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The GSK-3β gene, GSK3B, codes for an enzyme that is a target for the action of mood stabilizers, lithium and possibly valproic acid. In this study, the relationship between haplotypes consisting of single nucleotide polymorphisms (SNPs) of GSK3B −50T/C and −1727A/T and the effect of lithium was studied among Japanese bipolar disorder lithium nonresponders and responders. The distributions of the GSK3B haplotypes (−50T/C and −1727A/T) showed a trend for significant difference between the lithium nonresponders and responders (global P=0.07074). Haplotype 1 (T-A) was associated with a higher lithium response (haplotype-specific P=0.03477), whereas haplotype 2 (C-A) was associated with a lower lithium response (haplotype-specific P=0.03443). The pairwise D′ and r2 values between the 2 SNPs in this study were 1.0 and 0.097, respectively. The 2 SNPs showed weak linkage disequilibrium with each other.

In 181 healthy Japanese volunteers we examined the relationship between personality, sensitivity to pain and a single nucleotide polymorphism (rs3813034) in the 3′ untranslated region (3′ UTR) of the serotonin transporter (5-HTT) gene (SLC6A4). Pain sensitivity was assessed by using cold and pressure thresholds. Personality was assessed by the Temperament and Character Inventory (TCI). Males without the T allele (G/G) showed a significantly higher spiritual acceptance (ST3) score than those who had the T allele (T/T and T/G). Females with the T allele (T/T and T/G) showed significantly higher transpersonal identification (ST2) and self-transcendence (ST) scores than those without the T allele (G/G). As for pain sensitivity and its relationship with TCI, we found a low negative correlation between cold water stimulation, disorderliness (NS4) and novelty seeking (NS) in males, whereas in females we found a low positive correlation between cold water stimulation, self-acceptance (SD4) and pure-hearted principles (C5), as well as pressure stimulation and SD4. It is possible that the 5-HTT 3′ UTR gene polymorphism affects the character dimensions of Cloniger’s theory, and that there might be a low correlation between pain and a part of the personality.

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.

Aims: To clone, sequence and characterize the genetic organization of urease genes within urease-positive thermophilic Campylobacter (UPTC). Methods and Results: An approx. 5·1-kbp region encoding a urease gene operon was identified, when recombinant plasmid DNAs from a genomic DNA library of a Japanese isolate (CF89-12) of UPTC were analysed. Conclusions: Six closely spaced and putative open reading frames (ORFs) for ureA, ureB, ureE, ureF, ureG and ureH were detected. ATG codons initiated each ORF of the UPTC urease operon except for ureB and ureH, which commenced with the most probable TTG codon. Overlaps were detected between ureA and ureB and also between ureB and ureE. Probable ribosome-binding sites and a putative ρ-independent transcriptional termination region were identified. Two putative promoter structures, consisting of consensus sequences at the −35 like and −10 regions were also identified. Significance and Impact of the Study: Construction of a neighbour-joining tree based on the nucleotide sequence data of urease genes indicated that UPTC formed a cluster with some Helicobacter organisms separate from the other urease-producing bacteria, suggesting a commonly shared ancestry between UPTC and Helicobacter urease genes.

The 3,339 base pair (bp) sequences encoding a putative open reading frame (ORF), non-coding promoter and leader regions (approximately 320 bp), full-length 16S ribosomal RNA (rRNA) gene (approximate 1,540 bp) and part of the 16S-23S rDNA internal spacer region (ISR) were determined from genome DNA libraries of the Taylorella asinigenitalis (UK-1) isolate. The non-coding promoter and leader regions included antiterminators (boxB, boxA and boxC) immediately upstream of the 16S rRNA gene sequence. An approximately 680 bp region upstream of the non-coding promoter region appears to contain a putative ORF with high sequence similarity to GTP cyclohydrolase I. In addition, a typical order of intercistronic tRNA genes with the 48 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in a part of the 16S-23S rDNA ISR. The antiterminators of boxB and boxA were also identified in the ISR.A phylogenetic analysis based on the 16S rRNA gene sequence information clearly demonstrated that the five T. asinigenitalis isolates formed a cluster together with the three T. equigenitalis strains, more similar to Pelistega europaea than the other beta-Proteobacteria from the 13 species of 11 genera.

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.

An approximately 4.2 kbp region encoding 23S and 5S rRNA genes was identified when recombinant plasmid DNAs from two genomic DNA libraries and an inverse PCR product of Taylorella asinigenitalis UK-1 isolate were analyzed. Full-length genes of 23S rRNA (3,225 bp) and 5S rRNA (117 bp) of T. asinigenitalis are described. The present sequence analysis identified a non-coding hypothetically intrinsic transcription terminator region downstream of the 5S rRNA gene. The sequence, however, downstream of the 5S rRNA gene did not show any distal tRNA genes. Surprisingly, an intervening sequence (IVS) of 270 bp in length, including two specific tandem repeat units of 80 bp and one partial unit of 48 bp with unknown functions was identified in the first quarter of the 23S rRNA gene sequence. A second IVS of 70 bp in length was also identified in the central region of the 23S rRNA gene. In addition, by using PCR and sequencing procedures, two T. asinigenitalis isolates, UK-1 and UK-2, carried multiple IVSs in the first quarter and central regions. Moreover, the 23S rRNA fragmentation occurred in the UK-1 isolate. A phylogenetic analysis was first carried out based on the 23S rRNA sequence data from T. asinigenitalis UK-1 and 13 other beta-Proteobacteria. This is the first report of IVSs in the 23S rRNA gene from the beta-Proteobacteria.

Cloning, sequencing and molecular characterisation of a cryptic plasmid, pUPTC237, from a urease-positive thermophilic Campylobacter (UPTC) isolate obtained from the natural environment in Northern Ireland is reported in this study. Based on the determined DNA sequence, the pUPTC237 DNA was identified as a circular molecule of 3828 bp with a G+C content of 29.5%. As with other plasmid DNAs from Gram-negative bacteria, pUPTC237 contained an A+T-rich region (A+T content: 95%), followed by multiple direct tandem repeat units of 22 bp, characteristic of a replication origin and iteron sequence. A possible open reading frame (ORF)-1 was located upstream of the A+T-rich region and the iteron sequence that encoded a 460 amino acid protein similar to the mobilisation (mob) protein and two putative promoter structure sequences at the -35 and -10 regions and a possible ribosome binding site occurred upstream of the start codon for the ORF-1. Moreover, three possible ORFs (a short ORF-2 encoding 26 amino acids, similar to repA; an ORF-3 encoding 341 amino acids, similar to repB; and an ORF-4 encoding 96 amino acids with unknown function) were also identified. There are also two putative promoter structures for these three ORFs at the -35 and -10 regions upstream of the possible ORF-2. A possible transcription termination region was identified downstream of ORF-4. Northern blot hybridisation analysis suggested that these four ORFs constitute an operon and generate a messenger RNA (mRNA) transcript.

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.

Several studies have suggested that the norepinephrine transporter (NET) may play an important role in the pathogenesis of alcohol dependence. Therefore, in this study, we investigated whether the NET gene polymorphism is a susceptibility factor for alcohol dependence in 64 alcoholics and 73 healthy controls. In addition, we examined whether the combination of the NET and serotonin transporter genotypes are associated with alcohol dependence. The NET (1287G/A, -182T/C, and -3081A/T) and serotonin transporter (5-HTT3'UTR) genotypes were determined by the polymerase chain reaction (PCR)--restriction fragment length polymorphism (RFLP) method. No significant differences in genotype and allele frequencies of the NET and serotonin transporter gene polymorphisms were found between alcoholics and controls. The haplotype frequencies of the NET gene polymorphisms were not also significantly different between them. Furthermore, the combination of the NET and serotonin transporter genotypes had not significant effects on alcohol dependence. The present study suggests that the polymorphisms of 1287G/A, -182T/C and -3081A/T in NET gene are not.risk factors in alcohol dependence.

Degenerate PCR primers in silico based on the two urease structural genes, ureA and ureB, were designed for urease-positive thermophilic Campylobacter (UPTC). Resultant PCR amplification employing these primers generated an amplicon of approximately 2kb, which was cloned and sequenced in UPTC (n=12) isolated from various parts of Europe and Japan. Overall, sequence similarities were shown to be 96.7 to 99.9%. Following sequence alignment analysis, the approximate 1.96kb regions were deduced to consist of parts of ureA (about 570bps) and ureB (about 1390bps) with 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/Omega10bases) than in the ureB region (1/Omega15bases). A resulting dendrogram was constructed, which was based on the nucleotide sequence data of 12 UPTC isolates and demonstrated that the UPTC were genetically variable. They formed a major cluster with Helicobacter, separate from the other urease-producing bacteria examined, suggesting a shared ancestry between UPTC and Helicobacter.

Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.

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.

A restriction and genetic map of urease-positive thermophilic campylobacter (UPTC) CF89-12 genome DNA is constructed using a pulsed-field gel electrophoresis procedure after digestion with SalI and SmaI and Southern blot hybridisation. Each of the six gene fragments (flaA, glyA, lysS, recA, sodB and ureAB) selected are mapped in only a fragment on the restriction map. Three DNA fragments for rrn operon probes are mapped in multiple regions on the map. When two SmaI-digested neighbouring small fragments hybridised with rrn probes are cloned and sequenced, a total sequence length of 7487 bp is determined. In the sequence, part of the pnp gene (734 bp) bearing a p-independent transcriptional termination region, a cluster of five tRNA genes including the putative promoter region, a hypothetical Cj0171-like 507-bp sequence containing an internal termination codon, and a part of the rrn operon including the putative promoter region (4700 bp) are identified. The 507 bp sequence carried both putative transcriptional promoter sequences, including a ribosome binding site upstream of the ATG start codon and a characteristic G9 structure, and a possible p-independent transcriptional termination region. A hypothetical Cj0170-like 204-bp sequence containing an internal termination codon also occurred, overlapping partly with the Cj0171-like sequence. Based on nucleotide sequence alignment analysis between the UPTC rrn operon examined here and the previously reported one, two different 16S-23S ribosomal DNA (rDNA) internal spacer regions are shown to exist.

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.

In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C. coli and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894). Flagellin components, each consisting of a single peptide, with a heterogeneous molecular mass of approximately 52-63 kDa were demonstrated in the other four UPTC isolates (NCTC12892, NCTC12895, NCTC12896 and NI15F [from Northern Ireland]) and the two Japanese isolates of C. jejuni (JCM2013 and C. coli 27). The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C. jejuni and C. coli. Flagella were not detected by electron microscopy in the four flagellin-negative UPTC isolates but they were detected in the four flagellin-positive UPTC isolates and the two isolates of C. jejuni and C. coli. Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates.

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.

Macro-restriction analysis by means of double digestion using DraI and VspI demonstrated that they cleaved the genomic DNAs from Micrococcus luteus JCM1464T, JCM3347, and JCM3348 into four to five fragments in a distinguishable manner by pulsed-field gel electrophoresis (PFGE). Separate digestion with DraI and VspI cleaved the genomic DNA from M. luteus ATCC9341 into a relatively limited number of restriction fragments (six pieces). SspI and XbaI cleaved the genomic DNA from each of the four strains into restriction fragments in a distinctly different and distinguishable manner. Thus, PFGE profiles after digestion with these four restriction enzymes that recognize six specific base sequences demonstrated the heterogeneity at the whole genomic DNA level among the four strains of M. luteus. The size of the genomic DNA of M. luteus ATCC9341 was estimated to be approximately 2.3 Mb by the summing the lengths of the restriction fragments generated by each three restriction enzymes (DraI, SspI, and VspI) and averaging the results.

Formation of neurofibrillary tangles (NFTs) is the most common feature in several neurodegenerative diseases, including Alzheimer's disease (AD). Here we report the formation of filamentous tau aggregations having a beta-sheet structure in transgenic mice expressing mutant human tau. These mice contain a tau gene with a mutation of the frontotemporal dementia parkinsonism (FTDP-17) type, in which valine is substituted with methionine residue 337. The aggregation of tau in these transgenic mice satisfies all histological criteria used to identify NFTs common to human neurodegenerative diseases. These mice, therefore, provide a preclinical model for the testing of therapeutic drugs for the treatment of neurodegenerative disorders that exhibit NFTs.

Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T. equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T. equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T. equigenitalis.

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.

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.

5-Azacytidine (5AzC), a cytidine analogue, is thought to induce apoptosis in fetal neuronal cells and PC12 cells through DNA hypomethylation. However, apoptosis can be inhibited by adding protein synthesis inhibitors, indicatingde novoprotein synthesis may be partially responsible for apoptosis. Therefore, genes expressed just before apoptosis from 5AzC-treated PC12 cells were cloned. cDNA libraries were prepared from both 5AzC-treated and untreated PC12 cells and these libraries were subtracted. One clone overexpressed in 5AzC-treated PC12 cells was obtained, and was identified as the nearly full length (9 nt at 5′ end and 1 nt at 3′ end missing) rat ribosomal protein L4 (rpL4) gene. Time course study of Northern blot analysis in 5AzC-treated PC12 cells revealed that the peak of rat rpL4 gene expression preceded DNA fragmentation. COS-7 cells transfected with different amounts of cDNA from the subtracted clone expressed rat rpL4 dose-dependently. DNA fragmentation in the transfected COS-7 cells occurred proportional to the amount of the cDNA used for transfection. The present study indicates that rat rpL4 gene expression selectively increases in PC12 cells prior to 5AzC-induced apoptosis and that COS-7 cells transfected with and expressing the rat rpL4 gene also undergo apoptosis.

Families bearing mutations in the presenilin-1 (PSI) gene develop Alzheimer's disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co-immunoprecipitation with PS1 in transfected COS-7 cells indicates that PSI directly interacts with endogenous beta-catenin, and the interaction requires residues 322450 of PSI and 445-676 of beta-catenin. Both proteins are co-localized in the endoplasmic reticulum. Over-expression of PS1 reduces the level of cytoplasmic beta-catenin, and inhibits beta-catenin-T cell factor-regulated transcription. These results indicate that PSI plays a role as inhibitor of the beta-catenin signal, which may be connected with the AD dysfunction.

Families bearing mutations in the presenilin 1 (PS1) gene develop Alzheimer’s disease. Previous studies have shown that the Alzheimer-associated mutations in PS1 increase production of amyloid β protein (Aβ 1–42 ). We now show that PS1 also regulates phosphorylation of the microtubule-associated protein tau. PS1 directly binds tau and a tau kinase, glycogen synthase kinase 3β (GSK-3β). Deletion studies show that both tau and GSK-3β bind to the same region of PS1, residues 250–298, whereas the binding domain on tau is the microtubule-binding repeat region. The ability of PS1 to bring tau and GSK-3β into close proximity suggests that PS1 may regulate the interaction of tau with GSK-3β. Mutations in PS1 that cause Alzheimer’s disease increase the ability of PS1 to bind GSK-3β and, correspondingly, increase its tau-directed kinase activity. We propose that the increased association of GSK-3β with mutant PS1 leads to increased phosphorylation of tau.

(2006). Genetic heterogeneity of the cytolethal distending toxin B (cdtB) gene locus among isolates of Campylobacter lari. British Journal of Biomedical Science: Vol. 63, No. 4, pp. 179-181.

Presenilin 1 (PS 1) shows missense mutations in most early-onset familial Alzheimer's disease (FAD). Transfection of cDNA for wild type PS 1 into rat pheochromocytoma PC12 cells generated a 47 kDa full-size PS 1 protein, which was processed into a 28 kDa N-terminal fragment and a 19 kDa C-terminal fragment. We prepared selected Alzheimer-associated mutations (Gly384Ala, Leu392Val, and Cys410Tyr) of PS 1, which localized after a possible cleavage site. By transient expression in PC12 cells and rat glioma cell line, C6, we examined their influence on the processing of PS 1. Cys410Tyr inhibited proteolytic processing of PS 1, while Gly384Ala and Leu392Val did not. Thus, the Alzheimer related mutations can be divided into two groups in terms of their effect on the proteolytic cleavage of PS 1.

Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability. The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinical applications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation in mice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs. Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM and above inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuated the proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed the accumulation of lipid droplets and calcium depositions. These modifications of ADSCs by VPA were associated with a particular gene expression profile, viz., an increase in neuronal markers, that is, NSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but no changes in osteogenic markers, as estimated by reverse transcription-PCR analysis. These results suggested that VPA is a specific inducer of neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.

Integrin alpha 6 beta 1 is a major adhesion receptor for the basement membrane, specifically binding to laminin-1. To identify the peptide sequences recognized by alpha 6 beta 1, we screened a 15-mer phage display library by panning with alpha 6 beta 1 purified from human placenta. DNA sequencing of 73 randomly picked phage revealed that three clones were dominantly enriched after repeated panning with alpha 6 beta 1. None of the peptide sequences displayed on these phage showed significant homology to laminin-1. A synthetic peptide modeled after the sequence displayed by one of these phage, designated P3, was found to strongly inhibit the binding of laminin-1 to alpha 6 beta 1. This inhibitory effect of the P3 peptide seems to be specific for alpha 6 beta 1, since it did not affect the binding of fibronectin to integrin alpha 5 beta 1. A synthetic peptide with a scrambled P3 amino acid sequence barely inhibited the binding of laminin-1 to alpha 6 beta 1. When coated on a substratum after conjugation with bovine serum albumin, the P3 peptide was capable of promoting cell spreading in an alpha 6 beta 1-dependent manner, although the peptide with the scrambled sequence showed activity similar to that of a control peptide. These results taken together indicate that the P3 peptide is a novel ligand for integrin alpha 6 beta 1 with potent cell spreading activity.

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.

Author's addresses: 1. Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast, BT9 7AD, Northern Ireland. 2. Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, 1-17-71 Fuchinobe,Sagamihara,229,Japan.. Tel: +44 (28) 9026 3354 Fax: +44 (28) 2589 2887 E-mail: jemoore@niphl.dnet.co.uk to be a cause for concern in veterinary microbiology, especially due to its economic implications. Originally the organism was classified in the family Pasteurellaceae, as a species incertae sedis belonging to the genus Haemophilus. The taxonomic position of T. equigenitalis was clarified in 1983 by Sugimoto et al.3 employing DNA base composition and DNA-DNA hybridization data, which concluded that the organism which was originally named Haemophilus equigenitalis, should be transferred to a new genus Taylorella. More recently, there has activity with regard to the sequencing of the 16S rRNA for taxonomic purposes, as well as the description of a new species within this genus, T. asinigenitalis, originating from donkey jacks.4 We note with interest several anomalies between the originally published 16S rRNA gene of T. equigenitalis5 and the more recent submissions to GenBank, by several groups including ourselves, as shown in Table 1. Bacteria may contain several copies of their 16S rRNA gene and these may not all be identical, giving rise to cistonic SHERIDAN ANIMAL MEDICAL CENTER

As a help of the relationship between bipolar disorder and alcoholism, we studied the relationship between GSK-3 beta -50T/C polymorphism, which is reported to the relationship for bipolar disorder, and Japanese alcoholics. And we investigated the relationship between GSK-3 beta -50T/C polymorphism and DBI +529A/T polymorphisms, which is reported to one of the risk factor for alcoholism. We analyzed the GSK-3 beta genotype using a polymerase chain reaction (PCR), and DBI genotype using PCR with confronting two-pair novel primers (PCR-CTPP) in 75 health controls and 64 alcoholics. In this study, there was no significant difference in the frequency of GSK-3 beta -50T/C polymorphism between alcoholics and controls (p = 0.883), and there was no significant difference in the frequency of DBI +529A/T polymorphism (p = 0.131). Also, there was no relationship between GSK-3 beta -50T/C polymorphism and DBI +529A/T allele in alcoholism (p = 0.907). We suggested that bipolar disorder may not be one of the pathogenesis of alcoholism, and that there was no relationship between GSK-3 beta -50T/C polymorphism and DBI +529A/T polymorphism.

We investigated the relationship between the genetic polymorphism (s/l) of 5-HT2A receptors (A-1438G) and 5-HTT (s/l), and the drug effects among 31 patients who are administering olanzapine, as a help of the tailor made medical care in a time. As for the genetic polymorphism of 5-HT2AR, G/G group showed a significantly improvement tendency in the PANSS positive syndrome score in comparison with the group which did not have a G gene (AA and AG). It may be possible that this finding help to predict the drugs effect of olanzapine on the patients with excite state, who are used many antipsychotics together out of necessity.

Two types of tau isoform, three- and four-repeat tau, are found in neurofibrillary tangles—a pathological hallmark of tauopathies. Which isoform is deposited in the affected tissues depends on the tauopathy. To study how and which tau isoforms contribute to neuronal degeneration, we have developed and characterized two novel conformation-sensitive antibodies, T3R and T4R. Two closely related synthetic peptides, PGGGKVQIVYK and PGGGSVQIVYK, respectively, were designed as antigens. The isoform-specific residues, 305K in three-repeat tau or 305S in four-repeat tau, and the PHF6 motif (VQIVYK) were identified as critical sequences. Despite the high similarity of the antigens, there was no cross-reactivity between T3R and T4R. Furthermore, T3R and T4R showed reduced binding to the thioflavin-positive β-structural form of their target. These features may enable these antibodies to act as novel indicators that allow us to observe and evaluate conformational changes in each distinct isoform of tau.

Two flagellin gene (flaA and flaB) sequences and the adjacent gene loci of urease-positive thermophilic Campylobacter (UPTC)(1) were examined. The flagellin gene sequences (1.7 kb) and adjacent gene loci of the two UPTC isolates (89049 and A3), obtained from animal hosts, were very similar to those of C. lari RM2100, C. jejuni, and C. coli. However, the structure and loci of the two flagellin genes (1.46-1.47 kb) and the adjacent gene loci of a UPTC strain obtained from the natural environment (NCTC12892) clearly differed from those of C. lari RM2100 and UPTC obtained from animal hosts. The two flagellin genes of UPTC 89049 and A3 were located between topA /CLA0518 and CLA0521, whereas those of NCTC12892 were located between topA and CLA0521. The sequences involved in regulation of flagellin expression, like sigma(28), sigma(54) and transcription termination signals, were conserved in all isolates. The characteristic direct repeat sequences containing a complete repeat unit of 5'-TCTTTAAAACAAC-3' were located in the intergenic regions between flaA and flaB in UPTC89049 and A3, but not in NCTC12892. The deduced amino acid sequence alignment revealed that the two flagellin genes in NCTC12892 had a deletion of the variable region of flagellin, which was reported previously to be modified by pseudaminic acid in C. jejuni and C. coli.Consequently, these results may possibly suggest that the length of the flagellin is related to pathogenicity and colonization of Campylobacter.

The PCR amplicons (about 1450 bp in length) of flaA gene fragments of 11 isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from the natural environment not including wild birds in Northern Ireland were demonstrated to be shorter than those of C. jejuni 81116 and six isolates of C. jejuni and C. coli (about 1700 bp) isolated in Northern Ireland and Japan. When the nucleotide lengths of the possible open reading frame (ORF) of the flaA genes were determined, those from the 11 UPTC isolates were estimated to be 1464-1503 bp, and those from the six C. jejuni and C. coli isolates and C. jejuni 81116 strain to be 1716-1728 bp. Nucleotide sequence and deduced amino acid sequence alignments of the possible ORFs demonstrated that the ORFs from the 11 UPTC isolates lack about 80 amino acid residues, mainly from the approximate residue numbers 390-470 of the large variable region in the flaA protein of the seven isolates of C. jejuni and C. coli, and do not have any internal termination codons. High amino acid sequence similarity of both amino- and carboxy-termini of the ORFs of the flaA gene was demonstrated between the 11 isolates of UPTC and the 7 isolates of C. jejuni and C. coli. The 11 UPTC isolates examined were strongly suggested to possess a shorter flaA gene without any internal termination codons.

Background At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. Results Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. Conclusion High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184(T), Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184(T) and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184(T) and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.

Nucleotide sequencing after TA cloning of the amplicon of the almost-full length recA gene from three strains of UPTC (A1, A2, and A3) isolated from seagulls in Northern Ireland, the phenotypical and genotypical characteristics of which have been demonstrated to be indistinguishable, clarified nucleotide differences at three nucleotide positions among the three strains. In conclusion, the nucleotide sequences of the recA gene were found to discriminate among the three strains of UPTC, A1, A2, and A3, which are indistinguishable phenotypically and genotypically. Thus, the present study strongly suggests that nucleotide sequence data of the amplicon of a suitable gene or region could aid in discriminating among isolates of the UPTC group, which are indistinguishable phenotypically and genotypically.

Alzheimer's disease (AD) brains contain neurofibrillary tangles (NFTs) composed of abnormally hyperphosphorylated tau protein. Regional reductions in cerebral glucose metabolism correlating to NFT densities have been reported in AD brains. Assuming that reduced glucose metabolism might cause abnormal tau hyperphosphorylation, we inducedin vivoalterations of glucose metabolism in mice by starvation or intraperitoneal injections of either insulin or deoxyglucose. We found that the treatments led to abnormal tau hyperphosphorylation with patterns resembling those in early AD brains and also resulted in hypothermia. Surprisingly, tau hyperphosphorylation could be traced down to a differential effect of low temperatures on kinase and phosphatase activities. These data indicate that abnormal tau hyperphosphorylation is associated with altered glucose metabolism through hypothermia. Our results imply that serine-threonine protein phosphatase 2A plays a major role in regulating tau phosphorylation in the adult brain and providein vivoevidence for its crucial role in abnormal tau hyperphosphorylation in AD.

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11laminin-5laminin-1laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.

Aims: To clone and sequence the 16S–23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. Methods and Results: The primer pair for 16S–23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. Conclusions: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. Significance and Impact of the Study: A CCTCCT sequence was first detected at the 3′-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.

The mutation L271V in exon 8 of the presenilin-1 (PS-1) gene was detected in an Alzheimer's disease pedigree. Neuropathological examination of affected individuals identified variant, large, non-cored plaques without neuritic dystrophy, reminiscent of cotton wool plaques. Biochemical analysis of L271V mutation showed that it increased secretion of the 42-amino acid amyloid-beta peptide, suggesting a pathogenic mutation. Analysis of PS-1 transcripts from the brains of two mutation carriers revealed a 17-50% increase in PS-1 transcripts with deletion of exon 8 (PS-1deltaexon8) compared with unrelated Alzheimer's disease brains. Exon trapping analysis confirmed that L271V mutation enhanced the deletion of exon 8. Western blots of brain lysates indicated that PS-1deltaexon8 was overexpressed in an affected individual. Biochemical analysis of PS-1deltaexon8 in COS and BD8 cells indicate the splice isoform is not intrinsically active but interacts with wild-type PS-1 to generate amyloid-beta. Western blots of cell lysates immunoprecipitated with anti-Tau or anti-GSK-3beta antibodies indicated that PS-1deltaexon8, unlike wild-type PS-1, does not interact directly with Tau or GSK-3beta, potential modifiers of neuritic dystrophy. We postulate that variant plaques observed in this family are due in part to the effects of PS-1deltaexon8 and that interaction between PS-1 and various protein complexes are necessary for neuritic plaque formation.

Aims: To demonstrate two flaA-like sequences containing two internal termination codons (TAG) in two Japanese strains of urease-positive thermophilic Campylobacter (UPTC). Methods and Results: 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 strains of UPTC, CF89-12 and CF89-14. After molecular cloning and sequencing, the nucleotide sequences of the amplicons from the two strains were found to be 1461 bp in length and to have nucleotide sequence differences in relation to each other at four nucleotide positions, respectively. Conclusions: Nucleotide and amino acid sequence alignment and homology analysis demonstrated that the polymerase chain reaction (PCR) amplicons from the two Japanese strains have approx. 83% nucleotide and 80% amino acid sequence homology to the possible open reading frame of the flaA gene of UPTC NCTC 12892. Significance and Impact of the Study: Surprisingly, 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.

Two strains of urease-positive thermophilic Campylobacter (UPTC), designated YC98-1 and YC98-2, were identified by biochemical characterization after isolation from the intestinal contents of crows around Yokohama City, Japan, in 1998. The biochemical characteristics of these strains were identical to those of strains described previously. Pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI, SalI, and SmaI of the genomic DNA from the two strains indicated that respective PFGE profiles were distinctly different and distinguishable from each other. This is the first report of the isolation of UPTC from crows (Corvus levaillantii).