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Approximately 2.5 k base pair (bp) amplicons encoding a cdt gene operon of about 2.3 kbp and two partial and putative open reading frames (ORFs) were identified with all the six urease-negative (UN) Campylobacter lari isolates obtained from different sources and in different countries using a novel PCR primer pair constructed in silico. Three closely spaced and possible ORFs for cdtA, B and C and two putative promoters and a hypothetically intrinsic p-independent transcription terminator were found in the operon. Each ORF commenced with an ATG start codon and terminated with a TGA stop codon for cdtA and B and a TAA for C. A possible overlap was identified between the four nucleotides including the stop codon (TGA) of the cdtA and the four nucleotides including the start codon (ATG) of the B, and the non-coding region of six bp occurred between cdtB and C. The start codons for the three cdt genes were preceded by Shine-Dalgarno sequences. Although nucleotide sequence differences were identified at seven loci in the cdtA gene, six B and two C, among seven isolates, including C. lari RM2100 reference strain, no polymorphic sites were demonstrated to occur in the putative promoters, hypothetically intrinsic transcription terminators and three ribosome binding sites among the seven isolates. Although no PCR amplicons were generated with more than 10 isolates of urease-positive thermophilic Campylobacter (UPTC) using the primer pair, C. lari consisting of UN C. lari and UPTC was suggested to carry a cdt gene operon in the genome.

我々は,セロトニン受容体のような神経伝達物質受容体あるいはそのトランスポーター(例えばセロトニン・トランスポーター(5-HTT))の遺伝子多型とオランザピンのような非定型抗精神病薬との関係を調査した。その結果,5-HTTのs対立遺伝子をもつ患者ともたない患者との間で,統合失調症陰性症状に対するオランザピンの効果に,統計学的な有意差が見られた。一方,アルツハイマー病患者に認められる精神神経症状に関わる5HT2A受容体と神経機能に重要な役割を持つダウの関係を明らかにするための予備段階として,2つのダウ・アイソフォーム(T3RとT4R)を明確に識別できる特性の高いポリクローン抗体を作製した。これらの抗体を用いて5HT2A受容体とタウとの関係を明らかにするために免疫組織化学的解析等を進める予定である。, We investigated the relationships between the effects of atypical antipsychotics such as olanzapine and neurotransmitter receptor and transporter gene polymorphisms such as serotonin receptor and transporter (5-HTT). There was a significant difference in the effect of olanzapine on the schizophrenia negative syndrome among the patients with and without the s allele of 5-HTT. Two types of tau isoform are found in neurofibrillary tangles-a pathological hallmark of tauopathies. To study how and which tau isoforms contribute to neurobal degeneration, we have developed two novel conformation-sensitive antibodies. There was no cross-reactivity between T3R (tau isoform, three-repeat tau) and T4R (four-repeat tau), and T3R and T4R showed reduced binding to the thioflavin-positive β-structural form of their target.

Degenerate PCR primers were designed in silico based on two urease structural genes, namely ureA and ureB, for urease-positive thermophilic Campylobacter (UPTC) organisms. Resultant PCR amplification employing these primers generated an amplicon of approximately 2kb, which was cloned and sequenced, in UPTC organisms (n=12), isolated from various parts of Europe and Japan. Overall, sequence similarities were shown to be 96.7 to 99.9% and following sequence alignment analysis, the approximate 1.96kb regions from the 12 isolates were deduced to consist of parts of ureA (about 570 bps) and ureB (about 1390 bps) which contain an overlapping region between the ureA and ureB gene loci. Although a total of 144 heterogeneous sites of all substitutions were located throughout this region, the substitution ratio was higher in the ureA region (1/〓 10 bases) than in the ureB region (1/〓 15 bases). A resulting dendrogram was constructed, which was based on the nucleotide sequence data of 12 UPTC isolates examined and demonstrated that the UPTC organisms were genetically variable. UPTC organisms formed a major cluster with Helicobacter organisms, separate from the other urease-producing bacteria examined, suggesting a shared ancestry between UPTC organisms and Helicobacter.

解析したC. lari15株での配列の類似性は,98.2-100%であった。そして,驚くべきことに,今回の研究で配列決定されたUPTC株の1つでイギリスの海水中の二枚貝から分離されたNCTC12896株の16SrDNAの配列とレファレンス株として用いたカナダのヒト臨床由来のUN C. lari 11760株のそれとは全く同一であった。更に,UPGMA法を用いてリファレンス株を含めて系統樹を作成したところ,C. lariは,他の高温性C. jejuni及びC. coliとは離れて,いくつかの小さなクラスターからなる大きな1つのクラスターを形成することが明らかとなった。以上の様にC.lariの代表的な2つのtaxonであるUPTCと UNC. lariを16SrDNAの配列情報で分子識別できないことが初めて明かとなった。, When the sequences of nearly full-length of 16S rDNA of the 15 isolates of two representative taxons of Campylobacter lari consisting of 12 urease-positive thermophilic Campylobacter (UPTC) and three urease-negative Campylobacter lari (UN C. lari) were analyzed, the 12 isolates of UPTC showed 99.0-100% sequence similarities, with major hypervariable region of the 15 heterogeneous sites being from nucleotide position 620 to 1110 to each other, the three of UN C. lari showed 99.8-99.9% similarities with the three heterogeneous sites. However, the 15 isolates revealed to share relatively lower sequence similarities of 98.2-98.9%. Alternatively, a total of 40 heterogeneous sites of the seven isolates of UN C. lari including the other four reference strains were located throughout the sequences of the 16S rDNA. Surprisingly, however, one of the UPTC isolates examined showed the identical 16S rDNA sequence to that of a reference UN C. lari strain. The phylogenetic tree constructed by the mean of UPGMA method demonstrated that the UPTC isolates including a reference strain examined formed a cluster having a genetic hypervariablity separate from the isolates of UN C. lari, except for the two isolates described above. Other thermophilic Campylobacters formed some clusters apparently separate from these two clusters. Thus, the present results clearly demonstrated that the species of C. lari is genetically diverse and the two representative taxons of UPTC and UN C. lari cannot be reliably discriminated by use of 16S rDNA sequence data.

日本の河川水から分離されたウレアーゼ陽性高温性カンピロバクター(UPTC)株2株,CF89-12,CF89-14,から,いずれも内部に2つの終止コドン(TAG)を持つflaA-様配列(flaAの偽遺伝子)を単離した。これら2つのflaA偽遺伝子は更にC. jejuniで報告されているflaA(約1,700bp)よりも短く,その全長はいずれも1461bpであった。なお,これらUPTC2株からはいずれもプラスミドは分離されておらず,それ故にこれら2つの偽遺伝子はゲノムDNA上にコードされていることが示唆された。, A primer pair of A1 and A2, which ought to generate a product of approx. 1700 bp of the flaA gene for Campylobacter jejuni, was used to amplify products of approx. 1450 bp for two Japanese isolates of UPTC, CF89-12 and CF89-14. After molecular cloning and sequencing, the nucleotide sequences of the amplicons from the two isolates were found to be 1461 bp in length and to have nucleotide sequence differences in relation to each other at four nucleotide positions, respectively. Nucleotide and amino acid sequence alignment and homology analysis demonstrated that the polymerase chain reaction (PCR) amplicons from the two Japanese isolates have approx. 83% nucleotide and 80% amino acid sequence homology to the possible open reading frame of the flaA gene of UPTC NCTC 12892. Surprisingly, however, both PCR amplicons from the Japanese UPTC have two internal termination codons (TAG) at nucleotide positions from 775 to 777 and 817 to 819, respectively.

アルツハイマー病の主要な病理所見の一つである神経原線維変化の出現頻度は,痴呆の重症度と強く相関している。Glycogen synthase kinase-3β(以下GSK-3β)によるタウのリン酸化は神経原線維変化において観察されるらせん状の繊維状構造物(PHF)形成の原因と考えられている。GSK-3βは特に脳内で多く発現されており,加齢あるいはアルツハイマー病発症に伴って発現量が増加することが報告されている。また,統合失調症患者の前頭野におけるGSK-3βmRNAのレベルが健常者と比べて約40%減少しているとする報告もある。このように脳の高次機能に重要な役割を果たしていると考えられるGSK-3βの活性調節については,リン酸化を介した翻訳後修飾を中心に詳細に研究されている。一方,発現調節に関する研究は少ない。そこでアルツハイマー病や統合失調症とGSK-3βの発現調節との関係を明らかにすることを視野に入れ,GSK-3β遺伝子のプロモーター活性の分子メカニズムに関する研究を行った。転写開始点より上流を欠失させた5'欠失変異体,5'UTR欠失変異体及び5'UTR置換変異体をルシフェラーゼ遺伝子発現ベクター(pGL3)に挿入したプラスミドを用いたレポータージーンアッセイを行った。その結果,GSK-3β遺伝子のbasal levelの基本転写に関わるシスエレメントが転写開始点より上流にあり,5'UTRに細胞特異的な転写活性に関わるシスエレメントが存在することが示唆された。以上の結果より,本研究において,GSK-3β遺伝子の発現調節機序を解明する上で重要な知見が得られたと考えている。, Glycogen synthase kinase3β (GSK3β) phosphorylates tau, one of neuronal microtubule associated proteins. In Alzheimer's disease, highly phosphorylated tau that deposition neurons may contribute to neurodegeneration. In some case of schizophrenia patients, mRNA level of GSK3β in brain is lower than control brain (Kozlovskyetal., 2003). These suggest that GSK3β may have an important role in developing these diseases and the regulation of GSK3β gene expression may be associated with them. To understand how the expression of GSK3β gene is regulated in neuron, we used the reporter gene assay system (pGL3vector). DNA fragment containing human GSK3β promoter region was sub cloned into cloning site at upstream of luciferase gene. A set of 5'-deletion mutant of the promoter was generated by PCR based mutagenesis. Each construct was introduced transiently into neuronal cells (SHSY) and nonneuronal cells (COS7 and HEK293). The promoter activity of a deletion mutant (-1540～-436) harboring the SP1 sites, the core promoter region (Lau et al. 1999) and 5'-UTR was the highest among mutants used here. Although Luciferase activity was higher in neural cells than in non-neuronal cells, the mutant (-1540～-436) showed the highest activity in any type of cells. These results indicate existence of the neuron specific trans-acting factor for regulating GSK3β promoter activity. The length of 5'-UTR in human GSK3β gene is unusually long (952bp). To study how the long 5'-UTR contributes to GSK3β expression, we generated deletion mutants lacked a portion of 5'-UTR. Activities of the deletion mutants (+234～+952, +556～+952) were higher than that of wild type. In addition, effect of the region deleted (+234～+952) was studied by comparing 5'-UTR sequences of some other animals. The promoters harboring the 5'-UTR of bovine or swine were more active than of human or rat. Although 5'-UTR may be mainly involved in translation, it is possible that this region regulates GSK3β gene transcription.

本研究においてデザインされた,recA遺伝子のほぼ全長に渡る領域を増幅するためのPCRプライマーを用いて得られたPCR産物の配列情報は,UPTC及び他の高温性カンピロバクターのDNAレベルでの分子識別に大変有用であることが強く示唆された。更に,本研究の主目的であるUPTCのゲノムDNA上の多遺伝子配列情報に基づく分子識別にrecA遺伝子を対象とすることは極めて有用であることが初めて明らかとなった。, In the present study, for the first time, nucleotide sequencing after TA cloning of almost the full-length (about 1,050 bp in length) of the recA gene of two isolates of UPTC, NCTC12894 and CF89-12 and a strain of C. lari JCM2530T amplified using a degenerate primer pair was carried out. Nucleotide sequence comparison analysis of the possible open reading frame (ORF) demonstrated that the two isolates of UPTC showed about 94% sequence homology of the recA gene to each other and that they also showed 90-92% and about 84% homology of the recA gene to C. lari and C. jejuni, respectively. The two isolates of UPTC showed 50-60% homology of the recA gene to the other family Enterobacteriaceae. In conclusion, this study presents novel sequence data on recA for C. lari and UPTC, which may aid in the phylogenetic positioning of the UPTC group within the genus Campylobacter and may aid in the discriminating of isolates of the UPTC group.

神経原線維変化の形成に重要な関わりを持つタウタンパク質の2つの型のアイソフォーム(3リピートタウと4リピートタウ)の識別を可能にする抗体T3RおよびT4Rを得た。今回作製した抗体は,他のグループが作成したものと比較して特異性が高いことに加え,タウの微小菅結合領域の高次構造の変化を検出するのに利用可能であることが明らかとなった。微小管結合領域はタウの機能に重要な部分であり,抗体T3RおよびT4Rは痴呆症発症におけるタウの関わりを詳細に解析する上で利用価値の高い抗体だと考えられる。, Neurofibrillary tangle is one of major pathological hallmarks of Alzheimer's disease. In this pathological change, abnormal intracellular aggregates composed of hyperphosphorylated tau proteins are observed. Recently, familial missense mutations in tau gene were identified by reverse genetic analysis of FTDP-17 families. This suggests that tau is possibly involved in developing "tauopathy". Although tau is known as an axonal microtubule associated protein that stabilized the structure solubility of microtubules and promotes polymerization of tubulines into the microtubules, role (s) of tau in pathological events remains unclear. To elucidate role (s) of tau in the neurodegeneration, we made antibodies (T3R and T4R) recognizing specifically tau isoforms, three and four repeat tau, respectively. By enzyme linked immunosolvent assay and western blot analysis, it was revealed that the affinities of these antibodies were depend on coformations of their epitopes. In addition, a C-terminal portion of tau contributed to forming tertiary structure of these epitopes. T3R and T4R might be the biochemical markers for monitoring the change in local conformation of the repeat domain. T3R and T4R generated in this study may be powerful probe for the biochemical and pathological diagnostics of dementia.

遺伝子の働きを多様にする重要なステップであるRNAスプライシングの様子を蛍光・発光タンパクを利用して可視化・定量化する技術を開発し改良を加えた。微小管結合タンパク質タウのエキソン(2, 3, 10)を持つスプライシングベクターを作製し，各エキソンの選択的スプライシングについて詳細な解析を進めている。, Alternative splicing that contributes to proteomic diversity is a mechanism by which multiple transcripts are generated from a transcript from single gene. This post-transcriptional processing may occur in over 70 % of human genes. Abnormal splicing is associated with some disease, such as Alzheimer's disease (AD) and FTDP-17. Microtubule associated protein tau (MAPT) is produced as six alternative splicing isoforms in brain. Abnormality in this splicing has been thought as a causative event through which AD and FTDP-17 might be developed and/or progressed. Hence a change in alternative splicing must be worth monitoring for diagnostics and therapy of the diseases. In this study, we tried to develop a method for visualizing alternative splicing of tau pre-mRNA. Using the splicing cassette vector. pGL3intl and pGL3intl-MCS. we have developed, we constructed plasmids containing tau exon (exon 2, 3 or 10) and observed the alternative splicing of each exon in cultured cells such as COS7. SH-SY5Y or HEK293.

ショウジョウバエ卵巣は,多細胞生物の器官構築の過程を研究するための良いモデルシステムとなっている。我々の研究室で単離した卵巣形態に異常の生じる突然変異体のうち,主にLar変異体について間接蛍光抗体法で解析した。共焦点レーザー顕微鏡の観察から,Lar変異体では,卵巣小菅が個々に分解せず,Larはショウジョウバエの卵巣において接着班の分解を介してASCsの移動に関与し,蛹の卵巣を卵巣小管に分離する過程を制御している可能性が示唆された。, The shape of an organ is controlled by the morphogenetic properties of the participating cells. The Drosophila ovary represents an excellent system to study such a reorganization process. The ovary possesses 15-20 ovarioles, repeated units that serve as an assembly line for oogenesis. In early pupal stages, the ovary is composed of different cell populations from the anterior to the posterior side: anterior somatic cells (ASCs) and terminal filament cells, somatic and germline cell precursors that are intermingled in the central part of the ovary, and basal cells. During later stages, ASCs migrate posteriorly and separate the central populations of cells into ovarioles. Here, we report that Lar gene is crucial for normal morphogenesis of Drosophila ovary. Lar mutant ovary fail to separate the central population of cells into ovarioles. In wild-type, LAR is expressed in the ASCs, intermingled cells and basal cells. These three cell types share a common feature of cell motility during ovariole formation. Lar encodes a membrane protein containing immunoglobulin-like domains, fibronectin type III repeats and PTPase domains. Previous studies have demonstrated that LAR associates with focal adhesions (FAs) in human cells and plays a role in disassembly of FAs (Serra-Pages, 1995). Considering that the cyclical regulation of FAs formation and disassembly is critical in the control of cell movement, we propose that Lar may be involved in ASCs movement by disassembling FAs, resulting in proper separation of ovarioles in pupal ovary.

神経原線維変化の形成に重要な関わりを持つタウタンパク質の2つの型のアイソフォーム(3リピートタウと4リピートタウ)のmRNAレベルを識別定量する条件を検討した。本法は,神経細胞変性過程におけるタウ遺伝子発現およびアイソフォーム発現パターンの動態を追跡する上で有効な手段であると考えられる。, Neurofibrillary tangle composed of hyperphosphorylated tau aggregation in neuron is one of major pathological hallmarks of Alzheimer's disease or other neurodegenerative disease. Recently, familial mutations in tau gene were identified by reverse genetic analysis of FTDP-17 families. Some of these mutation lead amino acid substitution and the other affect alternative splicing of the exon10. This suggests that tau is possibly involved in developing "tauopathy". Although tau is known as an axonal microtubule associated protein that stabilized the structure solubility of microtubules and promotes polymerization of tubulines into the microtubules, role(s) of tau in pathological events remains unclear. To elucidate how tau protein and its isoforms are involved in the neurodegeneration, we developed quantitative method for tau isoforms, three and four repeat tau, respectively. Real Time PCR using Taq Man probe (ABI) three and four repeat tau mRNA could be quantified with high sensitivity (> 10pg of total RNA). In addition, a little cross reactivity between three and four repeat tau mRNA was observed. These results suggested that tau mRNA in small number of neuronal cells (～10 cells) of mouse or human brain could be quantified by our method. Combining with isoform specific antibodies T3R and T4R previously generated, the measurement may be useful for studying the molecular mechanism in developing dementia.