Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity

Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N(2)-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.     

Abiotic stress-inducible receptor-like kinases negatively control ABA signaling in Arabidopsis

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.     

A novel composite retrotransposon derived from or generated independently of the SVA (SINE/VNTR/Alu) transposon has undergone proliferation in gibbon genomes

The superfamily Hominoidea (hominoids) comprises two families: Hominidae (hominids) and Hylobatidae (gibbons, also called small apes). The SVA transposon is a composite retrotransposon that occurs widely in hominoids and is considered to have been generated by stepwise fusions of three genetic elements: SINE-R, a variable number of tandem repeat (VNTR) sequence, and Alu. We identified a novel transposon whose basic structure is the same as that of SVA, with one prominent difference being the presence of part of prostaglandin reductase 2 (PTGR2) in place of SINE-R. We designate this composite transposon as PVA and propose two possible mechanisms regarding its generation. One is the derivation of PVA from SVA: the SINE-R region of SVA was replaced with a PTGR2 fragment by template switching. The other is the formation of PVA independently of SVA: a PTGR2 fragment was fused to an evolutionary intermediate comprising the VNTR and Alu regions. The nucleotide sequence of the junction between the VNTR and PTGR2 regions supports the second hypothesis. We identified PVA in the white-cheeked gibbon Nomascus leucogenys by analysis of genome sequence databases, and subsequent experimental analysis revealed its presence in all four gibbon genera. The white-cheeked gibbon harbors at least 93 PVA copies in its haploid genome. Another SVA-like composite transposon carrying parts of the LINE1 and Alu transposons in place of SINE-R, designated as LAVA, has recently been reported. The significance of the discovery of PVA is that its substituted fragment originates not from a transposon but from a single-copy gene. PVA should provide additional insights into the transposition mechanism of this type of composite transposon; the transposition activity is conferred even if the substituted fragment is not related to a transposon.     

Evolution of sex chromosomes ZW of Schistosoma mansoni inferred from chromosome paint and BAC mapping analyses

Chromosomes of schistosome parasites among digenetic flukes have a unique evolution because they exhibit the sex chromosomes ZW, which are not found in the other groups of flukes that are hermaphrodites. We conducted molecular cytogenetic analyses for investigating the sex chromosome evolution using chromosome paint analysis and BAC clones mapping. To carry this out, we developed a technique for making paint probes of genomic DNA from a single scraped chromosome segment using a chromosome microdissection system, and a FISH mapping technique for BAC clones. Paint probes clearly identified each of the 8 pairs of chromosomes by a different fluorochrome color. Combination analysis of chromosome paint analysis with Z/W probes and chromosome mapping with 93 BAC clones revealed that the W chromosome of Schistosoma mansoni has evolved by at least four inversion events and heterochromatinization. Nine of 93 BAC clones hybridized with both the Z and W chromosomes, but the locations were different between Z and W chromosomes. The homologous regions were estimated to have moved from the original Z chromosome to the differentiated W chromosome by three inversions events that occurred before W heterohcromatinization. An inversion that was observed in the heterochromatic region of the W chromosome likely occurred after W heterochromatinization. These inversions and heterochromatinization are hypothesized to be the key factors that promoted the evolution of the W chromosome of S. mansoni.     

Silencing of miR-370 in Human Cholangiocarcinoma by Allelic Loss and Interleukin-6 Induced Maternal to Paternal Epigenotype Switch

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor arising from the biliary tract epithelium. Interleukin-6 (IL-6) is a major mediator of inflammation and contributor to carcinogenesis within the biliary tree. Previous studies suggested that enforced IL-6 contributes to cholangiocarcinogenesis through hypermethylation of several genes implicated in CCA. However, the precise mechanisms of IL-6 effects in CCA remain unclear. We now demonstrate that microRNA (miR)-370 is underexpressed in a large cohort of human CCA vs. normal liver tissues. In addition, we show that IL-6 induces a time-dependent silencing of miR-370. In addition, demethylation of CCA cells results in upregulation of miR-370. Furthermore, we demonstrate that miR-370 is imprinted, and that the Intergenic Differentially Methylated Region (IG-DMR) responsible for imprinting regulation of this genomic locus is hypermethylated in response to IL-6 treatment. In addition, the IG-DMR is hypermethylated in human CCA specimens compared to normal matched controls, in the same location as the IL-6 induced hypermethylation. Finally, miR-370 was found to regulate WNT10B in luciferase as well as western blotting experiments. Our data indicate that the paternal allele of miR-370 is normally silenced through genomic imprinting and that the overexpression of IL-6 in CCA effectively suppresses the expression of miR-370 from the maternal allele, lending support to the theory that miR-370 silencing in human CCA follows a classic two-hit mechanism.     

Single nucleotide polymorphisms in Cryptomeria japonica: their discovery and validation for genome mapping and diversity studies

In order to develop a large set of single-nucleotide polymorphisms (SNPs) in Cryptomeria japonica, for a wide range of applications, we adopted a systematic EST (expressed sequence tags) re-sequencing approach. We examined a group of four genotypes comprising parents of a mapping population as well as representatives of two main lines from natural populations. We sequenced 5,170 gene fragments, representing analysis of over 1.3?Mb of DNA sequences in C. japonica. This analysis leads to the discovery of 13,413 SNPs in 3,744 amplicons, with an average of one SNP for every 101.0?bp (one SNP for every 78.3?bp in introns and for every 106.7?bp in exon regions). Nucleotide diversity in C. japonica (???=?0.0045) was found to be similar to values recorded in highly polymorphic forest tree species such as pine. We also validated the use of the SNPs as molecular markers for genetic diversity studies using the high throughput SNP genotyping platform GoldenGate. From 1,536 candidate SNP sites tested, 1,164 (75.8?%) were confirmed to be polymorphic. We anticipate that the genome-wide SNP markers reported here will be useful for evaluating the species?? range-wide genetic structure and in marker-assisted selection used as part of the C. japonica tree improvement program.     

Oxovanadium complexes with quinoline and pyridinone ligands: syntheses of the complexes and effect of alkyl chains on their apoptosis-inducing activity in leukemia cells

Vanadium complexes with quinoline ligands (1b-g) and pyridinone ligands (2b-d) were synthesized, and the effect of the length and shape of alkyl chains on the antiproliferative activity toward U937 cells was studied. For the synthesis of the vanadium complexes, quinoline and pyridinone ligands were prepared and then treated with VOSO(4) or VO(acac)(2). The vanadyl(IV) complexes were characterized by IR, ESR, and UV-vis spectroscopy and elemental analyses. The antiproliferative activity of 1a-g toward U937 cells showed little dependence on the length and shape of the alkyl chain. In contrast, a good correlation was found between the IC(50) values and partition coefficients (logP) values of 2a-c. Among them, 2c showed the highest inhibitory activity, and its IC(50) value was smaller than that of cisplatin. The apoptosis-inducing ability of 2b and 2c was supported by annexin V-propidium iodide staining experiments and agarose gel electrophoresis analysis. Inhibitors of caspase-3, -8, and -9 did not affect the antiproliferative activity of 2c, indicating that the apoptosis induced by 2c was via a caspase-independent pathway.     

Dopamine D1 receptor-induced signaling through TrkB receptors in striatal neurons

In addition to its role as a neurotransmitter, dopamine can stimulate neurite outgrowth and morphological effects upon primary neurons. To investigate the signal transduction mechanisms used by dopamine in developing striatal neurons, we focused upon the effects of activating the dopamine D1 receptor. Using the D1 receptor agonist SKF38393, we found that Trk neurotrophin receptors were activated in embryonic day 18 striatal neurons. K-252a, a Trk tyrosine kinase inhibitor, and a dopamine D1 receptor antagonist could block the effects of SKF38393. The increase in TrkB phosphorylation was not the result of increased neurotrophin production. Induction of TrkB activity by SKF38393 was accompanied by the phosphorylation of several Trk signaling proteins, including phospholipase Cgamma, Akt, and MAPK. Biotinylation experiments followed by immunostaining by phospho-TrkB-specific antibodies indicated that the mechanism involved increased TrkB surface expression by dopamine D1 receptor activation. This increase in cell surface TrkB expression was dependent upon an increase in intracellular Ca(2+). These results indicate that stimulation of dopamine D1 receptors can be coupled to the neurotrophin receptor signaling to mediate the effects of dopamine upon striatal neurons.     

Micromechanical evaluation of mineralized multilayers

The biomechanical stability of osseointegrated implants is of particular importance, especially the stability which is achieved from structural manipulation at the interface between the implant surface and the bone tissues. Nanoscale β-tricalcium phosphate-immobilized titanium was prepared by discharge into a physiological buffered saline solution. Compared with hydroxyapatite, it has been shown to be effective in generating a bone-like chemical structure on the surface by cooperative interaction between osteoblastic cells and the β-tricalcium phosphate. The present study, after cell cultivation, investigates the nanostructures and biomechanical property differences of a mineralized layer formed on two samples of nano-calcium phosphate-immobilized titanium. A scanning probe microscope study revealed that the mineralized tissue formed on the β-tricalcium phosphate samples after 1 week of cell culture showed significantly higher roughness, compared with hydroxyapatite samples. Nanoindentation micromechanical evaluation of the in vitro generated multilayered structures exhibited thicker bone-like mineralized layers on the β-tricalcium phosphate samples. A successful modification of titanium implants through the cooperative interaction between osteoblastic cells and nano β-tricalcium phosphate is anticipated.     

Mutations in foregut SOX2+ cells induce efficient proliferation via CXCR2 pathway

Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2⁺ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as Kras^G12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2⁺ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and Kras^G12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.