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The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

The Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns (PAMPs). TLR2 is essential for the signaling of a variety of PAMPs, including bacterial lipoprotein/lipopeptides, peptidoglycan, and GPI anchors. TLR6 associates with TLR2 and recognizes diacylated mycoplasmal lipopeptide along with TLR2. We report here that TLR1 associates with TLR2 and recognizes the native mycobacterial 19-kDa lipoprotein along with TLR2. Macrophages from TLR1-deficient (TLR1(-/-)) mice showed impaired proinflammatory cytokine production in response to the 19-kDa lipoprotein and a synthetic triacylated lipopeptide. In contrast, TLR1(-/-) cells responded normally to diacylated lipopeptide. TLR1 interacts with TLR2 and coexpression of TLR1 and TLR2 enhanced the NF-kappaB activation in response to a synthetic lipopeptide. Furthermore, lipoprotein analogs whose acylation was modified were preferentially recognized by TLR1. Taken together, TLR1 interacts with TLR2 to recognize the lipid configuration of the native mycobacterial lipoprotein as well as several triacylated lipopeptides.

Gamma-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many medium-sized spherical somata emitting intense GFP fluorescence were observed in layer I.

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.

Two evolutionarily distant plant species, rice (Oryza sativa L.), a short-day (SD) plant, and Arabidopsis thaliana, a long-day plant, share a conserved genetic network controlling photoperiodic flowering. The orthologous floral regulators-rice Heading date 1 (Hd1) and Arabidopsis CONSTANS (CO)-integrate circadian clock and external light signals into mRNA expression of the FLOWERING LOCUS T (FT) group floral inducer. Here, we report that the rice Early heading date 1 (Ehd1) gene, which confers SD promotion of flowering in the absence of a functional allele of Hd1, encodes a B-type response regulator that might not have an ortholog in the Arabidopsis genome. Ehd1 mRNA was induced by 1-wk SD treatment, and Ehd1 may promote flowering by inducing FT-like gene expression only under SD conditions. Microarray analysis further revealed a few MADS box genes downstream of Ehd1. Our results indicate that a novel two-component signaling cascade is integrated into the conserved pathway in the photoperiodic control of flowering in rice.

Toll-like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub-types, including PDC, but that TLR3 is preferentially expressed by CD8 alpha(+) DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8 alpha(-) DC and PDC, but not CD8 alpha(+) DC as measured by survival ex vivo, up-regulation of surface markers and production of IL-12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non-plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8 alpha(+) DC in recognition of certain mouse pathogens.

Potential mechanisms of Al toxicity measured as Al-induced inhibition of growth in cultured tobacco cells (Nicotiana tabacum, nonchlorophyllic cell line SL) and pea (Pisum sativum) roots were investigated. Compared with the control treatment without Al, the accumulation of Al in tobacco cells caused instantaneously the repression of mitochondrial activities [monitored by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and the uptake of Rhodamine 123] and, after a lag of about 12 h, triggered reactive oxygen species (ROS) production, respiration inhibition, ATP depletion, and the loss of growth capability almost simultaneously. The presence of an antioxidant, butylated hydroxyanisol, during Al treatment of SL cells prevented not only ROS production but also ATP depletion and the loss of growth capability, suggesting that the Al-triggered ROS production seems to be a cause of ATP depletion and the loss of growth capability. Furthermore, these three late events were similarly repressed in an Al-tolerant cell line (ALT301) isolated from SL cells, suggesting that the acquisition of antioxidant functions mimicking butylated hydroxyanisol can be a mechanism of Al tolerance. In the pea root, Al also triggered ROS production, respiration inhibition, and ATP depletion, which were all correlated with inhibition of root elongation. Taken together, we conclude that Al affects mitochondrial functions, which leads to ROS production, probably the key critical event in Al inhibition of cell growth.

The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2′-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1n/n mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression. The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2′-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1n/n mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression. In mammalian embryogenesis the first cellular differentiation begins at the end of the third cleavage, which leads to compaction and formation of the blastocyst. The inner cell mass (ICM) 1The abbreviations used are: ICM, inner cell mass; ChIP, chromatin immunoprecipitation; RT, reverse transcription; dpc, days postcoitum; ES cells, embryonic stem cells; TS cells, trophoblast stem cells; T-DMR, tissue-dependent, differentially methylated region; 5-aza-dC, 5-aza-2′-deoxycytidine; TSA, trichostatin A; MeCP2, methyl-CpG-binding protein 2; DE, distal enhancer; PE, proximal enhancer. of the blastocyst is known to generate all fetal somatic cells and germ cells, whereas the outer cell layer, the trophectoderm, gives rise to the trophoblastic components of the placenta. The explant culture of ICM or epiblast cells produces pluripotent embryonic stem (ES) cell lines, which contribute to all of the ICM lineages in chimeric embryos (1Martin G.R. Proc. Natl. Acad. Sci. 1981; 78: 7634-7636Crossref PubMed Scopus (4355) Google Scholar, 2Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9424-9428Google Scholar). Trophoblast stem (TS) cell lines, which have the ability to differentiate into trophoblast lineage in vitro, have also been established from blastocyst or early postimplantation trophoblastic tissue (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). TS cells do not contribute to the embryo proper in chimeras but differentiate only into the trophoblastic cells of the placenta. We previously investigated the genome-wide DNA methylation status of CpG islands by RLGS (restriction landmark genomic scanning) of mouse stem cells, i.e. ES, embryonic germ, and TS cells, before and after differentiation, as well as germ cells isolated from testis and some somatic tissues (4Shiota K. Kogo Y. Ohgane J. Imamura T. Urano A. Nishino K. Tanaka S. Hattori N. Genes Cells. 2002; 7: 961-969Crossref PubMed Scopus (180) Google Scholar). These analyses revealed that genes having tissue-dependent, differentially methylated regions (T-DMRs) were numerous. The methylation pattern of T-DMRs was specific but varied according to cell lineage and tissue type (4Shiota K. Kogo Y. Ohgane J. Imamura T. Urano A. Nishino K. Tanaka S. Hattori N. Genes Cells. 2002; 7: 961-969Crossref PubMed Scopus (180) Google Scholar, 5Imamura T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics. 2001; 76: 117-125Crossref PubMed Scopus (97) Google Scholar). We have also shown that individual cloned mice have different methylation aberrations, mainly at tissue-specific methylated loci (6Ohgane J. Aikawa J. Ogura A. Hattori N. Ogawa T. Shiota K. Dev. Genet. 1998; 22: 132-140Crossref PubMed Scopus (61) Google Scholar). Considering that DNA methylation is involved in various biological phenomena, such as tissue-specific gene expression, cell differentiation, X-chromosome inactivation, genomic imprinting, changes in chromatin structure, and tumorigenesis (7Eden S. Hashimshony T. Keshet I. Cedar H. Thorne A.W. Nature. 1998; 394: 842Crossref PubMed Scopus (240) Google Scholar, 8Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2252) Google Scholar, 9Bird A. Wolffe A. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar, 10Cho J. Kimyra H. Mimami T. Ohgane J. Hattori N. Tanaka S. Shiota K. Endocrinology. 2001; 142: 3389-3396Crossref PubMed Scopus (54) Google Scholar), it is conceivable that the formation of specific DNA methylation patterns is one of the principal epigenetic events underlying mammalian development. In mice, Oct-4, a member of the POU transcription factors, is expressed in the oocyte and preimplantation embryo but is later restricted to only the ICM of the blastocyst (11Okamoto K. Okazawa H. Okuda A. Sakai M. Muramatsu M. Hamada H. Cell. 1990; 60: 461-472Abstract Full Text PDF PubMed Scopus (620) Google Scholar, 12Rosner M.H. Vigano M.A. Ozato K. Timmons P.M. Poirier P.W. Rigby P.W.J. Staudt L.M. Nature. 1990; 345: 686-692Crossref PubMed Scopus (771) Google Scholar, 13Schöler H.R. Ruppert S. Suzuki N. Chowdhury K. Gruss P. Nature. 1990; 344: 435-439Crossref PubMed Scopus (598) Google Scholar), indicating that expression is restricted to totipotent and pluripotent cells. In Oct-4-deficient embryos, the ICM loses pluripotency and the trophoblast cells no longer proliferate to form the placenta (14Nichols J. Zevnik B. Anastassiadis K. Niwa H. Klewe-Nebenius D. Chambers I. Schöler H.R. Smith A. Cell. 1998; 95: 379-391Abstract Full Text Full Text PDF PubMed Scopus (2703) Google Scholar). Expression of the Oct-4 gene is found in ES cells but not in TS cells (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar, 15Palmieri S.L. Peter W. Hess H. Schöler H.R. Dev. Biol. 1994; 166: 259-267Crossref PubMed Scopus (529) Google Scholar). Reduction in Oct-4 gene expression leads to the trans-differentiation of ES cells into TS cells under adequate culture conditions (16Niwa H. Miyazaki J. Smith A.G. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2926) Google Scholar), demonstrating that suppression of the Oct-4 gene is a critical step for the determination of the potency of these stem cells. The Oct-4 gene has a GC-rich and TATA-less minimal promoter (17Okazawa H. Okamoto K. Ishino F. Ishino-Kaneko T. Takeda S. Toyoda Y. Muramatsu M. Hamada H. 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Philipsen S. Schöler H.R. Cell. Mol. Biol. (Noisy-Le-Grand). 1999; 45: 709-716PubMed Google Scholar, 24Barnea E. Bergman Y. J. Biol. Chem. 2000; 275: 6608-6619Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 25Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Schöler H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). However, specific regulatory mechanisms conferring the developmental stage- and cell type-specific expression of the Oct-4 gene have not been conclusively revealed to date. In this report, to determine whether DNA methylation is involved in the regulation of Oct-4 gene expression, we investigated 1) the effect of a DNA methylation inhibitor on Oct-4 non-expressing cells, 2) the methylation status of Oct-4 enhancer/promoter locus in ES and TS cells, the effect of in vitro methylation on Oct-4 enhancer/promoter Oct-4 expression in the DNA and the chromatin structure of Oct-4 enhancer/promoter region in ES and TS cells. and were from The ES cell from mice E. H. N. J. Dev. Biol. 1994; Google Scholar), was by H. and in a W. A Scholar). The cells were by the of S. D. N. Nature. PubMed Scopus Google TS cells were from mice according to the previously (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). the of these TS cells, were on cells in the of and (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar). TS cells from mice were in the by in embryonic with and on culture cells were in with fetal to with 5-aza-2′-deoxycytidine methylation TS cells were for and for days in or with or trichostatin A inhibitor of cells were in with or for days with TSA, cells were for and to or with or for mice were from The Jackson were on a and to and of the on which a was found was as days Dnmt1n/n embryos and were from mice and at dpc, in and at of the and CpG the and CpG of the genomic DNA with a by the of DNA from cells and tissues of mice and Dnmt1n/n was as previously (6Ohgane J. Aikawa J. Ogura A. Hattori N. Ogawa T. Shiota K. Dev. Genet. 1998; 22: 132-140Crossref PubMed Scopus (61) Google Scholar). was with and in with was by and for at the and were to of and and the was at for in The DNA was the DNA and the was with at for The was by to a of The DNA was in and by as were with or to the methylation status of Oct-4 upstream only to by the from genomic DNA to or and from methylated DNA by the The of were by The were and by the of The were cloned into and were for Analysis of Oct-4 Expression by was with of first was with to genomic The of was into first with and for was with and reverse to Oct-4 whereas of was with and reverse In the of Oct-4, were under the of were under the of region of the Oct-4 gene including the proximal enhancer/promoter to was by from the genomic DNA of and the was cloned into of the reporter was the which found in methylated the was with P. D. A. S. A. Nucleic Acids Res. 1990; PubMed Scopus Google in the of at for of the methylation was by to cells at in a were for and were with reporter the of a having a was into the cells. In a a of of the reporter and of expression was into cells. The of were a according to the were in Chromatin chromatin immunoprecipitation assay was and Cell. 1998; Full Text Full Text PDF PubMed Scopus Google In cells were with and with for at to form was on and with the at was with of the DNA from the for The of was with The were used to the Oct-4 proximal promoter and mouse A and and and at of and of Oct-4 by with Oct-4 gene is expressed only in restricted of cells. The expression of Oct-4 was not in TS cells or in cells, by analyses the Oct-4 gene was in somatic tissues and cells not Oct-4 in TS cells with 5-aza-dC, inhibitor of DNA in a In the Oct-4 expression was also in TS cells with inhibitor of that Oct-4 gene activity is in the suppressed in TS cells by DNA methylation and chromatin The expression of Oct-4 in cells, was not by with or the with and the Oct-4 gene in cells DNA or was not to Oct-4 transcription in cells. the that the mechanism of the Oct-4 gene in Oct-4 non-expressing cells DNA this was the the region of the Oct-4 gene is to hypermethylated in cells or tissues in which the Oct-4 gene is DNA of the of the Oct-4 Oct-4 gene has a distal and proximal which the cell type-specific expression of the Oct-4 gene G. A. K. M. K. Schöler H.R. PubMed Google Scholar, T. N. M. K. K. M. K. Schöler H.R. Y. Dev. 1999; PubMed Google Scholar). The is in Oct-4 expression in preimplantation embryos, germ cells, ES and embryonic germ cells G. A. K. M. K. Schöler H.R. PubMed Google Scholar, T. N. M. K. K. M. K. Schöler H.R. Y. Dev. 1999; PubMed Google Scholar). is no CpG at the of the Oct-4 the promoter region is in CpG is one and and in the DE, whereas has a for and at and In the promoter is one at We the DNA methylation status of the Oct-4 regulatory region by on these in ES cells, TS cells, and In ES cells, for were not with indicating that the genomic DNA of ES cells was at including and after in ES cells. it is that the Oct-4 locus was hypomethylated a of regulatory regions in ES cells In in TS cells all were to The of CpG methylation was that the Oct-4 upstream region of TS cells was methylated that of ES cells in the in which the Oct-4 gene is also not all of were indicating that the Oct-4 is methylated the regulatory regions in this tissue and is a the and distal regions of the Oct-4 gene the was hypomethylated in ES cells that express the Oct-4 whereas TS cells and the which do not express Oct-4, have methylated DNA of the Oct-4 investigated the methylation status of CpG upstream of the Oct-4 gene the promoter region to from the as by genomic CpGs in this and the CpG is in regions upstream of the Oct-4 gene in In ES cells, the of CpG methylation was only and the region is In the region was methylated in TS cells, the methylation of we that the promoter region of Oct-4 is hypomethylated in ES cells but hypermethylated in TS cells and these with of the it is that Oct-4 gene expression is DNA gene of Oct-4 by DNA promoter activity of Oct-4 the region was G. A. K. M. K. Schöler H.R. PubMed Google Scholar). We the reporter with the region of Oct-4 the and promoter regions to The Oct-4 reporter activity to that of the The activity of Oct-4 was by in vitro DNA DNA methylation at the Oct-4 regulatory region in Oct-4 of the reporter with expression also caused These that the transcription of the Oct-4 gene was CpG Expression of Oct-4 in has been in Dnmt1n/n mice, which activity compared with type E. R. Cell. Full Text PDF PubMed Scopus Google Scholar). We that the Oct-4 gene in mice the Oct-4 promoter region is also in these We the methylation status of the Oct-4 promoter region and the expression of Oct-4 in the placenta of Dnmt1n/n mice at E. R. Cell. Full Text PDF PubMed Scopus Google Scholar). methylated CpGs in the Oct-4 promoter region only of the CpGs in the placenta of Dnmt1n/n mice, whereas methylated CpGs in the placenta of type the expression of Oct-4 was in the Dnmt1n/n placenta but not in type placenta Expression of the Oct-4 gene was in type embryos to the of germ cells that express the Oct-4 gene. expression of Oct-4 in the mutant embryo to in the DNA in the expression of Oct-4 gene. Chromatin of the Oct-4 in ES and TS of CpG with the of transcription A. Cell. Full Text PDF PubMed Scopus Google and of and the transcription A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). DNA methylation chromatin by with and A. Nature. 1998; PubMed Scopus Google Scholar). we the status in ES cells and TS cells at the and promoter region of the Oct-4 gene by chromatin immunoprecipitation assay that and We of from the Oct-4 and promoter regions assay of in ES cells was that of TS cells and that the status of in the Oct-4 region was in ES cells in TS cells. Furthermore, the status of in ES cells was revealed to that of TS cells, indicating that the Oct-4 promoter region was in ES cells compared with TS cells and the of at the Oct-4 and promoter was in ES cells in TS cells. DNA methylation status is linked to the chromatin structure of the Oct-4 upstream We and on the (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar, K. Okazawa H. Okuda A. Sakai M. Muramatsu M. Hamada H. Cell. 1990; 60: 461-472Abstract Full Text PDF PubMed Scopus (620) Google Scholar, 12Rosner M.H. Vigano M.A. Ozato K. Timmons P.M. Poirier P.W. Rigby P.W.J. Staudt L.M. Nature. 1990; 345: 686-692Crossref PubMed Scopus (771) Google Scholar, 13Schöler H.R. Ruppert S. Suzuki N. Chowdhury K. Gruss P. Nature. 1990; 344: 435-439Crossref PubMed Scopus (598) Google Scholar, 15Palmieri S.L. Peter W. Hess H. Schöler H.R. Dev. Biol. 1994; 166: 259-267Crossref PubMed Scopus (529) Google that Oct-4 is not in TS cells, cell lines, the and somatic In Oct-4 was expressed in ES cells, that Oct-4 expression is restricted to totipotent and pluripotent cells such as the preimplantation embryo and ICM of the blastocyst (3Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1099) Google Scholar, K. Okazawa H. Okuda A. Sakai M. Muramatsu M. Hamada H. Cell. 1990; 60: 461-472Abstract Full Text PDF PubMed Scopus (620) Google Scholar, 12Rosner M.H. Vigano M.A. Ozato K. Timmons P.M. Poirier P.W. Rigby P.W.J. Staudt L.M. Nature. 1990; 345: 686-692Crossref PubMed Scopus (771) Google Scholar, 13Schöler H.R. Ruppert S. Suzuki N. Chowdhury K. Gruss P. Nature. 1990; 344: 435-439Crossref PubMed Scopus (598) Google Scholar, 15Palmieri S.L. Peter W. Hess H. Schöler H.R. Dev. Biol. 1994; 166: 259-267Crossref PubMed Scopus (529) Google Scholar). the upstream region of the Oct-4 gene was hypomethylated in ES cells but was hypermethylated in TS cells and the of DNA methylation and TS cells and cells to express the Oct-4 gene Furthermore, in vitro methylation severely suppressed the Oct-4 promoter activity in the reporter assay. In with these Oct-4 was in the placenta of Dnmt1n/n mice, which have minimal activity of DNA E. R. Cell. Full Text PDF PubMed Scopus Google Scholar). a Oct-4 expression and epigenetic by DNA methylation and chromatin was in the mammalian all cells differentiate changes in DNA the differentiation of a cell type is with the activation of a of genes and the of the Oct-4 gene is a for regulation DNA methylation early In the the that the Oct-4 gene has T-DMRs and that DNA methylation is critical for the regulation of Oct-4 gene expression. and DNA methylation pattern is specific as reported previously T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics. 2001; 76: 117-125Crossref PubMed Scopus (97) Google Scholar). differentiation with the and methylation of the genomic DNA to form the tissue type-specific methylation pattern T. Ohgane J. Ito S. Ogawa T. Hattori N. Tanaka S. Shiota K. Genomics. 2001; 76: 117-125Crossref PubMed Scopus (97) Google Scholar, J. Aikawa J. Ogura A. Hattori N. Ogawa T. Shiota K. Dev. Genet. 1998; 22: 132-140Crossref PubMed Scopus (61) Google Scholar). The Oct-4 gene is one of the genes by DNA as the expression of Oct-4 is severely suppressed in cells and Oct-4 gene expression in cell In the that gene regulatory mechanisms DNA methylation and changes in chromatin structure for development. The Oct-4 enhancer/promoter region was hypomethylated in the placenta of Dnmt1n/n several and have been reported in into or DNA on in vitro M. E. Cell. 1999; 99: Full Text Full Text PDF PubMed Scopus Google Scholar). The of leads to embryonic after E. R. Cell. Full Text PDF PubMed Scopus Google Scholar), whereas the mice of and for a longer of M. E. Cell. 1999; 99: Full Text Full Text PDF PubMed Scopus Google Scholar, D. B. Science. 2001; PubMed Scopus Google Scholar, K. M. H. E. 2002; PubMed Google Scholar), that activity is and the into and DNA in The of of with J. N. S. 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A polymer-stabilized liquid-crystal blue phase with a Kerr constant 170 times larger than that of nitrobenzene is shown to be capable of microsecond electro-optical switching over a wide temperature range in flat Kerr cells, without need for additional surface processing to generate alignment. The Figure shows a polarizing optical micrograph, under crossed polarizers, of an in-plane switched Kerr cell containing the polymer-stabilized blue phase.

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNAs) can function as powerful immune adjuvants by activating APC. Compared with conventional phosphorothioate-backbone CpG DNAs, another type of CpG DNAs, called an A or D type (A/D-type), possesses higher ability to induce IFN-alpha production. Conventional CpG DNAs can exert their activity through Toll-like receptor 9 (TLR9) signaling, which depends on a cytoplasmic adapter, MyD88. However, it remains unknown how A/D-type CpG DNAs exhibit their immunostimulatory function. In this study we have investigated murine dendritic cell (DC) responses to these two distinct CpG DNAs. Not only splenic, but also in vitro bone marrow-derived, DCs could produce larger amounts of IFN-alpha in response to A/D-type CpG DNAs compared with conventional CpG DNAs. This IFN-alpha production was mainly due to the B220(+) DC subset. On the other hand, the B220(-) DC subset responded similarly to both CpG DNAs in terms of costimulatory molecule up-regulation and IL-12 induction. IFN-alpha, but not IL-12, induction was dependent on type I IFN. However, all activities of both CpG DNAs were abolished in TLR9- and MyD88-, but were retained in DNA-PKcs-deficient DCs. This study demonstrates that the TLR9-MyD88 signaling pathway is essential for all DC responses to both types of CpG DNAs.

The adzuki bean beetle, Callosobruchus chinensis, is triple-infected with distinct lineages of Wolbachia endosymbiont, wBruCon, wBruOri, and wBruAus, which were identified by their wsp (Wolbachia surface protein) gene sequences. Whereas wBruCon and wBruOri caused cytoplasmic incompatibility of the host insect, wBruAus did not. Although wBruCon and wBruOri were easily eliminated by antibiotic treatments, wBruAus persisted over five treated generations and could not be eliminated. The inheritance pattern of wBruAus was, surprisingly, explained by sex-linked inheritance in male-heterozygotic organisms, which agreed with the karyotype of C. chinensis (2n = 20, XY). Quantitative PCR analysis demonstrated that females contain around twice as much wsp titer as males, which is concordant with an X chromosome linkage. Specific PCR and Southern blot analyses indicated that the wBruAus-bearing strain of C. chinensis contains only a fraction of the Wolbachia gene repertoire. Several genome fragments of wBruAus were isolated using an inverse PCR technique. The fragments exhibited a bacterial genome structure containing a number of ORFs typical of the alpha-proteobacteria, although some of the ORFs contained disruptive mutations. In the flanking region of ftsZ gene, a non-long terminal repeat (non-LTR) retrotransposon sequence, which is typical of insects but not found from bacteria, was present. These results strongly suggest that wBruAus has no microbial entity but is a genome fragment of Wolbachia endosymbiont transferred to the X chromosome of the host insect.

Recent studies have showed that inflammatory responses occur in inner ear under various damaging conditions including noise-overstimulation. We evaluated the time-dependent expression of proinflammatory cytokines in noise-exposed rat cochlea. Among several detected cytokines, real-time RT-PCR showed that interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) were significantly induced 3 hr after noise exposure, and quickly downregulated to the basal level. Tumor necrosis factor-alpha (TNF-alpha) was also slightly upregulated immediately after noise exposure. Immunohistochemical analysis showed that IL-6 expression was distinctively induced within the lateral side of the spiral ligament. Sequential expression analysis showed that IL-6 immunoreactivity was initially found in the cytoplasm of lateral wall cells, including Type IV and III fibrocytes, and expanded broader throughout the lateral wall, finally to the stria vascularis. Because of the negative Iba-1 staining, IL-6 expression in the early-phase was not due to macrophage or microglia activation. IL-6 was also detected in spiral ganglion neurons at 12 and 24 hr after noise exposure. Our data demonstrates the production of proinflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, in early phase of noise overstimulated cochlea. IL-6 expression was observed in the spiral ligament, stria vascularis, and spiral ganglion neurons. These cytokines, produced by the cochlear structure itself in response to noise exposure, may initiate an inflammatory response and have some role in the mechanism of noise-induced cochlear damage.

We compared volatiles from lima bean leaves (Phaseolus lunatus) infested by either beet armyworm (Spodoptera exigua), common armyworm [Mythimna (Pseudaletia) separata], or two-spotted spider mite (Tetranychus urticae). We also analyzed volatiles from the leaves treated with jasmonic acid (JA) and/or methyl salicylate (MeSA). The volatiles induced by aqueous JA treatment were qualitatively and quantitatively similar to those induced by S. exigua or M. separata damage. Furthermore, both S. exigua and aqueous JA treatment induced the expression of the same basic PR genes. In contrast, gaseous MeSA treatment, and aqueous JA treatment followed by gaseous MeSA treatment, induced volatiles that was qualitatively and quantitatively more similar to the T. urticae-induced volatiles than those induced by aqueous JA treatment. In addition, T. urticae damage resulted in the expression of the acidic and basic PR genes that were induced by gaseous MeSA treatment and by aqueous JA treatment, respectively. Based on these data, we suggest that in lima bean leaves, the JA-related signaling pathway is involved in the production of caterpillar-induced volatiles, while both the SA-related signaling pathway and the JA-related signaling pathway are involved in the production of T. urticae-induced volatiles.

Endogenous genetic factors and environmental signals control the time of flowering in plants. One of the environmental signals is photoperiod. Genetic control mechanisms for the photoperiodic response of flowering of long-day plants (LDPs) have been extensively analyzed through the use of

We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is approximately 32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene.

A backcrossed population (BC(4)F(2)) derived from a cross between a japonica rice variety, Nipponbare, as the recurrent parent and an indica rice variety, Kasalath, as the donor parent showed a long-range variation in days to heading. Quantitative trait loci (QTL) analysis revealed that two QTL, one on chromosome 3, designated Hd6, and another on chromosome 2, designated Hd7, were involved in this variation; and Hd6 was precisely mapped as a single Mendelian factor by using progeny testing (BC(4)F(3)). The nearly isogenic line with QTL (QTL-NIL) that carries the chromosomal segment from Kasalath for the Hd6 region in Nipponbare's genetic background was developed by marker-assisted selection. In a day-length treatment test, the QTL-NIL for Hd6 prominently increased days to heading under a 13.5-hr day length compared with the recurrent parent, Nipponbare, suggesting that Hd6 controls photoperiod sensitivity. QTL analysis of the F(2) population derived from a cross between the QTL-NILs revealed existence of an epistatic interaction between Hd2, which is one of the photoperiod sensitivity genes detected in a previous analysis, and Hd6. The day-length treatment tests of these QTL-NILs, including the line introgressing both Hd2 and Hd6, also indicated an epistatic interaction for photoperiod sensitivity between them.

Multifunctionalization of carbon nanotubules is easily achieved by attaching functional molecules that provide specific advantages for microscopic applications. We fabricated a double photodynamic therapy (PDT) and photohyperthermia (PHT) cancer phototherapy system that uses a single laser. Zinc phthalocyanine (ZnPc) was loaded onto single-wall carbon nanohorns with holes opened (SWNHox), and the protein bovine serum albumin (BSA) was attached to the carboxyl groups of SWNHox. In this system, ZnPc was the PDT agent, SWNHox was the PHT agent, and BSA enhanced biocompatibility. The double phototherapy effect was confirmed in vitro and in vivo. When ZnPc-SWNHox-BSA was injected into tumors that were subcutaneously transplanted into mice, the tumors almost disappeared upon 670-nm laser irradiation. In contrast, the tumors continued to grow when only ZnPc or SWNHox-BSA was injected. We conclude that carbon nanotubules may be a valuable new tool for use in cancer phototherapy.

We studied neurogliaform neurons in the stratum lacunosum moleculare of the CA1 hippocampal area. These interneurons have short stellate dendrites and an extensive axonal arbor mainly located in the stratum lacunosum moleculare. Single-cell reverse transcription-PCR showed that these neurons were GABAergic and that the majority expressed mRNA for neuropeptide Y. Most neurogliaform neurons tested were immunoreactive for alpha-actinin-2, and many stratum lacunosum moleculare interneurons coexpressed alpha-actinin-2 and neuropeptide Y. Neurogliaform neurons received monosynaptic, DNQX-sensitive excitatory input from the perforant path, and 40 Hz stimulation of this input evoked EPSCs displaying either depression or initial facilitation, followed by depression. Paired recordings performed between neurogliaform neurons showed that 85% of pairs were electrically connected and 70% were also connected via GABAergic synapses. Injection of sine waveforms into neurons during paired recordings resulted in transmission of the waveforms through the electrical synapse. Unitary IPSCs recorded from neurogliaform pairs readily fatigued, had a slow decay, and had a strong depression of the synaptic response at a 5 Hz stimulation frequency that was antagonized by the GABA(B) antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP55845). The amplitude of the first IPSC during the 5 Hz stimulation was also increased by CGP55845, suggesting a tonic inhibition of synaptic transmission. A small unitary GABA(B)-mediated IPSC could also be detected, providing the first evidence for such a component between GABAergic interneurons. Electron microscopic localization of the GABA(B1) subunit at neurogliaform synapses revealed the protein in both presynaptic and postsynaptic membranes. Our data disclose a novel interneuronal network well suited for modulating the flow of information between the entorhinal cortex and CA1 hippocampus.

The intracellular distribution of organelles is a crucial aspect of effective cell function. Chloroplasts change their intracellular positions to optimize photosynthetic activity in response to ambient light conditions. Through screening of mutants of Arabidopsis defective in chloroplast photorelocation movement, we isolated six mutant clones in which chloroplasts gathered at the bottom of the cells and did not distribute throughout cells. These mutants, termed chloroplast unusual positioning (chup), were shown to belong to a single genetic locus by complementation tests. Observation of the positioning of other organelles, such as mitochondria, peroxisomes, and nuclei, revealed that chloroplast positioning and movement are impaired specifically in this mutant, although peroxisomes are distributed along with chloroplasts. The CHUP1 gene encodes a novel protein containing multiple domains, including a coiled-coil domain, an actin binding domain, a Pro-rich region, and two Leu zipper domains. The N-terminal hydrophobic segment of CHUP1 was expressed transiently in leaf cells of Arabidopsis as a fusion protein with the green fluorescent protein. The fusion protein was targeted to envelope membranes of chloroplasts in mesophyll cells, suggesting that CHUP1 may localize in chloroplasts. A glutathione S-transferase fusion protein containing the actin binding domain of CHUP1 was found to bind F-actin in vitro. CHUP1 is a unique gene identified that encodes a protein required for organellar positioning and movement in plant cells.