Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.     

Adaptive introgression of anticoagulant rodent poison resistance by hybridization between old world mice

Polymorphisms in the vitamin K 2,3-epoxide reductase subcomponent 1 (vkorc1) of house mice (Mus musculus domesticus) can cause resistance to anticoagulant rodenticides such as warfarin [1-3]. Here we show that resistant house mice can also originate from selection on vkorc1 polymorphisms acquired from the Algerian mouse (M. spretus) through introgressive hybridization. We report on a polymorphic introgressed genomic region in European M. m. domesticus that stems from M. spretus, spans >10 Mb on chromosome 7, and includes the molecular target of anticoagulants vkorc1 [1-4]. We show that in the laboratory, the homozygous complete vkorc1 allele of M. spretus confers resistance when introgressed into M. m. domesticus. Consistent with selection on the introgressed allele after the introduction of rodenticides in the 1950s, we found signatures of selection in patterns of variation in M. m. domesticus. Furthermore, we detected adaptive protein evolution of vkorc1 in M. spretus (Ka/Ks = 1.54-1.93) resulting in radical amino acid substitutions that apparently cause anticoagulant tolerance in M. spretus as a pleiotropic effect. Thus, positive selection produced an adaptive, divergent, and pleiotropic vkorc1 allele in the donor species, M. spretus, which crossed a species barrier and produced an adaptive polymorphic trait in the recipient species, M. m. domesticus.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Transformation of human umbilical mesenchymal cells into neurons in vitro

Neuronal transplantation has provided a promising approach for treating neurodegenerative diseases. Recently, efforts have been directed at in vitro induction of various stem cells to transform into neurons. We report the first successful quantities in an in vitro attempt at directing the transformation into neurons of human umbilical mesenchymal cells, which are capable of rapid proliferation in vitro and are easily available. When cultured in neuronal conditioned medium, human umbilical mesenchymal cells started to express neuron-specific proteins such as NeuN and neurofilament (NF) on the 3rd day and exhibited retraction of the cell body, elaboration of processes, clustering of cells and expression of functional mRNA responsible for the synthesis of subunits of the kainate receptor and glutamate decarboxylase on the 6th day. Between the 9th and 12th days, the percentage of human umbilical mesenchymal cells expressing NF was as high as 87%, while functionality was demonstrated by glutamate invoking an inward current. At this stage, cells were differentiated into mature neurons in the postmitosis phase.     

Extract‐SAGE: An integrated platform for cross­analysis and GA­based selection of SAGE data

Serial analysis of gene expression (SAGE) is a powerful quantification technique for gene expression data. The huge amount of tag data in SAGE libraries of samples is difficult to analyze with current SAGE analysis tools. Data is often not provided in a biologically significant way for cross‐analysis and ‐comparison, thus limiting its application. Hence, an integrated software platform that can perform such a complex task is required. Here, we implement set theory for cross‐analyzing gene expression data among different SAGE libraries of tissue sources; up‐ or down‐regulated tissue‐specific tags can be identified computationally. Extract‐SAGE employs a genetic algorithm (GA) to reduce the number of genes among the SAGE libraries. Its representative tag mining will facilitate the discovery of the candidate genes with discriminating gene expression.     

The type 2 dengue virus envelope protein interacts with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9)

Dengue viruses are mosquito-borne flaviviruses and may cause the life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its envelope protein is responsible mainly for the virus attachment and entry to host cells. To identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, we have performed a screening with the yeast-two-hybrid-based “Functional Yeast Array”. Interestingly, the small ubiquitin-like modifier-1 conjugating enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, we have demonstrated that it indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nuclear membrane and co-localized with Flag-Ubc9 proteins around the nuclear membrane in the cytoplasmic side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus propagation.     

Polar positional information in Escherichia coli spherical cells

Shigella surface protein IcsA and its cytoplasmic derivatives are localized to the old pole of rod-shaped cells when expressed in Escherichia coli. In spherical mreB cells, IcsA is targeted to ectopic sites and close to one extremity of actin-like MamK filament. To gain insight into the properties of the sites containing polar material, we studied the IcsA localization in spherical cells. GFP was exported into the periplasm via the Tat pathway and used as a periplasmic space marker. GFP displayed zonal fluorescence in both mreB and rodA-pbpA spherical E. coli cells, indicating an uneven periplasmic space. Deconvolution images revealed that the cytoplasmic IcsA fused to mCherry was localized outside or at the edge of the GFP zones. These observations strongly suggest that polar material is restricted to the positions where the periplasm possesses particular structural or biochemical properties.     

Phylogeny and biogeography of the freshwater crab genus Johora (Crustacea: Brachyura: Potamidae) from the Malay Peninsula, and the origins of its insular fauna

The phylogeny and biogeography of the Malayan freshwater crab genus Johora was studied using two mitochondrial genes, 16S rRNA (560 bp) and cytochrome oxidase subunit I (COI) (616 bp), and one nuclear gene, histone 3 (H3) (328 bp). Johora is shown to be monophyletic and composed of three clades that correspond with the topography of the Malay Peninsula. The three clades were estimated to be of similar age ( c . 11 million years ago (mya)). The Malayan island of Pulau Tioman (with five species) was determined to have been colonised independently by two separate clades (at c . 11 and 5 mya, respectively), one of which evolved semiterrestrial habits, possibly in response to competition by the second. A partitioned Bremer support (PBS) analysis reveals that most of the support for the phylogenetic tree comes from the COI gene fragment and that the nuclear protein-encoding genes H3 is useful for reconstructing the relationships of Johora .     

Effects of extremely low frequency (ELF) electric fields on cell growth and DNA repair in human skin fibroblasts

A number of physical and chemical agents in the environment have been studied for their ability to induce or alter DNA repair mechanisms in human cells. We have investigated the effects of 60 Hz, 1000 V/cm electric fields on DNA repair in normal human fibroblasts in vitro. An examination was done on the ability of electric fields suspected to cause damage which could be repaired by thymine dimer excision and measurable by the bromodeoxyuridine photolysis assay. The thymine dimer assay with enzyme-sensitive site analysis was used to measure the cells' capacity for removing ultraviolet light (u.v.)-induced pyrimidine dimers; during exposure to electric field 24 hr before u.v. irradiation; 24 hr after u.v. irradiation; and up to 48 hr continuously after u.v. irradiation. Cell growth and cell survival following electric field exposure were also studied. Within the limits of these experiments, it was found that exposure to such electric fields did not alter cell growth or survival, and no DNA repair or alteration in DNA excision repair capacity was observed as compared with unexposed control cultures.