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A newly built ship must conduct sea trial to understand its performance including speed, maneuverability and so on. The result of the speed trial is very important for the ship owner, because the ship speed is directly related to profit. In this case, current becomes an important factor because the ship speed is changed by the effect of the current. To remove this effect, International Towing Tank Committee and International Organization for Standardization heve presented procedures of removing this effect. However, these procedures require two or three round–trips against one ship speed. To solve this difficulty, we attempt to estimate herein the current from the ship motion based on successive data assimilation. The proposed method is verified by twin experiments. Consequently, the proposed method is found to estimate the current well.

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An ISOL-based radioactive nuclear beam (RNB) facility, Tokai Radioactive Ion Accelerator Complex (TRIAC), has been jointly developed by High Energy Accelerator Research Organization (KEK) and Japan Atomic Energy Agency (JAEA). The facility started to supply RNBs for experiments in 2005 and RNBs including fission fragments with energies up to 1.1MeV/A are available in the present. Several experimental studies were performed successfully using 8Li beams with various energies.

Exaggerated activation of the renin-angiotensin system via tissue angiotensin II (Ang II) type 1 receptor (AT1R) signaling exerts detrimental effects on cardiovascular, renal and endocrine systems to provoke hypertension and related target organ damage. On the other hand, accumulated research evidence of both basic and clinical studies shows that physiological AT1R signaling also plays an indispensable role for the normal organ development such as the kidney and the maintenance of cardiovascular and renal homeostasis. Such functional diversity of AT1R signaling prompts us to seek a new strategy of selective modulation of AT1R signaling in pathophysiology. In the course of an investigational search for a means to functionally and selectively modulate AT1R signaling for that purpose, a molecule directly interacting with the carboxyl-terminal cytoplasmic domain of AT1R was identified by employing yeast two-hybrid screening of a mouse kidney cDNA library and named AT1R-associated protein (ATRAP). The results of functional analysis showed that ATRAP promotes constitutive AT1R internalization in cultured cells and inhibits Ang II-mediated pathological response in mouse distal convoluted cells. The ATRAP is expressed in a variety of tissues including the kidney where ATRAP is abundantly distributed in epithelial cells along the renal tubules. The results employing genetic engineered mice with modified ATRAP expression showed that ATRAP plays a key role in the regulation of renal sodium handling and the modulation of blood pressure in response to pathological stimuli such as chronic Ang II infusion, and suggest ATRAP to be a target of interest.

AbstractSouthern hybridisation shows that urease-negative (UN) Campylobacter lariJCM2530Tcarries two putative major outer membrane protein (MOMP) genes. Sequences of approximately 2.1 kbp, encoding non-coding (NC) regions, with possible open reading frames (ORFs) for MOMP (porAlor porA2)of approximately 1.2 kbp, NC regions and partial and putative Cla_0435 or Cla_1109 ORFs were identified in all five UN C. lariisolates examined, following polymerase chain reaction (PCR) cloning and sequencing. Each putative MOMP structural gene carried start and stop codons and ribosome binding sites of 1236–1278 bp in length. The putative σ70transcriptional promoter and the hypothetical ρ-independent transcription terminator structures were also seen. Using Northern hybridisation, there was in vivomonocistronic MOMP gene transcription. In addition, in a Japanese urease-positive thermophilic Campylobacter(UPTC) CF89–12 strain, the porA1gene locus, including an extra gene (approximately 2000 bp in length) was identified. The extra gene may occur within the porA1gene locus in the eight UPTC isolates of the 23 C. lariisolates examined. Thus, a genetic heterogeneity occurred within the porA1gene locus from some of the C. lariorganisms including the UPTC CF89–12.

AbstractRecombinant full-length urease gene cluster and seven 100% deletion recombinant variants of urease subunits genes, (ureG, ureH, ureA, ureB, ureC, ureEand ureF)were constructed in vitrofrom the Campylobacter sputorumbiovar paraureolyticusLMG17591 strain and expressed in Escherichia coliJM109 cells. A urease-positive reaction (1.885 μmol/min/mg protein) in the log-phase cultured E. colicells transformed with pGEM-T vector carrying the recombinant full-length urease genes cluster was detected. Among the seven 100% deletion recombinant variants, each of the ureG-, ureH(D)-, ureA-, ureB-, ureC-, ureE-and ureF-deletion variants showed no change in assay of the urease reaction, and similarly as in the E. colicell lysate with pGEM-T vector only. Recombinant full-length urease gene cluster and 100% deletion recombinants of the ureEgene in the transformed and log-phase cultured E. colicells from the C. sputorumshowed positively accelerated urease activities when cultured in the medium containing NiCl2(750 μmol/L), but no activity was accelerated in the C. sputorumcultured in NiCl2. In addition, thiourea (20 mmol/L) completely inhibited urease activities from all C. sputorumexamined. The putative recombinant urease subunits A and C were immunologically identified by Western blot analysis with polyclonal anti-urease α (A) and β (B), raised against Helicobacter pylori.

AbstractTwo examples of Campylobacter upsaliensisRM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod)enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res,internal methylase gene and mod,in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the-10-like structure and a semiconserved T-rich region and a putative intrinsic ρ-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensisisolates generated two expected amplicons of the resand modgene segments, and using another primer pair, the same number of isolates also generated an amplicon of the resand modgene segments cluster, including the third internal methylase gene. Thus, C. upsaliensisisolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensisisolates and very different from other thermophilic campylobacters.

AbstractThe methionine sulphoxide reductase A (msrA)gene and its adjacent genetic loci from urease-negative (UN) Campylobacter lariRM2100 and urease-positive thermophilic Campylobacter(UPTC) CF89–12 strains appear to be composed of a msrAstructure gene (507 base pairs [bp]) and another five-gene cluster (approximately 6300 bp) in the same strand and direction. A primer pair (F1/R4-msrA) for polymerase chain reaction (PCR) amplification was designed to generate a product of approximately 900 bp of the msrAgene, including its adjacent genetic loci for the thermophilic Campylobacterorganisms and generate an amplicon with 16 C. lariisolates (n=4 for UN C. lari;n=12 for UPTC). Following direct nucleotide sequencing, sequence analysis and nucleotide sequence alignment analysis, the putative full-length msrAgene from the 16 C. lariisolates showed high nucleotide sequence similarities (91.8–100%) to each other and relatively low similarity (69.3–71.8%) to three reference C. jejuniand C. colistrains. In addition, the msrAgene was transcribed in both the UPTC CF89–12 and NCTC12893 cells using reverse transcription PCR. An immunoreactively positive signal was identified in the UPTC CF89–12 and NCTC12893 cells with anti-UPTC MsrA synthetic peptide antibodies.

AbstractA recombinant molecule of the full-length urease gene operon was constructed in vitrofrom the Japanese ureasepositive thermophilic Campylobacter(UPTC) CF89-12 isolate and expressed in Escherichia colicells. Several large deletion recombinant variants of urease subunit genes were also constructed and expressed in E. colicells. A positive urease reaction with the log-phase cultured E. coliJM109 cells in the NiCl2-containing medium transformed with pGEM-T vector carrying the recombinant molecule of the fulllength operon was detected with isopropyl-β-Dthiogalactoside. Among the several deletion recombinant variants, each ureA-, ureB-, ureE-, ureF-, ureG- and ureH-large deficient, only ureE-large deletion variant (63% deficient) showed a positive urease reaction (approximately 15-fold). In addition, a ureE-complete deletion recombinant variant (100% deficient) constructed also showed a positive reaction of urease (approximately 18-fold). Recombinant urease subunits A and B were immunologically identified by Western blot analysis with anti-urease α (A) and β (B) raised against Helicobacter pylori.

ABSTRACTInadvertent exposure of bacterial pathogens to X-ray radiation may be an environmental stress, where the bacterium may respond by increasing mutational events, thereby potentially resulting in increased antibiotic resistance and alteration to genotypic profile. In order to examine this, four clinical pathogens, including the Gram-negative organisms Escherichia coliO157:H7 NCTC12900 and Pseudomonas aeruginosaNCTC10662, as well as the Grampositive organisms Staphylococcus aureusNCTC6571 and Enterococcus faeciumwere exposed to X-rays (35,495 cGy/cm2) over a seven-day period. Antibiotic susceptibility was assessed before, during and after exposure by examining susceptibility, as quantified by E-test with six antibiotics, as well as to a further 11 antibiotics by measurement of susceptibility zone sizes (mm). Additionally, the DNA profile of each organism was compared before, during and after exposure employing the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC PCR). Results indicated that exposure of these organisms to this amount of X-ray radiation did not alter their antibiotic susceptibility, nor their genomic DNA profile. Overall, these data indicate that exposure of bacteria to X-ray radiation does not alter the test organisms' antibiotic susceptibility profiles, nor alter genomic DNA profiles of bacteria, which therefore does not compromise molecular epidemiological tracking of bacteria within healthcare environments in which patients have been exposed to X-ray radiation.

At present, there are various types of manufacturing systems from the traditional centralized system to the autonomous distributed system which has high flexibly for changes in the kinds and/or amounts of products. To support production planning for such a variety and, as a result, to prepare an adequate production scenario which can be well reviewed not only from an economical but also from an environmental view, IT support tools are necessary. In this paper, an agent oriented digital factory is proposed as an IT support tool for production planning. A trial implementation of the digital factory is also reported.

AbstractThe present study examines the expression of cytolethal distending toxin (cdt) gene encoding a cytotoxin in Campylobacter lari(n=6 urease-negative [UN] C. lari; n=4 urease-positive thermophilic Campylobacter[UPTC]). When reverse transcription polymerase chain reaction (RT-PCR) was carried out with 10 C. lariisolates using a primer pair to amplify the cdtBgene transcript segment, an approximate 260 bp RT-PCR amplicon was generated with all the isolates. In addition, cdtA, cdtBand cdtCgene operon was identified to be polycistronicly transcribed in the C. laricells. The cdtBgene translation in the C. laricells was also confirmed by Western blot analysis. Thus, the cdtgene operon in C. lariorganisms, including UN C. lariand UPTC, was expressed at the transcriptional and translational levels in the cells. The present results suggest that all three cdtgenes may be functional in the cells.

AbstractThis study aims to characterise biochemically urease from an atypical Campylobacterlari, namely urease-positive thermophilic Campylobacter(UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, Gand H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF89- 12 showed enzyme activity over a broad pH range (pH 6–10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20–60°C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease α (A) and α (B) raised against Helicobacter pylori.

AbstractThe primer pair (C412F/C1228R) constructed previously for the polymerase chain reaction (PCR) identification of the genus Campylobacterusing an approximate 800 base pair (bp) 16S rRNA gene target segment proved to be useful for the identification of a total of 49 Campylobacter lariisolates including urease-positive thermophilic Campylobacter(UPTC) organisms (n=25). When the primer pair (CLF/R) developed previously for the PCR identification of C. larispecies using an approximate 250 bp glyAsegment was employed, 27 C. lariisolates, including all the UPTC isolates, were identified to be PCR-negative (55%). Therefore, this PCR procedure developed for the molecular identification of C. lariwas shown to be unreliable for C. lariidentification. Nucleotide sequencing analysis clarified the reason(s) why PCR-negative examples occurred in many C. lariisolates, including UPTC isolates. The primer pair target sequences in the C. lari-specific PCR-negative isolates apparently varied at the 3’ end region, as compared with C. lari-specific PCR-positive isolates. Thus, the multiplex PCR assay developed previously was shown to be unreliable for the molecular identification of C. larisubspecies organisms.

AbstractA degenerate polymerase chain reaction (PCR) primer pair (f-ClflaC/r-ClflaC) was constructed in silicoto amplify flaCand its adjacent genetic loci from Campylobacter lariisolates. Approximately 1.45 kbp amplicons, including the sequences encoding the flaCstructural gene of 750 bp, putative promoter, r-independent intrinsic terminator regions and partial sequences of two putative open reading frames (ORFs), immediately upstream and downstream of the gene, were identified in 16 C. lariisolates (four ureasenegative [UN] C. lari; 12 urease-positive thermophilic campylobacters [UPTC)]). All 16 flaCstructural genes commenced with an ATG start codon and terminated with a TAA stop codon and probable ribosome-binding sites were identified in all 16 isolates. These probably indicate a monocistronic operon structure for the flaCgene in C. lariisolates. In addition, the putative flaCgene ORFs were deduced to be similar in 747 bp among all 26 thermophilic Campylobacterisolates examined, resulting in a similar calculated molecular weight of approximately 26.6–26.9 kDa. The flaCfrom C. lariwas different from the flaA-like sequence and the shorter flaAof UPTC isolates found previously. Reverse transcription PCR and Northern blot hybridisation analyses identified flaCtranscription in C. laricells. The transcription initiation site for the flaCgene was also determined by primer extension analysis. A dendrogram constructed, based on the nucleotide sequence information of flaCfrom 17 C. lariisolates, demonstrated that the C. lariisolates were genetically variable and formed two minor clusters for UN C. lariand UPTC.

AbstractPolymerase chain reaction (PCR) amplification was performed on 20 isolates of five Campylobacterspecies using a degenerate primer pair designed in silicoto generate a product of the luxSgene or its homologue from Campylobacterorganisms. Although the primer pair successfully amplified products of approximately 500 base pairs (bp) with the eight isolates of C. jejuniand C. coliand some of C. upsaliensisand C. fetus, it failed to amplify fragments with all four isolates of C. lari(two ureasenegative C. lari; two urease-positive thermophilic campylobacters). When Southern blot hybridisation analysis was carried using the mixed luxSgene fragments prepared from the C. jejuni, C. coli, C. upsaliensisand C. fetusstrains as a probe, all C. jejuni, C. coli, C. upsaliensisand C. fetusisolates gave positive signals, but no positive signal was detected with any C. lariisolate. These results clearly indicate that C. jejuni, C. coli, C. upsaliensisand C. fetuscarry the luxSgene or its homologue. However, no luxSgene or its homologue was identified to occur in the C. larigenome. Although autoinducer-2 assays were positive in C. jejuni, C. coli, C. upsaliensisand C. fetusisolates, it was negative with all the C. lariisolates examined. In addition, a biofilm formation assay demonstrated that biofilm formation in the C. larispecies does not appear to correlate with the occurrence of the luxSgene because biofilm formation occurred among some isolates of C. lari.

We obtained three conducting crystals based on a [ FeIII(Pc)(CN)2] molecular unit. All crystals showed a large anisotropic negative magnetoresistance arising from the $\pi $-d interaction self-contained in the [ FeIII(Pc)(CN)2] unit. The anisotropy is attributable to the anisotropic g-tensor in the [ FeIII(Pc)(CN)2] unit. We also obtained a thin film containing [ FeII(Pc)(CN)2] . The film exhibits photocurrent response for the UV irradiation. These features suggest [ M(Pc)(CN)2] molecular unit is a well-designed one for a building block of molecular devices. Key words.Molecular electronics – phthalocyanine – $\pi $-d system – thin film.

AbstractTwo sets of PCR primers are constructed to clone the cytochrome P450structural gene, including putative promoter and terminator structures, and its adjacent genetic loci in Campylobacter lariisolates. The putative open reading frames (ORFs) of the P450genes from 11 C. lariisolates (n=5 for urease-negative (UN) C. lari; n=6 urease-positive thermophilic campylobacters [UPTC]) examined consisted of 1365 or 1371 bases (455 or 457 amino acid residues), differing from those of the other thermophilic campylobacters (1359 [453] for C. jejuniand C. upsaliensis; 1368 [456] for C. coli). Each of the putative ORFs from the 11 isolates examined was also shown to carry start and stop codons and ribosome binding sites. Two putative promoter structures, consisting of sequences at the –35- and –10-like regions were also identified upstream of the ORFs. A single copy of the P450gene in the genome was identified with UN C. lariJCM2530Tand UPTC CF89-12, based on Southern blot hybridisation analysis. In addition, when reverse transcription polymerase chain reaction (RT-PCR) analyses were carried out, the transcription of the P450structural gene in C. lariorganisms in vivowas confirmed. The transcription initiation site for the gene was also determined. High nucleotide sequence similarities (95.2–98.8%) of the full-length P450structural gene were shown with each of the 12 C. lariisolates. The UN C. lariand UPTC organisms showed similar findings with the neighbour-joining method, based on the sequence information of the P450structural gene.

AbstractA polymerase chain reaction (PCR) method was carried out on 21 isolates of atypical Campylobacter sputorum(n=14) and C. curvus(n=7) using a primer pair to amplify the helix 11 region within 16S ribosomal RNA (rRNA) gene sequences. Following sequencing and alignment analysis, 14 C. sputorum(100%) and six C. curvus(86%) isolates were shown to carry intervening sequences (IVSs) in this region. Interestingly, the nucleotide sequences of all the IVSs were identical among the 14 C. sputorumisolates (n=5 C. sputorumbiovar [bv] paraureolyticus; n=5 bv fecalis; n=4 bv sputorum). In addition, two different nucleotide lengths and sequences of IVSs were identified among the six C. curvusisolates. On the first prediction of the secondary structure model of the IVSs in 16S rRNA genes, stem and loop structures were identified. In the purified RNA fractions from the 20 Campylobacterisolates carrying IVSs, no 16S rRNA was evident. Instead, other smaller RNA fragments were identified. Thus, the primary 16S rRNA transcripts may have been fragmented in the 20 isolates. This is the first demonstration of atypical C. sputorumand C. curvusisolates carrying IVSs in the helix 11 region in 16S rRNA genes.

AbstractMolecular and comparative analyses of the full-length cytolethal distending toxin (cdt) gene operon and its adjacent genetic loci (2.7–9.4 kilo base pairs in length) are carried out with 12 urease-positive thermophilic Campylobacter(UPTC) isolates using several polymerase chain reaction (PCR) primer pairs. Three putative open reading frames (ORFs) for cdtA, cdtBand cdtC, two putative promoters and a hypothetically intrinsic ρ-independent transcription terminator were identified in all the operons of the 12 UPTC isolates examined. Although the number of amino acid residues slightly varied for the putative cdtAand cdtCORFs, those for the cdtBwere similar among all the UPTC isolates, as well as the six urease-negative (UN) C. lariexamined previously. Regarding the cdtgenes in UPTC CF89-12, each ORF commenced with an ATG start codon and terminated with a TAG stop codon for cdtAand cdtBand a TAA for cdtC.Start and stop codons of the three ORFs for the other 11 UPTC isolates were identical to those from the UPTC CF89-12 isolate except for the TTG start codon for cdtCin the two isolates (NCTC12892 and 12893) and the TGA stop codon for cdtAin five isolates (A1, A2, A3, 89049 and 92251). Two putative promoter structures, consisting of sequences at the –35-like (TTAATA) and –10-like (TATTAA) regions, as well as the start codon (ATG), were identified for the transcriptional promoter, immediately upstream of the cdtAgene in all the 12 isolates, Although the genetic heterogeneity of the cdtBgene locus occurred in all 28 C. lariisolates (n=16 UN C. lari; n=12 UPTC) examined, all nine amino acid-specific DNase residues were completely conserved in all their cdtBgenes. Variable gene insertions with heterogeneous order and combinations occurred between cdtCand lpxBgenes in the all UPTC organisms examined.

AbstractFollowing TA cloning and sequencing with a novel in silicodesigned polymerase chain reaction (PCR) primer pair (f-ClvacJ/r-ClvacJ), approximately 750 base pairs (bp) of promoter and structural gene regions for vacJand its adjacent genetic loci (approximately 1.14 kbp) were identified in 20 isolates of Campylobacter lari(urease-negative C. lari[n=7]; urease-positive thermophilic Campylobacter[n=13]). The nucleotide sequences of an approximately 70-bp non-coding region, including the typical promoter structure, showed sequence differences at 12 loci among 21 isolates including C. lariRM2100. The putative σ70promoter region upstream of the putative open reading frame (ORF), a start codon TTG and a probable ribosome binding site, AGGA, for the vacJgene were also identified in all 21 C. lariisolates examined. Each ORF for the vacJterminated with a TAA stop codon. No hypothetical transcriptional terminators were identified within the amplicons. The putative ORFs of the vacJgene from 21 C. lariisolates consisted of 684 bases, similarly differing from those of the other thermophilic campylobacters (696 bases for C. jejuniRM1221 and NCTC11168 and C. coliRM2228; 690 for C. upsaliensisRM3195). Reverse transcription PCR analysis confirmed the transcription of the vacJgene in the C. laricells. A neighbour joining tree suggested a strong molecular discrimination efficacy between UPTC and UN C. lariemploying vacJnucleotide sequence information. The vacJgene homologue from C. lariorganisms appears not to be a lipoprotein signal peptide or a signal peptide in silico.

AbstractThis study aims to clarify the molecular characteristics of the urease gene operon from urease-positive thermophilic campylobacters (UPTC) obtained from different sources and in various countries. Sequence heterogeneity was observed for the promoter structures at the -35-like region among the 12 isolates examined. The most probable TTG start codon was suggested for the ureB and ureH genes, and for the ureA, E, F and G genes, ATG was suggested among all the isolates examined. Overlap was detected between ureA and ureB and between ureB and ureE among all the isolates examined. UPTC is the first example of an overlap between the two structural genes ureA and ureB. When the completely sequenced open reading frames (ORFs) for ureE, ureF, ureG and ureH were identified, non-coding regions between ureE and ureF, ureF and ureG, and ureG and ureH were also demonstrated. All six start codons of the six urease genes were demonstrated to be preceded by Shine-Dalgarno sequences among all the isolates examined. The Cys-His sequence corresponding to urease active sites were aligned perfectly and fully conserved among the three UPTC isolates examined. A putative and intrinsic -independent transcriptional terminator was identified to be identical among all the isolates examined. A partial and putative ORF of about 200 bp in length showing high sequence similarity to GTP cyclohydrolase I was observed downstream of ureH.

AbstractPolymerase chain reaction (PCR) amplicons (approximately 2.5 kbp) encoding a cdtgene operon and two partial and putative open reading frames (ORFs) were identified in six urease-negative (UN) Campylobacter lariisolates using a new PCR primer pair constructed in silico.Three closely spaced and putative ORFs for cdtA, cdtBand cdtC,two putative promoters and a hypothetically intrinsic ρ-independent transcription terminator were found in the operon. Each ORF commenced with an ATG start codon and terminated with a TGA stop codon for cdtAand cdtBand a TAA for cdtC. Interestingly, an overlap of four nucleotides was detected between cdtAand cdtBand the non-coding region of six base pairs occurring between cdtBand cdtC.The start codons for the three cdtgenes were preceded by Shine-Dalgarno sequences. Although nucleotide sequence differences were identified at seven loci in the cdtAgene, six in cdtBand two in cdtCamong the seven isolates (including C. lariRM2100), no polymorphic sites occurred in the putative promoters, hypothetically intrinsic transcription terminator and the three ribosome binding sites among the seven isolates. All nine amino acid residues specific for both Escherichia coli cdtBand mammalian DNase I were completely conserved in the cdtBgene locus in the 26 C. lariisolates, as well as in C. jejuniand C. coli. No PCR amplicons were generated with urease-positive thermophilic campylobacters (UPTC; n=10) using the primer pair.

AbstractNucleotide sequences of approximately 3.1 kbp consisting of the full-length open reading frame (ORF) for grpE, a non-coding (NC) region and a putative ORF for the fulllength dnaKgene (1860 bp) were identified from a ureasepositive thermophilic Campylobacter(UPTC) CF89-12 isolate. Then, following the construction of a new degenerate polymerase chain reaction (PCR) primer pair for amplification of the dnaKstructural gene, including the transcription terminator region of C. lariisolates, the dnaKregion was amplified successfully, TA-cloned and sequenced in nine C. lariisolates. The dnaKgene sequences commenced with an ATG and terminated with a TAA in all 10 isolates, including CF89-12. In addition, the putative ORFs for the dnaKgene locus from seven UPTC isolates consisted of 1860 bases, and the four urease-negative (UN) C. lariisolates included C. lariRM2100 reference strain 1866. Interestingly, different probable ribosome binding sites and hypothetically intrinsic ρ-independent terminator structures were identified between the seven UPTC and four UN C. lariisolates, respectively. Moreover, it is interesting to note that 20 out of a total of 28 polymorphic sites occurred among amino acid sequences of the dnaKORF from 11 C. lariisolates, identified to be alternatively UPTC-specific or UN C. lari-specific. In the neighbour-joining tree based on the nucleotide sequence information of the dnaKgene, C. lariforms two major distinct clusters consisting of UPTC and UN C. lariisolates, respectively, with UN C. laribeing more closely related to other thermophilic campylobacters than to UPTC.

Sorption of Co(II) on the biogenic Mn oxide produced by a Paraconiothyrium sp.-like strain was investigated. The biogenic Mn oxide, which was characterized to be poorly crystalline birnessite (Na4Mn(III) 6Mn(IV) 8O27 ·9H2O) bearing Mn(III) and Mn(IV) in the structure, showed approximately 6.0-fold higher efficiency for Co(II) sorption than a synthetic Mn oxide. XP-spectra of Co 2p for the biogenic and synthetic Mn oxides after Co(II) sorption indicate that Co was immobilized as Co(III) on the surface of Mn oxides, clearly suggesting that redox reaction occurs between Co(II) ions and each Mn oxides. The Co(II) ions would be initially sorbed on the vacant sites of the surface of biogenic Mn oxide, and then oxidized to Co(III) by neighbor Mn(III/IV) atoms to release Mn(II). For the synthetic Mn oxide, release of Mn(II) was negligibly small because the oxidant is only Mn(IV) in ramsdellite (γ-MnO2). The Mn(II) release from the biogenic Mn oxide during Co(II) adsorption would be not only from weakly bounded Mn(II), but also from redox reaction between Mn(III/IV) and Co(II) ions.

AbstractCloning, 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.

Small GTPases, which are binary switches regulating various signal transduction cascades, function not only to relay signals but also to integrate them from multiple signalling branches. For example, RalA activity is regulated by at least three signalling cascades involving Ras, Rac or PI3K (phosphoinositide 3-kinase). To untangle such complicated regulatory mechanisms, we have been developing probes for GTPases, kinases and phosphatidylinositols based on the principle of FRET (fluorescence resonance energy transfer). We demonstrated previously that, upon EGF (epidermal growth factor) stimulation, Ras activity increases diffusely in the plasma membrane, whereas RalA activity increases predominantly in lamellipodial protrusions. Here, we show that the level of PtdIns(3,4,5)P3 is increased diffusely in the plasma membrane, whereas, in the central region, the level of PtdIns(3,4)P2 is increased more in the nascent lamellipodia than in the plasma membrane. The distribution and time course of Akt activation are similar to those of increased PtdIns(3,4)P2 levels. These observations suggest that the increase in PtdIns(3,4)P2 and the subsequent activation of Akt may be responsible for the localized activation of RalA. Thus the signals from Ras and PI3K converge at the level of Ral GEFs (guanine nucleotide-exchange factors), and this convergence restricts the area of RalA activation.

Several clinical cohort and case-control studies have suggested a link between diabetes and colon cancer. Otsuka Long-Evans Tokushima Fat (OLETF) rats spontaneously develop type 2 diabetes mellitus and Long-Evans Tokushima Otsuka (LETO) rats are non-diabetic. The relationship between type 2 diabetes mellitus and colon cancer was examined in these rats. The carcinogen 1, 2-dimethylhydrazine was administered subcutaneously once weekly for 10 weeks, and the animals were killed and necropsied in week 29. All OLETF rats and 80% of the LETO rats developed cancer. The number of colon cancers per rat was significantly greater in the diabetic than in the non-diabetic rats. Although the tumours tended to be larger in diabetic rats, the difference was not statistically significant. No significant differences were observed in the depth of invasion or histological type of cancer in the two groups. Type 2 diabetes mellitus may enhance the generation and growth of colon cancer.

Objective:The main objective was to study the relationships of the molecular defects in 38 dysfibrinogens with their fibrin networks. Methods and results:Scanning electron microscopic analyses revealed that all the fibrins formed under the same conditions had networks composed of either normal thickness fibers or thin fibers, accompanied by a variety of alterations in the network structure and characteristics. We classified these fibrin networks into five classes, designated normal, less-ordered, porous A, porous B and lace-like networks. The dysfibrinogens with defects in fibrinopeptide A release or the E:D binding sites formed normal or less-ordered networks, while those with defects in the D:D association formed porous A networks composed of many tapered terminating fibers, despite having fibers of normal width, and containing many pores or spaces. The porous B and lace-like networks were composed of highly branched thin fibers because of defects in the lateral association among protofibrils, and the major difference between them was the porosity of the porous B networks. All the porous B networks were easily damaged by mechanical stress, whereas the lace-like networks retained high resistance to such stress, indicating that the network strength was not dependent on the fiber width, but on the porosity that led to fragility of the network. Conclusion:Impairment of the D:D association is the major disturbing factor that leads to the formation of porous fibrin networks. The porosity may be introduced by severe impairment of the D:D association, as well as the lateral association, as has often been observed by extra glycosylation or defects in Ca2+binding.

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184T, 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 NCTC11184T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNAIle-tRNAAla-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.

AbstractA 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, sodBand ureAB) selected are mapped in only a fragment on the restriction map. Three DNA fragments for rrnoperon probes are mapped in multiple regions on the map. When two SmaI-digested neighbouring small fragments hybridised with rrnprobes are cloned and sequenced, a total sequence length of 7487 bp is determined. In the sequence, part of the pnpgene (734 bp) bearing a ρ-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 rrnoperon 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 ρ-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 rrnoperon examined here and the previously reported one, two different 16S-23S ribosomal DNA (rDNA) internal spacer regions are shown to exist.

The efficacy of 5-fluorouracil (5-FU) treatment and the incidence of adverse events differ among patients and depend to some extent on individual variations in drug catabolism. This feasibility study aimed to determine the optimum conditions for a 5-FU oral load test, which would allow the simple evaluation of individual differences in 5-FU catabolism. Patients with colon cancer were given oral 5-FU (200 mg/day) for 3 days (n= 36) or a single 100 mg dose (n= 14). Serum concentrations of uracil, dihydrouracil, 5-FU and 5-fluoro-5, 6-dihydrouracil were measured before and after 5-FU administration. The results suggested that a decline in 5-FU metabolism was associated with continuous administration and increasing age. We conclude that a continuous load of 5-FU is necessary in order to predict the efficacy and side-effects of the drug. The 3-day regimen, with its ease of administration, merits further study to assess its possible clinical application.

The crystal structures of LaGaO3, LaGa0.9Mg0.1O2.95, and La0.9Sr0.1Ga0.9Mg0.1O2.9 have been investigated by high-temperature neutron powder diffraction in order to clarify a mechanism for high oxide ion conduction properties in LaGaO3-based compounds. Parent phase LaGaO3 crystallized in the orthorhombic (Pbnm) up to 150 °C and the rhombohedral (R&thremacr;c) at between 160 and 800 °C. LaGa0.9Mg0.1O2.95 crystallized in the orthorhombic (Ibmm) up to 340 °C and the rhombohedral (R&thremacr;c) at between 350 and 800 °C. On the other hand, La0.9Sr0.1Ga0.9Mg0.1O2.9 crystallized in the monoclinic (I2/a) up to 400 °C and the rhombohedral (R&thremacr;c) at between 410 and 800 °C. The correlations between the crystal structures and conduction properties of LaGaO3-based materials were discussed on the basis of the analytical results.

Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.

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 indis- tinguishable phenotypically and genotypically. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Fibrinogen Lima is an abnormal fibrinogen with an Aα Arg141→Ser substitution resulting in an extra N‐glycosylation at Aα Asn139, which seems to be responsible for the impairment of fibrin polymerization. We have studied the polymerization and properties of clots made from both plasma and purified fibrinogen of both the homozygous and heterozygous forms. The clot permeation studies with both plasma and purified protein revealed a normal flux through the network for the heterozygous form but very decreased permeation in the homozygous form. Consistent with turbidity results, the clot network of the homozygous form, seen by scanning electron microscopy, was tight and composed of thin fibers, with many branch points, while the appearance of clots from the heterozygous form was similar to that of control clots, but in both cases the fibers were more curved than those of control clots. The rheological properties of clots from the homozygous form were also altered, with rigidity being increased in plasma clots, but decreased in the purified system, a consequence of the balance between numbers of branch points and fiber curvature. From these results it seems that the extra carbohydrate moiety, located in the α coiled‐coil region close to the βC domains, impairs the protofibril lateral association process, giving rise to thinner, more curved fibers, with the structural anomalies being most pronounced in the clots from the homozygous plasma. These studies support a model for fibrin polymerization in which the βC–βC interactions are involved in lateral aggregation.

High-spin states in ¹⁴⁵Tb have been investigated by means of in-beam γ-ray spectroscopy techniques with the ¹¹⁸Sn(³²S, 1p4n) reaction. Excitation functions, X–γ–t and γ–γ–t coincidences and γ-ray anisotropies were measured. A level scheme of ¹⁴⁵Tb was established up to E_x≈ 7 MeV. The level structure shows characteristics of a spherical nucleus. Based on the systematics of level structure in the odd-A N = 80 isotones, the level structure below 2 MeV excitation is interpreted by coupling an h_11/2 proton to the excitations in the even–even ¹⁴⁴Gd core. Above 2 MeV excitation, most of the yrast levels are interpreted with multi-quasiparticle shell-model configurations.

AbstractIn 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 jejuniand C. coliand 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. coli27). The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C. jejuniand 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. jejuniand C. coli. Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates.

AbstractSquamous cell carcinoma (SCC) is the most common form of oral malignancy and is often preceded by premalignant lesions, some of which are more likely to progress to carcinoma than others. In this study, a panel of monoclonal antibodies (AE1/AE3, cytokeratin [CK] 14, Ki-67 and p53) is applied to 10 cases of human oral tissue in each of six categories to establish staining patterns indicative of which lesions are more likely to progress to malignancy. The six tissue categories are normal tissue; abnormal benign lesions; mild, moderate and severe dysplasia; and SCC. A statistical analysis of Ki-67 and p53 immunoexpression is performed. The results showed that AE1/AE3 and CK 14 expression was reduced as a late event in oral carcinogenesis, particularly in poorly differentiated SCC. Expression of Ki-67 and p53 proved to be a weak but statistically significant predictor of malignant progression in oral tissue.

A film was prepared from an n-butyl methacrylate/methacrylamide ethylethyleneurea (EU) copolymer [P(BMA–EU)] emulsion produced by an emulsifier-free emulsion copolymerization. The wet adhesion of the emulsion film on an alkyd resin was significantly improved by copolymerization with a small amount of EU (0.5–1.0 mol %). A sodium dodecyl sulfate emulsifier, postadded to the emulsifier-free emulsion, reduced the wet adhesion. The wet adhesion of a film prepared from a poly(n-butyl methacrylate) (PBMA)/P(BMA–EU) composite emulsion produced by an emulsifier-free seeded emulsion copolymerization with PBMA seed particles was higher than that of a P(BMA–EU) film with the same EU content. The localization of EU and the cleanliness at the particle surface were also key factors in the improvement of the wet adhesion of the polymer emulsion film. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 1825–1829, 2003

Congenital hypofibrinogenemia, fibrinogen Tottori II, caused by a nonsense mutation in the fibrinogen Bβ chain gene, was found in a 68-year-old Japanese female. The plasma fibrinogen level was 99.2 mg dL−1as determined by the thrombin time method. No overt molecular abnormalities were observed in purified patient fibrinogen by SDS–PAGE analysis. After sequencing all exons and exon–intron boundaries of three fibrinogen genes, we found a heterozygous single point mutation of T→G at position 3356 of the patient fibrinogen Bβ chain gene. This nucleotide mutation results in a nonsense mutation (TAT sequence for Bβ 41Tyr to TAG sequence for a translation termination signal). The mutation was confirmed by polymerase chain reaction-restriction fragment length polymorphism analysis, since this nucleotide mutation results in a new NheI recognition sequence at this position. These data indicated that the nonsense mutation of the fibrinogen Bβ chain gene caused a truncated fibrinogen Bβ chain, which may not be assembled in the fibrinogen molecule.

The crystal structures of lanthanum gallate perovskite compounds, LaGaO3, LaGa0.8Mg0.2O2.9, and La0.8Sr0.2Ga0.8Mg0.2O2.8 which is well-known as a superior oxide ion conductor, have been analyzed by neutron powder diffraction to clarify the effect of the substitutions on the perovskite structures. The crystal structure of Mg²⁺-substituted LaGaO3, LaGa0.8Mg0.2O2.9, belongs to the same orthorhombic space group, Pbnm, as that of LaGaO3, and the oxygen vacancies were found to be created at the planar O2 atom sites of GaO6 octahedron. On the other hand, the crystal structure of doubly Sr²⁺- and Mg²⁺-substituted LaGaO3, La0.8Sr0.2Ga0.8Mg0.2O2.8, belongs to the cubic space group Pm&thremacr;m and the oxygen vacancies were found to be in disorder. The distortion in the perovskite structures and the oxide ion conduction mechanism are discussed on the basis of the analytical results, together with the results of X-ray diffraction and total conductivity up to 800 °C.

Macro-restriction analysis by means of double digestion using DraI and VspI demonstrated that they cleaved the genomic DNAs from Micrococcus luteus JCM1464^T, 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.