MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain-of-function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD-1). We discovered a number of miRNAs that increased or decreased cell viability in DLD-1. They included known oncogenic miRNAs such as miR-372 and miR-373, and tumor suppressive miRNAs such as miR-124a, but also some for which this information was novel. Among them, miR-491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl-X L was a direct target of miR-491, and its silencing contributed to miR-491-induced apoptosis. Moreover, treatment of miR-491 suppressed in vivo tumor growth of DLD-1 in nude mice. Our study provides a new regulation of Bcl-X L by miR-491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl-X L .MicroRNAs (miRNAs) are small noncoding RNAs of around 22 nucleotides that are highly conserved across metazoans 1,2 and over 500 human miRNAs have been described.3 miRNAs induce post-transcriptional gene repression by inhibiting translation and/or stabilization of their target mRNAs, and are thus involved in many biological processes including cell proliferation, differentiation, and apoptosis.

OBJECTIVE Aging of the population may lead to epidemiological changes with respect to chronic subdural hematoma (CSDH). The objectives of this study were to elucidate the current epidemiology and changing trends of CSDH in Japan. The authors analyzed patient information based on reports using a Japanese administrative database associated with the diagnosis procedure combination (DPC) system. METHODS This study included patients with newly diagnosed CSDH who were treated in hospitals participating in the DPC system. The authors collected data from the administrative database on the following clinical and demographic characteristics: patient age, sex, and level of consciousness on admission; treatment procedure; and outcome at discharge. RESULTS A total of 63,358 patients with newly diagnosed CSDH and treated in 1750 DPC participation hospitals were included in this study. Analysis according to patient age showed that the most common age range for these patients was the 9th decade of life (in their 80s). More than half of patients 70 years old or older presented with some kind of disturbance of consciousness. Functional outcomes at discharge were good in 71.6% (modified Rankin Scale [mRS] score 0-2) of cases and poor in 28.4% (mRS score 3-6). The percentage of poor outcomes tended to be higher in elderly patients. Approximately 40% of patients 90 years old or older could not be discharged to home. The overall recurrence rate for CSDH was 13.1%. CONCLUSIONS This study shows a chronological change in the age distribution of CSDH among Japanese patients, which may be affecting the prognosis of this condition. In the aging population of contemporary Japan, patients in their 80s were affected more often than patients in other age categories, and approximately 30% of patients with CSDH required some help at discharge. CSDH thus may no longer have as good a prognosis as had been thought.

fIn the fission yeast Schizosaccharomyces pombe, centromeric heterochromatin is maintained by an RNA-directed RNA polymerase complex (RDRC) and the RNA-induced transcriptional silencing (RITS) complex in a manner that depends on the generation of short interfering RNA. In association with the telomerase RNA component (TERC), the telomerase reverse transcriptase (TERT) forms telomerase and counteracts telomere attrition, and without TERC, TERT has been implicated in the regulation of heterochromatin at locations distinct from telomeres. Here, we describe a complex composed of human TERT (hTERT), Brahma-related gene 1 (BRG1), and nucleostemin (NS) that contributes to heterochromatin maintenance at centromeres and transposons. This complex produced doublestranded RNAs homologous to centromeric alpha-satellite (alphoid) repeat elements and transposons that were processed into small interfering RNAs targeted to these heterochromatic regions. These small interfering RNAs promoted heterochromatin assembly and mitotic progression in a manner dependent on the RNA interference machinery. These observations implicate the hTERT/BRG1/NS (TBN) complex in heterochromatin assembly at particular sites in the mammalian genome.T elomeres and centromeres are both tightly condensed heterochromatic areas within the genome, and the maintenance of heterochromatin is important for overall genome stability. In Schizosaccharomyces pombe, heterochromatin near centromeres is maintained by the RNA-directed RNA polymerase complex (RDRC) and the RNA-induced transcriptional silencing (RITS) complex (1, 2). Specifically, inhibition of RNA-dependent RNA polymerase (RdRP) activity leads to loss of small interfering RNAs (siRNAs) that are associated with the RITS complex and correlates with loss of transcriptional silencing and heterochromatin at centromeres (3). In addition, when RdRP activity is inhibited, siRNAs that are usually associated with the RITS complex are lost (4). These observations implicate RdRPs as a component of a loop coupling heterochromatin assembly to siRNA production.In Caenorhabditis elegans, the Argonaute CSR-1, the RdRP EGO-1, and the Dicer-related helicase DRH-3 localize to chromosomes and are required for proper chromosome segregation, and in the absence of these factors, chromosomes fail to properly align in mitotic phase (5, 6). Moreover, a conserved germ line-specific nucleotidyltransferase, CDE-1, localizes specifically to mitotic chromosomes in embryos in a manner that requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1 (5, 6). Although it is clear that RdRP and components of the RNA interference (RNAi) machinery are necessary to regulate heterochromatin in S. pombe and C. elegans, it is believed that heterochromatin is regulated in mammals through different mechanisms (7).Telomerase is a ribonucleoprotein complex that elongates telomeres. Human telomerase reverse transcriptase (hTERT) acts as an RNA-dependent DNA polymerase (RdDP) and synthesizes telomere DNA from a nonco...

Mouse embryonic stem (ES) cells can self-renew in the presence of leukemia inhibitory factor (LIF). Several essential transcription factors have been identified for the self-renewal of mouse ES cells, including STAT3, Oct-3/4, and Nanog. The molecular mechanism of ES cell self-renewal, however, is not fully understood. In the present study, we identified Eed, a core component of Polycomb repressive complex 2, as a downstream molecule of STAT3 and Oct-3/4. Artificial activation of STAT3 resulted in increased expression of Eed, whereas expression of a dominant negative mutant of STAT3 or suppression of Oct-3/4 expression led to down-regulation of Eed. Reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that STAT3 and Oct-3/4 directly bind to the promoter region of Eed, suggesting that Eed is a common target molecule of STAT3 and Oct-3/4. We also found that suppression of STAT3, Oct-3/4, or Eed causes induction of differentiation-associated genes as well as loss of Lys 27 -trimethylated histone H3 at the promoter regions of the differentiation-associated genes. Suppression of STAT3 and Oct-3/4 also resulted in the absence of Eed at the promoter regions. These results suggest that STAT3 and Oct-3/4 maintain silencing of differentiation-associated genes through up-regulation of Eed in self-renewing ES cells. Embryonic stem (ES)5 cells are derived from the inner cell mass of the mammalian blastocyst and have two major characteristics, pluripotency and self-renewal (1, 2). Previous studies have identified several essential transcription factors for the self-renewal of mouse ES cells, such as Oct-3/4, Nanog, and STAT3 (3). Oct-3/4 is a POU-family transcription factor involved in inner cell mass formation (4). A precise level of Oct-3/4 expression is required for maintenance of ES cells: repression of Oct-3/4 leads to trophectodermal differentiation, and overexpression of Oct-3/4 stimulates differentiation, mainly to extraembryonic endoderm (5). Nanog is a homeodomain transcription factor whose overexpression sustains ES cell self-renewal (6). Targeted disruption of the nanog gene results in ES cell differentiation, primarily along the primitive endoderm lineage, suggesting that Nanog prevents ES cells from endoderm differentiation (7).The pluripotency and self-renewal of mouse ES cells can be maintained by the presence of leukemia inhibitory factor (LIF). LIF stimulation leads to the activation of transcription factor STAT3. Previously, using a fusion protein consisting of STAT3 and the ligand-binding domain of estrogen receptor (STAT3ER), we demonstrated that the self-renewal of ES cells can be maintained by activation of STAT3ER with a synthetic estrogen receptor ligand, 4-hydroxytamoxifen (4HT), even in the absence of LIF (8). Another study showed that expression of a dominant negative mutant of STAT3 causes differentiation of ES cells (9). These observations indicate that the activation of STAT3 is essential and sufficient for the self-renewal of mouse ES cells.Despite having ...

A mutation that confers white plumage with black eyes was identified in the Minohiki breed of Japanese native chicken (Gallus gallus domesticus). The white plumage, with a few partially pigmented feathers, was not associated with the tyrosinase gene, and displayed an autosomal recessive mode of inheritance against the pigmented phenotype. All F1 offspring derived from crosses with mottled chickens (mo/mo), which show characteristic pigmented feathers with white tips, had plumage with a mottled-like pattern. This result indicates that the white plumage mutation is a novel allele at the mo locus; we propose the gene symbol mow for this mutant allele. Furthermore, the F1 hybrid between the mow/mow chicken and the panda (s/s) mutant of Japanese quail (Coturnix japonica), whose causative gene is the endothelin receptor B2 (EDNRB2) gene, showed a mow/mow chicken-like plumage, suggesting the possibility that the mutations in parental species are alleles of the same gene, EDNRB2. Nucleotide sequencing of the entire coding region of EDNRB2 revealed a non-synonymous G1008T substitution, which causes Cys244Phe amino acid substitution in exon 5 (which is part of the extracellular loop between the putative fourth and fifth transmembrane domains of EDNRB2) in the mutant chicken. This Cys244Phe mutation was also present in individuals of four Japanese breeds with white plumage. We also identified a non-synonymous substitution leading to Arg332His substitution that was responsible for the mottled (mo/mo) plumage phenotype. These results suggest that the EDN3 (endothelin 3)–EDNRB2 signaling is essential for normal pigmentation in birds, and that the mutations of EDNRB2 may cause defective binding of the protein with endothelins, which interferes with melanocyte differentiation, proliferation, and migration.

During early development in vertebrates, pluripotent cells are generated from the neural crest and migrate according to their presumptive fate. In birds and mammals, one of the progeny cells, melanoblasts, generally migrate through a dorsolateral route of the trunk region and differentiate to melanocytes. However, Silky is an exceptional chicken in which numerous melanoblasts travel via a ventral pathway and disperse into internal organs. Finally, these ectopic melanocytes induce heavy dermal and visceral melanization known as Fibromelanosis (Fm). To identify the genetic basis of this phenotype, we confirmed the mode of inheritance of Fm as autosomal dominant and then performed linkage analysis with microsatellite markers and sequence-tagged site markers. Using 85 backcross progeny from crossing Black Minorca chickens (BM-C) with F 1 individuals between White Silky (WS) and BM-C Fm was located on 10.2-11.7 Mb of chicken chromosome 20. In addition, we noticed a DNA marker that all Silky chickens and the F 1 individuals showed heterozygous genotyping patterns, suggesting gene duplication in the Fm region. By quantitative real-time PCR assay, Silky line-specific gene duplication was detected as an 130-kb interval. It contained five genes including endothelin 3 (EDN3), which encoded a potent mitogen for melanoblasts/melanocytes. EDN3 with another three of these duplicated genes in Silky chickens expressed almost twofold of those in BM-C. Present results strongly suggest that the increase of the expression levels resulting from the gene duplication in the Fm region is the trigger of hypermelanization in internal organs of Silky chickens.

We prepared five pairs of hydrogenous and deuterated ring polystyrene samples over a wide range of molecular weights (10 kg/mol ≤ M w ≤ 400 kg/mol) and investigated their chain conformations in bulk by small-angle neutron scattering (SANS) measurements. From the SANS profiles obtained, we estimated the radii of gyration R g of the ring polymers by the Guinier approximation. R g can be related to the degree of polymerization N as R g ∼ N 0.47 . This scaling exponent ν = 0.47 ± 0.01 is evidently smaller than that for the Gaussian chains (ν = 0.50) but higher than previous experimental reports (ν = 0.42−0.43). Then our data were compared with various simulation and experimental data by introducing the entanglement degree of polymerization N e for linear polymers as a normalized parameter. R g of three smaller rings, i.e., R-10, R-30, and R-70, where the numbers denote molecular weights in kg/mol unit, are in good agreement with simulation results, while two larger rings, R-100 and R-400, exhibit higher R g values than the simulations. Considering that the latter two higher molecular weight samples include maximum 3% of linear contamination, their effects on chain dimension were calculated. As a result, it has been confirmed that 3% of linear contaminations can overestimate R g of rings as much as 6% for R-100 and 12% for R-400. Thus, R g for pure large rings should be considerably lower than the present experimental values. We conclude Flory's exponent v in R g ∼ N v for rings may not be constant but rather show molecular weight dependence due to their topological constraint.

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