Effective procedure to develop alternative annotations of bacterial tRNA genes by means of deductive inference on the basis of characteristic tandems of tRNA genes

In a series of analysis of genomic DNA sequences, we have established an induction-deduction method to dig up hidden tRNA and rRNA genes from bacterial genome DNA sequences by means of a concept of a characteristic tRNA-gene tandem we have ldeveloped, and are accumulating information on positions of putative tRNA and rRNA genes to be proposed as alternative annotations to the DDBJ/GenBank/EMBL Database. We have searched the DNA sequences near the existing tRNA genes as golcondas for tRNA genes, and found mord than fifty genes, e.g. tRNA-Ser and tRNA-Met in [AB013377], and 5S rRNAs in [AE014192], [AE017000], and others. A part of miserable states of the Database was partly introduced, and it is discussd how such status will be disso1ved. In addition, we proposed some ideas to maintain and improve the DDBJ/GenBank/EMBL database

アヒル消化管における内分泌細胞の分布

食道から直腸末端までのアヒル消化管における内分泌細胞の存在と分布を,鉛ヘマトキシリン,塩酸トルイジンブルー,Sevier and Mungerの鍍銀法およびMassonの銀親和反応を用いて検索した。 鉛ヘマトキシリンと塩酸トルイジンブルーで染まる細胞は食道を除く消化管の全部位でみられ,好銀性細胞は腺胃粘膜,腺胃腺および腸管の全部位に,銀親和性細胞は腸管にのみみられた。 内分泌細胞の分布密度は,筋胃幽門部が最も多く,次が腺胃腺であり,腸は十二指腸,空腸,回腸,盲腸,結直腸の5部位とも同程度で幽門部の約1/2,腺胃粘膜および胃峡部は腺胃腺の1/10,筋胃中央部は前2部位よりも少なく最少であった。 筋胃幽門部は,筋胃と十二指腸を分けるわずかな粘膜ひだから筋胃側5㎜位の部分であり,この部に非銀親和性で非好銀性の内分泌細胞が高密度に存在することは興味ある所見である。 これら内分泌細胞の染色性および分布から,アヒル消化管における4種類以上の内分泌細胞の存在が推測された。The four histological methods, previously known to be useful in selective detection of endocrine cells, were applied to the duck digestive tracts, from oesophagus to colorectum. Cells stained with lead-hematoxylin and HC1-toluidine blue were observed in all regions of the duck digestive tracts with the exception of the oesophagus. Argyrophil cells were observed in proventricular mucosa and glands, and in the intestine. Argentaffin cells were observed only in the intestine. The frequency of endocrine cells in the duck digestive tracts was highest in the restricted region of gizzard mucosa where was called the pyloric region in this paper, next in the proventricular glands, equally about half of the frequency in the pyloric region in the five regions of the intestine, one tenth of that of the proventricular glands in the proventricular and isthmus mucosa, and the smallest frequency was noted in the central part of gizzard mucosa. The pyloric region was about 5mm anterior to the narrow mucosal fold separating the gizzard from the intestine. It was an interesting to find that the endocrine cells which were nonargentaffin and nonargyrophil were densely present in this region. From the staining properties and the distribution of the endocrine cells, the possibility of existence of four and more types of endocrine cells in the duck digestive tracts was discussed

Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology

<p>Abstract</p> <p>Background</p> <p>Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine <it>PRNP</it> (b<it>PRNP</it>) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down b<it>PRNP</it> were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of <it>PRNP</it>, and lengths of siRNAs.</p> <p>Results</p> <p>Four siRNA expression plasmid vectors were used: three harboring different cloning sites were driven by the human U6 promoter (hU6), and one by the human tRNA^Valpromoter. Six target sites of bovine <it>PRNP </it>were designed using an algorithm. From 1 (22 mer) to 9 (19, 20, 21, 22, 23, 24, 25, 27, and 29 mer) siRNA expression vectors were constructed for each target site. As targets of siRNA, the entire b<it>PRNP </it>coding sequence was connected to the reporter gene of the fluorescent EGFP, or of firefly luciferase or <it>Renilla </it>luciferase. Target plasmid DNA was co-transfected with siRNA expression vector DNA into HeLaS3 cells, and fluorescence or luminescence was measured. The activities of siRNAs varied widely depending on the target sites, length of the siRNAs, and vectors used. Longer siRNAs were less effective, and 19 mer or 21 mer was generally optimal. Although 21 mer GGGGAGAACTTCACCGAAACT expressed by a hU6-driven plasmid with a <it>Bsp </it>MI cloning site was best under the present experimental conditions, the corresponding tRNA promoter-driven plasmid was almost equally useful. The effectiveness of this siRNA was confirmed by immunostaining and Western blotting.</p> <p>Conclusion</p> <p>Four siRNA expression plasmid vectors, six target sites of b<it>PRNP</it>, and various lengths of siRNAs from 19 mer to 29 mer were examined to establish optimal conditions for knocking down of b<it>PRNP </it>in vitro. The most effective siRNA so far tested was 21 mer GGGGAGAACTTCACCGAAACT driven either by a hU6 or tRNA promoter, a finding that provides a basis for further studies in vivo.</p

IL-17A produced by αβ T cells drives airway hyper-responsiveness in mice and enhances mouse and human airway smooth muscle contraction.

Emerging evidence suggests that the T helper 17 (T(H)17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle

Potential Role of Free Fatty Acids in the Pathogenesis of Periodontitis and Primary Sjögren's Syndrome

Clinical studies have shown that metabolic disorders such as type 2 diabetes and dyslipidemia are associated with increased risk of oral-related diseases, such as periodontitis and Sjögren’s syndrome. Although changes in the immune system are critical in both of these metabolic disorders and oral-related diseases, the mechanism underlying the interaction between these diseases remains largely unknown. Obesity and type 2 diabetes are known to be associated with higher concentrations of free fatty acids in blood. Among free fatty acids, saturated fatty acids such as palmitic acid have been demonstrated to induce inflammatory responses mainly via the innate immune systems, and to be involved in the pathogenesis of type 2 diabetes in tissues such as adipose tissue, liver, pancreas, and skeletal muscle. Here, we highlight recent advances in evidence for the potential involvement of palmitic acid in the pathogenesis of periodontitis and Sjögren’s syndrome, and discuss the possibility that improvement of the lipid profile could be a new strategy for the treatment of these diseases

Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks

Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM