaffishes and also provide a reanalysis of previously published morphological data. Our results confirm: (1) that the Polycentridae and Nandidae are only remotely related, and hence, the classic leaffishes are diphyletic; (2) that the Polycentridae is monophyletic, with new skeletal synapomorphies being congruent with molecular data in placing the enigmatic Afronandus – a taxon that thus far has never been included in any molecular study – as sistergroup to the remaining genera; (3) the monophyly of the Nandidae + Badidae and their inclusion into a larger monophyletic group – along with the Pristolepididae, Anabantoidei and Channoidei – comprising the Labyrinthici sensu Rosen & Patterson. We also review the morphological and molecular evidence for both the conflicting placement of Pristolepis and the putative sistergroup relationship between the labyrinth fishes (Anabantoidei) and snakeheads (Channoidei).     

Species are hypotheses: avoid connectivity assessments based on pillars of sand

Connectivity among populations determines the dynamics and evolution of populations, and its assessment is essential in ecology in general and in conservation biology in particular. The robust basis of any ecological study is the accurate delimitation of evolutionary units, such as populations, metapopulations and species. Yet a disconnect still persists between the work of taxonomists describing species as working hypotheses and the use of species delimitation by molecular ecologists interested in describing patterns of gene flow. This problem is particularly acute in the marine environment where the inventory of biodiversity is relatively delayed, while for the past two decades, molecular studies have shown a high prevalence of cryptic species. In this study, we illustrate, based on marine case studies, how the failure to recognize boundaries of evolutionary‐relevant unit leads to heavily biased estimates of connectivity. We review the conceptual framework within which species delimitation can be formalized as falsifiable hypotheses and show how connectivity studies can feed integrative taxonomic work and vice versa. Finally, we suggest strategies for spatial, temporal and phylogenetic sampling to reduce the probability of inadequately delimiting evolutionary units when engaging in connectivity studies.     

中国野生百合叶表皮特征及系统学意义

利用光学显微镜和扫描电镜对20个野生百合种或变种的叶表皮进行了系统观察和比较分析,结果表明:(1)20种野生百合气孔器均分布于叶片远轴面,气孔器的长轴与远轴面细胞长轴方向一致,均位于远轴面细胞的端点处。气孔器形状从长椭圆形到近圆形不等;保卫细胞均为肾形,无副卫细胞,保卫细胞表面角质层均具条纹;表皮细胞角质层近于光滑或具条纹,均具蜡质,蜡质纹饰呈膜片状晶体或颗粒状;气孔密度、气孔器大小、近轴面远轴面细胞、垂周壁样式差异较大。(2)聚类分析表明,兰州百合、川百合、卷丹百合、紫斑百合、乳头百合、宝兴百合、山丹百合、大理百合、绿花百合、毛百合和有斑百合聚为类群Ⅰ;淡黄花百合、岷江百合和玫红百合3个野生种聚为类群Ⅱ;青岛百合单独为类群Ⅲ;宜昌百合、紫脊百合、通江百合、百合和野百合聚为类群Ⅳ,聚类结果总体上支持形态学分类的结果。(3)叶表皮形态对于区分野生百合不同种具有重要的分类学价值。     

The Endosomal Sorting Complex Required for Transport Pathway Mediates Chemokine Receptor CXCR4-promoted Lysosomal Degradation of the Mammalian Target of Rapamycin Antagonist DEPTOR

G protein-coupled receptor (GPCR) signaling mediates many cellular functions, including cell survival, proliferation, and cell motility. Many of these processes are mediated by GPCR-promoted activation of Akt signaling by mammalian target of rapamycin complex 2 (mTORC2) and the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase 1 (PDK1) pathway. However, the molecular mechanisms by which GPCRs govern Akt activation by these kinases remain poorly understood. Here, we show that the endosomal sorting complex required for transport (ESCRT) pathway mediates Akt signaling promoted by the chemokine receptor CXCR4. Pharmacological inhibition of heterotrimeric G protein Gα_i or PI3K signaling and siRNA targeting ESCRTs blocks CXCR4-promoted degradation of DEPTOR, an endogenous antagonist of mTORC2 activity. Depletion of ESCRTs by siRNA leads to increased levels of DEPTOR and attenuated CXCR4-promoted Akt activation and signaling, consistent with decreased mTORC2 activity. In addition, ESCRTs likely have a broad role in Akt signaling because ESCRT depletion also attenuates receptor tyrosine kinase-promoted Akt activation and signaling. Our data reveal a novel role for the ESCRT pathway in promoting intracellular signaling, which may begin to identify the signal transduction pathways that are important in the physiological roles of ESCRTs and Akt.     

Systematics of the Southeast Asian mongooses (Herpestidae,Carnivora): solving the mystery of the elusive collared mongoose and Palawan mongoose

Although recent molecular studies have clarified the phylogeny of mongooses, the systematics of the Southeast Asian species was incomplete as the collared mongoose Urva semitorquata and some debatable taxa (Hose's mongoose, Palawan mongoose) were missing in the analyses. We sequenced three mitochondrial (cytochrome b, ND2, control region) and one nuclear (beta‐fibrinogen intron 7) fragments of the Southeast Asian mongooses to clarify the systematic position of the different species and populations occurring in this region. Our results showed that the collared mongoose is closely related to the crab‐eating mongoose Urva urva, these two species forming a sister‐group to the short‐tailed mongoose Urva brachyura. Despite Sumatran collared mongooses having a peculiar orange phenotype, we showed that they exhibited very little genetic divergence to individuals from Borneo. In contrast, the populations of the short‐tailed mongoose from Borneo were strongly divergent to those from Peninsular Malaysia and Sumatra, and these might represent separate species. Within the crab‐eating mongoose, we observed little geographical genetic structure. Our study suggests that Hose's mongoose is not a valid species. The Palawan mongooses did not cluster with the other populations of the short‐tailed mongoose; they were closer to the collared mongoose and should be included in this species. © 2014 The Linnean Society of London