The mitogen-activated protein kinase (MAPK) pathways are signal transduction mechanisms that regulate many cellular processes in eukaryotic organisms, from yeasts to mammals. Multiple MAPKs regulate eukaryotic gene expression in response to various extracellular stimuli through phosphorylation of transcription factors. We have been studying the 1 MAPK, a homologue of the mammalian ERK/MAPK in fission yeast. The 1 MAPK regulates cell integrity and cell morphology. We have previously demonstrated that Atf1, a transcription factor downstream of the stress-activated MAPK pathway, serves also as a target of the 1 MAPK signaling in fission yeast. Here, we identified ecm33⁺ gene, encoding a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of 1 and Atf1. The gene expression of ecm33⁺ is regulated by two transcription factors Atf1 and Mbx1. We also developed an in vivo real-time monitoring system of Atf1 or Mbx1 transcriptional activity, which enables to monitor the activation of the 1 MAPK pathway by various stimuli. Finally, we demonstrated that Ecm33 is involved in the negative regulation of the 1 MAPK signaling through the control of Ca²⁺ homeostasis. The ecm33 deleted cells displayed Ca²⁺ sensitivity and increased phosphorylation levels of 1 MAPK. In addition, the Ecm33 overproducing cells displayed phenotypes closely similar to those of the knockout cell. Collectively, Ecm33 plays a role in the negative feedback regulation of 1 cell integrity signaling.

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An E. coli mutant (M8328) was isolated by NTG mutagenesis from E. coli KMS3207 which has an inducible N-acetylneuraminate lyase (NPL). The mutant was resistant to catabolite repression and produced NPL constitutively. To improve the productivity of NPL, a hybrid plasmid, 6, carrying the npl gene was introduced into the mutant, M8328. The NPL activity level was increased more than 5-fold in the 6-harboring mutant compared with that in the mutant, when the cells were grown in inducer (N-acetylneuraminic acid)-free medium. The NPLs produced by the mutant and the 6-harboring cells were structurally and immunologically identical with that purified from the parent strain (KMS3207).

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To evaluate the utility of polyphosphate kinase gene (ppk)-specified polyphosphate in mercury remediation, a fusion plasmid, 27, with ppk from Klebsiella aerogenes and mercury transport genes, merT and merP, from Pseudomonas K-62, was constructed. The transcription and translation of ppk, merT and merP were found to be mercury-inducible. The ppk-specified polyphosphate was identified in cells preinduced by Hg²⁺, but not in cells without mercury induction, suggesting that the synthesis of polyphosphate is regulated by merR. The hyper-sensitive phenotype to Hg²⁺, shown by bacteria with pMRD141, which contains merT and merP, was almost completely restored to its original levels when the ppk was introduced into the plasmid, suggesting that the Hg²⁺-toxicity was reduced by the polyphosphate, probably via chelation formation. Bacteria with 27 accumulated approximately 6-fold more mercury than the bacteria with cloning vector, pUC119. These results clearly demonstrate that the polyphosphate is capable of retaining mercury in the cells without taxing the cells. Based on the results obtained in the present study, the fusion plasmid 27 may serve as a strategy for mercury remediation.

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通常チョコレートとカルシウム添加チョコレートを各々60%含む半合成飼料をラットに与え,それぞれの油脂の吸収率を求めた.その結果,カルシウム添加チョコレートの油脂及びカルシウムの吸収率が,通常チョコレートに比べて低くなることが観察された.これはココアバターの主成分であるステアリン酸やパルミチン酸などの長鎖飽和脂肪酸の吸収が,チョコレートにカルシウムを添加することによって抑制されたためであろうと考えられた.

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To establish the mode of gene expression specified by transposon, we investigated the correlation among the homology of the DNA sequence, the extent of transposon-specific transcription, the specific activity of penicillinase (PCase) per cell, and the transposition frequency by using two ampicillin resistance transposons (TnAs), Tn2601 and Tn2602. Although both the TnAs specify the so-called type I PCase, Tn2602 always conferred 10-to 20-fold higher PCase activity per cell than Tn2601 regardless of the kind of replicon carrying TnA. The transposition frequency of Tn2602 also was 8 to 50 times higher than that of Tn2601 in all combinations of donor and recipient plasmids examined. As a result, the transposability expressed by the TnA was thought to correlate with the productivity of PCase in the cell specified by the corresponding TnA. The level of TnA-specific transcription of Tn2602 was noticeably higher than that of Tn2601, whereas the two TnAs shared a high degree (more than 90%) of DNA sequence homology. These results suggest that the difference in rates of transcription of the two transposons plays a key role in determining the difference in the productivity of PCase and the transposition-protein (s) of Tn2601 and Tn2602.

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A cell line (JINET) which has a limited life-time in culture was established from a cynomolgus monkey kidney tissue. JINET cells lost their rapidly growing potentials at around the 40th passage level. The cell line has been maintained over 3 years by recovering cells from stocks frozen at young generations when the cells lost the rapidly growing potentials at each series of serial cultivations.
The cell line showed a high susceptibility to wide varieties of viruses almost comparable to that of primary cynomolgus monkey kidney cell cultures. Possible availability of this cell line as a substitute for primary monkey kidney cell cultures in virus researches is discussed.

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Quite recently [IEEE Commu. Letters, Vol.14, No.1, 2010], Choi et al. proposed a handover authentication scheme using credentials based on chameleon hashing, claiming to provide several security features including Perfect Forward/Backward Secrecy (PFS/PBS). This paper examines the security of the scheme and shows that the scheme still fails to achieve PFS/PBS unlike their claims.

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Mitogen-activated protein kinase (MAPK) is a highly conserved serine/threonine kinase that regulates multiple cellular processes such as cell proliferation, differentiation, apoptosis, and inflammation. Rnc1 has been identified as a regulator of

1 MAPK signaling, a homologue of extracellular signal-regulated kinase (ERK)-1 MAPK in mammals. Rnc1 encodes a K-homology (KH)-type RNA-binding protein (RBP). Previously, it was reported that Rnc1 acts as a negative regulator of 1 MAPK signaling through the mRNA stabilization of Pmp1, the MAPK phosphatase for 1 in our laboratory. We analyzed the spatial regulation of Rnc1 and discovered that Rnc1 is exported from the nucleus by the mRNA-export system. The nuclear export of Rnc1 is important for exerting its function to stabilize Pmp1 mRNA. Therefore, the spatial regulation of Rnc1 affects MAPK signaling activity. We also reported that Nrd1, an RRM-type RBP, plays a critical role in cytokinesis by binding to and stabilizing myosin mRNA. Notably, Rnc1 and Nrd1 localize to stress granules (SGs) in response to various environmental stresses. Moreover, SG formation is inhibited in the Nrd1 or Rnc1 deletion cells, whereas the overproduction of Nrd1 or Rnc1, as well as that of mammalian RBP TIA-1, induces granule formation. These data show that Nrd1 and Rnc1 regulate SG formation as a novel SG component. Alterations of SG formation are linked to neurodegenerative diseases and resistance to anti-cancer drugs, thus conferring remarkable clinical importance to SGs. This review discusses the spatial regulation of RBPs or SG formation as novel targets for drug discovery.

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A new hybrid anthracycline antibiotic was produced by heterologous expression of dnrK encoding carminomycin 4-O-metyltransferase in an epelmycin-producing Streptomyces violaceus. 100 was constructed by insertion of Steptomyces peucetius dnrK gene in Steptomyces-expresion vector pIJ6021 and introduced to the epelmycin producer. The transformant produced a hybrid anthracycline antibiotic together with host epelmycins when cultured in antibiotic production medium in the presence of thiostrepton. The hybrid anthracycline was determined to be 7-O-L-rhodosaminyl-4-O-methyl-ε-rhodomycinone (4-O-memylepelmycin D). However, the attempts on production of hybrid 4-O-methyl aclarubicin and 4-O-methyl-1-deoxyobelmycin by the transformants of aclarubicin and 1-deoxyobelmycin producers with 100 were unsuccessful.

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MK is a gene that is activated by retinoic acid in embryonal carcinoma (EC) cells and is expressed temporarily during the mid-gestation period of mouse embryogenesis. Midkine, the product of the gene is a novel heparin-binding growth/differentiation factor with neurite outgrowth and neurotrophic activities. The regulatory DNA element in the retinoic acid-induced expression of the MK gene has been investigated. The 1.9 kb 5'-flanking region of the MK gene can mediate retinoic acid-responsive gene expression in F9 and HM-1 EC cells. Analysis of this region by deletion mutagenesis in F9 EC cells shows that there is a retinoic acid-responsive enhancer (designated as REM1) around 900 by upstream from the transcription start site. This enhancer is composed of two sequence elements, which are located between -1006 and -895 and between -901 and -794. The core element of the upstream region (-971 to -955), whose deletion abolished the retinoic acid responsiveness, contained a sequence highly homologous to a binding site for retinoic acid receptors. Binding of a retinoic acid receptor heterodimer to this core element was verified by gel shift assay. Thus, retinoic acid and the receptor complex can directly induce the expression of a growth/differentiation factor gene.

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カルシニューリン（CN）は，酵母からヒトに至るまで高度に保存されたCa²⁺/カルモジュリン依存性タンパク質脱リン酸化酵素であり，免疫抑制薬FK506，シクロスポリンAの標的分子である．免疫抑制薬，シクロスポリンAおよびFK506は，イムノフィリンと結合し，さらにCNと複合体を形成し，CNの酵素活性を阻害することにより免疫抑制効果を発揮する．CNは免疫応答や心臓発生，さらには神経可塑性（LTP，LTD）など，多種多様な生体機能に関与する事が明らかにされてきたが，従来の生化学的手法や細胞生物学的手法のみでは，これらの機構を分子レベルで解明することは，困難であった．我々は，哺乳動物に極めて近い細胞内情報伝達系を持つモデル生物である分裂酵母においてもCNがFK506の標的分子であることに着目し，CNと機能的に関連する因子を遺伝学的アプローチにより同定し，機能解析を行うことで，CNを介するシグナル伝達経路を分子レベルで解明しようと考えた．その結果，分裂酵母CN遺伝子はCl⁻ホメオスタシスに必須であること，CNシグナル経路は哺乳動物のERKと相同な経路である1 MAPキナーゼ経路と拮抗的にCl⁻ホメオスタシスを制御することを明らかとした．さらに分裂酵母CN遺伝子をノックアウトしても致死ではないことに着眼し，CN遺伝子破壊と合成致死になる変異体のスクリーニングを行った．その結果，分裂酵母CN遺伝子は，phosphatidylinositol-4-phosphate 5-kinaseなどの遺伝子とともに，細胞質分裂などの生理現象の制御に関わっていることが明らかとなってきた．本論文では，分裂酵母モデル系を用いた筆者らの研究を中心に，CNの細胞機能，およびその作用経路について述べる．

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The rates of the nitrosation of phenyl cyclohexanecarboxylate (abbreviated as PCC) with nitrosyl hydrogensulfate (NHS) in 0-7% oleum to form ε-caprolactam have been investigated by measuring the volume of evolved carbon dioxide. The rate equation of the reaction is written as follows : Rate=(κ_1e+κ_2esC_so3+κ_2enC_NHS)C_PCC A probable mechanism which involves the rate-determining steps of formation of pentamethylene ketene () by deprotonation from cyclohexanecarbonyl cation formed by elimination of phenolate anion and sulfur trioxide from an adduct of PCC and SO₃ (κ₁, main path, 90.4%), and by the elimination of SO₃, phenol and proton from the adduct (κ_2es, 5.3%) followed by a rapid attack of NHS on the double bond of , and a rate-determining formation of cyclohexanone oxime hydrogensulfate from the adduct of NHS and PCC (κ_2en, 4.3%) was discussed and postulated.

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Luciferases are widely used for the quantitative monitoring of gene expression in a variety of organisms. We successfully expressed novel red- and green-emitting luciferases of Phrixothrix railroad worms in mammalian cells in combination with the Kozak sequence and the CAG promoter. The characteristic properties of these luciferases indicate that they are appropriate reporter genes for the simultaneous monitoring of two gene expressions.

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We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney () cells as well as in FL cells in response to human recombinant IFN-αA treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-αA induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration.

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Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that are activated by diverse stimuli such as growth factors, cytokines, neurotransmitters and various cellular stresses. MAPK cascades are generally present as three-component modules, consisting of MAPKKK, MAPKK and MAPK. The precise molecular mechanisms by which these MAPK cascades transmit signals is an area of intense research, and our evolving understanding of these signal cascades has been facilitated in great part by genetic analyses in model organisms. One organism that has been commonly used for genetic manipulation and physiological characterization is the nematode Caenorhabditis elegans. Genes sequenced in the C. elegans genome project have furthered the identification of components involved in several MAPK pathways. Genetic and biochemical studies on these components have shed light on the physiological roles of MAPK cascades in the control of cell fate decision, neuronal function and immunity in C. elegans.

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This report describes efficient plasmid uptake by the thermophile Geobacillus kaustophilus HTA426 by means of a ternary conjugation system, which was used to construct thermophile DNA libraries for G. kaustophilus and to identify the genes for orotidine-5'-phosphate decarboxylase by in vivo functional screening. The results indicate that the conjugation system is useful in constructing G. kaustophilus libraries, which are practical in identifying thermophile genes.

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To directly examine the cellular roles of the retinoblastoma gene product (pRb), we constructed an effective antisense Rb RNA vector that expressed an antisense Rb-1 sequence directed toward the 3' untranslated region of Rb-1 mRNA. In this study, we introduced the antisense Rb vector into mouse fibroblast SV-T2 cells expressing large amounts of SV40 LT, which inhibits the biological functions of pRb through direct binding. Only a small number of drug-resistant colonies were obtained after drug selection. Co-transfection of SV-T2 cells with pRb expression vectors and the antisense Rb vector resulted in an apparent increase in the number of drug-resistant colonies, suggesting that a decrease in cellular pRb content induced cell death in the cells. The cell death in SV-T2 cells was also abrogated by simultaneous transfection with antisense p53 RNA expression vector. These results indicate that a decrease in the amount of biologically active pRb by both the effects of the transfected antisense Rb RNA and the large amount of endogenous SV40 LT induces cell death in SV-T2 cells through a p53-dependent pathway. The antisense Rb and p53 vectors used in this study are useful to examine the biological functions of these genes in established cell lines.

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Two human interferons (IFNs), natural lymphoblastoid IFN-α (Lb-IFN-α) and recombinant IFN-α (Re-IFN-αA) were compared for their induction of 2'-5' oligoadenylate (2-5A) synthetase and pharmacokinetic behaviors in cynomolgus monkeys. In in vitro experiments, (1) the enzyme activity induced in an epithelial cell strain from human amniotic membrane (FL) cells was much higher than that in primary culture of cynomolgus monkey kidney () cells, and (2) Lb-IFN-α induced higher levels of the enzyme than did Re-IFN-αA in both cells. In in vivo experiments, (1) no difference was found in clearance from the blood between the two IFNs, but the half life of the enzyme induced in peripheral leukocytes by Lb-IFN-α was about twice longer than that by Re-IFN-αA, and (2) the enzyme levels detected in various tissues were IFN-dose dependent. These results indicate that natural Lb-IFN-α is more effective in inducing 2-5A synthetase than Re-IFN-αA, and that the enzyme activity of various tissues as well as peripheral blood lymphocytes (PBL) increased in the cynomolgus monkeys administered with human IFNs.

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