Characterisation of antibiotic resistance of Salmonella isolated from dog treats in Japan.

Dog treats might be contaminated with Salmonella. In Canada and the USA, outbreaks of human salmonellosis related to exposure to animal-derived dog treats were reported. Consequently, surveillance data on Salmonella contamination of dog treats have been gathered in many countries, but not in Japan. In the current study, we investigated whether dog treats in Japan were contaminated with Salmonella. Overall, 303 dog treats (of which 255 were domestically produced) were randomly collected and the presence of Salmonella investigated. Seven samples were positive for Salmonella enterica subsp. enterica. Among these isolates, three were identified as serovar 4,5,12:i:-; two were serovar Rissen; and two were serovar Thompson. All serovar 4,5,12:i:- and Thompson isolates were resistant to one or more drugs. Two serovar Rissen isolates were fully susceptible to all tested antimicrobial agents. All Salmonella isolates were susceptible to cefotaxime, ciprofloxacin and nalidixic acid. The gene blaTEM was detected in two serovar 4,5,12:i:- isolates. The blaCTX-M and blaCMY genes were not detected in any isolates. This study demonstrated that dog treats in Japan could constitute a potential source of dog and human Salmonella infections, including multidrug-resistant Salmonella isolates.

Nationwide surveillance of salmonella in the faeces of pigs in Japan.

The prevalence of faecal carriage of salmonella in 5393 pigs reared on 218 pig farms located in 31 of 47 prefectures in Japan over the period July 2003 to June 2005 was investigated. We isolated 172 strains belonging to 20 serovars and one untypable Salmonella enterica from 169 pig faecal samples (3.1%) collected from 48 farms (22.0%). The most prevalent type of S. enterica was untypable O4,12:d:- which lacks phase 2 flagellar antigen, representing 29.1% (50/172) of all isolates. Of 26 S. enterica serovar Typhimurium isolates, 16 strains appeared to be definitive phage type 104 (DT104) by polymerase chain reaction.

Bacteriological characteristics of Staphylococcus aureus isolates from humans and bulk milk.

The aim of this study was to clarify the epidemiological association and bacteriological characteristics of human and animal Staphylococcus aureus isolates. Pulsed-field gel electrophoresis showed that pulsotypes (PT) of isolates from bulk milk differed from PT from human isolates, suggesting that there is no epidemiological association between isolates from these 2 sources. The absence of a common PT could result from the lack of contact between the sources. Methicillin-resistant S. aureus from human secretions and S. aureus from bulk milk in Japan consisted of 1 and 2 dominant clusters, respectively, whereas methicillin-susceptible S. aureus from humans consisted of assorted clusters. Isolates belonging to the dominant clusters showed the coagulase serotype, the capsule serotype, detection of exotoxin genes, and antimicrobial susceptibility. Isolates from bulk milk did not show the penicillin-binding protein 2a gene, and 252 of 275 isolates belonging to the 2 dominant clusters of bulk milk were susceptible to ampicillin, cefazolin, erythromycin, chloramphenicol, oxacillin, and vancomycin. Moreover, the LukM/LukF'-PV leukotoxin gene was detected in 233 of 275 isolates belonging to the dominant clusters in bulk milk isolates. These results support the hypothesis that a number of factors play a role in the adaptation of S. aureus isolates to specific hosts.

Rapid and effective detection of anthrax spores in soil by PCR.

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.

Molecular cloning of bovine (Bos taurus) cDNA encoding a 94-kDa glucose-regulated protein and developmental changes in its mRNA and protein content in the mammary gland.

We isolated and sequenced cDNA clones encoding a 94-kDa glucose-regulated protein (GRP94) from a cDNA library constructed using bovine (Bos taurus) mammary gland poly(A)(+) RNA. The coding nucleotide sequence and the deduced amino acid sequence of bovine GRP94 shared 94.2-88.4% and 98.1-96.5% identity with those of other mammalian species, respectively. The primary structure contained a carboxyl-terminal signal sequence for retention in the endoplasmic reticulum, six potential sites for N-linked glycosylation and two potential adenosine 5'-triphosphate binding sites, similar to other mammalian and avian GRP94 homologues. In Northern blot hybridization using a cDNA probe from the bovine GRP94 cDNA sequence, a transcript 3.0 kb in size was detected. We measured the amounts of GRP94 and its mRNA in mammary glands from cows at various developmental stages of hormonally induced lactation. The highest level of GRP94 mRNA, determined by dot blot analysis, was detected in the developing stage. In contrast to the mRNA level, the amount of protein, determined by immunoblot analysis using rabbit antiserum raised against GRP94 purified from bovine brain, was higher in lactating stages than in others. The increased level of GRP94 mRNA during the developing stage and the maintenance of GRP94 protein during lactation suggest that the synthesis of GRP94 is regulated during mammary development and differentiation, and also that the protein is involved in a function related to lactation.

A simple and sensitive detection system for Bacillus anthracis in meat and tissue.

AIMS: To detect and isolate Bacillus anthracis from meat and tissue by rapid and simple procedures. METHODS AND RESULTS: Bacillus anthracis Pasteur II cells were added to 1 g lymph node and pig meat, which were then cut into small pieces and suspended in PBS. Aliquots were spread on Bacillus cereus selective agar (BCA) plates to isolate B. anthracis cells, and incubated in trypticase soy broth. The enrichment culture was used for nested PCR with B. anthracis specific primers, which were to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSION: One cell of B. anthracis was detected by nested PCR from 1 g of the samples, and was also isolated on BCA plates according to colony morphology within two days. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be useful for detecting animals with latent anthrax, and meat contaminated with B. anthracis, rapidly and simply.

Detection of anthrax spores from the air by real-time PCR.

AIMS: To detect and isolate Bacillus anthracis from the air by a simple and rapid procedure. METHODS AND RESULTS: One hundred litres of air were filtered through an air monitor device. After the membrane was suspended in PBS, spores of B. anthracis were added. The suspension was plated on Bacillus cereus selective agar (BCA) plates to detect B. anthracis colonies. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using a Light Cycler system and anthrax-specific primers. CONCLUSION: A single cell of B. anthracis was detected by real-time PCR within 1 h and was also isolated on a BCA plate within two d. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR and a Light Cycler provides a flexible and powerful tool to prevent epidemics.

Growth inhibition due to complementation of transforming growth factor-beta receptor type II-defect by human chromosome 3 transfer in human colorectal carcinoma cells.

The transforming growth-beta receptor type II (TGF-beta RII) gene is one of the target genes of the DNA mismatch repair (MMR) defect. The human colorectal carcinoma cell line HCT116 has mutations in the hMLH1 gene and in the microsatellite region of the TGF-beta RII gene, both located on the short arm of chromosome 3. Introduction of the wild-type hMLH1 gene on transferred human chromosome 3 restores many characteristics of MMR-deficiency in HCT116. In this study, we determined whether transfer of chromosome 3 into HCT116 also complements the TGF-beta RII gene defect. We compared in vitro growth characteristics between HCT116 and HCT116 with a transferred chromosome 3 (HCT116 + ch3). The growth was suppressed in HCT116 + ch3 compared with parental HCT116. This suppression was abolished by frequent replacement with fresh medium, suggesting that the autocrine TGF-beta-TGF-beta RII system may be responsible for growth suppression. To explore this possibility, we determined several characteristics essential for the autocrine system. We found that HCT116 + ch3 expresses wild-type as well as mutated TGF-beta RII mRNA. In addition, phosphorylation of TGF-beta RI and growth inhibition were observed in HCT116 + ch3 but not in HCT116 by exposure to exogenous TGF-beta. The amount of TGF-beta1 in HCT116 + ch3 cultures was remarkably less than that in the HCT116, suggesting that TGF-beta produced by HCT116 + ch3 cells may be consumed by the cells. The conditioned medium from HCT116 cultures inhibits HCT116 + ch3 growth. This inhibition was neutralized by the anti-TGF-beta antibody. Taken together, these results strongly suggest that the TGF-beta RII gene defect in HCT116 is complemented by a wild-type gene on the transferred chromosome 3 and that HCT116 + ch3 gained the ability to respond to TGF-beta. Simultaneous complementation of defects of a responsible gene and a major target gene by the chromosome transfer is useful to prove the inactivated phenotypes acquired during colorectal tumorigenesis.

Inhibitory effect of theophylline on recombinant GABA(A) receptor.

Convulsions due to systemic toxicity are a major and frequently fatal side effect of theophylline. The cause of theophylline-induced convulsions is not clear, but antagonism of the inhibitory nervous system may be implicated, so we examined the effects of theophylline on GABA-induced currents using recombinant GABA(A) receptor (GABA(A)-R). Theophylline dose-dependently inhibited GABA-induced currents: the IC50 value was 1841+/-63 microM and Hill coefficient 1.09+/-0.03. The inhibitory action of theophylline on GABA-induced currents was competitive and voltage dependent. The inhibition of GABA-induced currents by theophylline may be a primary mechanism underlying theophylline-induced convulsions.

A malonylated anthocyanin and flavonols in blue Meconopsis flowers.

The structures of the major anthocyanin and two flavonols from the blue flowers of Meconopsis were identified by NMR spectroscopy as being cyanidin 3-O-[(6-O-malonyl-2-O-B-D-xylopyranosyl)-beta-D-glucopyranoside]-7-O-beta-D-glucopyranoside, kaempferol 3-O-(6-O-beta-D-glucopyranosyl)-beta-D-glucopyranoside and kaempferol 3-O-(6-O-beta-D-glucopyranosyl)-beta-D-galactopyranoside respectively.

Molecular typing and epidemiological study of Salmonella enterica serotype Typhimurium isolates from cattle by fluorescent amplified-fragment length polymorphism fingerprinting and pulsed-field gel electrophoresis.

One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.

Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence.

We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.

4-Aminoquinolines: novel nociceptin antagonists with analgesic activity.

Small-molecule nociceptin antagonists were synthesized to examine their therapeutic potential. After a 4-aminoquinoline derivative was found to bind with the human ORL(1) receptor, a series of 4-aminoquinolines and related compounds were synthesized and their binding was evaluated. Elucidation of structure-activity relationships eventually led to the optimum compounds. One of these compounds, N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride (11) not only antagonized nociceptin-induced allodynia in mice but also showed analgesic effect in a hot plate test using mice and in a formalin test using rats. Its analgesic effect was not antagonized by the opioid antagonist naloxone. These results indicate that this nociceptin antagonist has the potential to become a novel type of analgesic that differs from mu-opioid agonists.

[Comparison of propofol and isoflurane anesthesia on postoperative nausea, vomiting and pruritus induced by epidural morphine].

We compared propofol-nitrous oxide anesthesia (Group P) with isoflurane-nitrous oxide anesthesia (Group I) on the incidence of postoperative nausea, vomiting and pruritus induced by epidural morphine. Twenty-eight patients for thoracotomy for lung surgeries were randomly assigned either to Group P or Group I. All patients were administrated epidural morphine (4-7 mg.day-1) during and after the operation. The incidence of nausea, vomiting and pruritus was evaluated at the postoperative early (< 9 hour) and late (> 9 hour) periods. In the late postoperative period, in Group P the incidence of nausea and vomiting tended to be low compared with Group I, but the difference was not statistically significant. The incidence of pruritus was not different between the two groups in both early and late periods.

The quantitative analysis of three action modes of volatile anesthetics on purple membrane.

We quantitatively assessed the spectroscopic changes of purple membrane in relation to the concentrations of a volatile anesthetic. As reported previously, volatile anesthetics show three modes of action on purple membrane. By using an anesthetic for which the concentration in solution could be determined spectroscopically and by applying modified analytical methods regarding the M-intermediate lifetime, we were able to clarify the quantitative relation between anesthetic concentration and each mode of action, a relation which in the past has only been described qualitatively. We also determined through the measurement of transient pH changes with pyranine that the proton pump efficiency per photochemical cycle in an action mode induced with low concentrations of anesthetic does not change from that of the native state. Moreover, we dynamically obtained the individual M-bacteriorhodopsin difference spectrum of each state at room temperature using our flash photolysis system equipped with a wavelength-tunable dye laser. These results demonstrated again that we should clearly distinguish different action modes of anesthetics according to their concentrations.

Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei.

Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein. Using an Escherichia coli expression library of the C. chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein. DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da. Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains. The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114. However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC. Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice. These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.

bis(2-(Acylamino)phenyl) disulfides, 2-(acylamino)benzenethiols, and S-(2-(acylamino)phenyl) alkanethioates as novel inhibitors of cholesteryl ester transfer protein.

A series of bis(2-(acylamino)phenyl) disulfides, 2-(acylamino)benzenethiols, S-(2-(acylamino)phenyl) alkanethioates, and related compounds were synthesized, and their inhibitory effect on cholesteryl ester transfer protein activity in human plasma was evaluated. This study elucidated the structural requirements for inhibitory activity and determined that the optimum compound was S-(2-((1-(2-ethylbutyl)cyclohexane)carbonylamino)phenyl) 2-methylpropanethioate (27) (JTT-705). This compound achieved 50% inhibition of CETP activity in human plasma at a concentration of 9 microM and 95% inhibition of CETP activity in male Japanese white rabbits at an oral dose of 30 mg/kg. It increased the plasma HDL cholesterol level by 27% and 54%, respectively, when given at oral doses of 30 or 100 mg/kg once a day for 3 days to male Japanese white rabbits.

The alpha and gamma subunit-dependent effects of local anesthetics on recombinant GABA(A) receptors.

Although convulsions due to local anesthetic systemic toxicity are thought to be due to inhibition of GABA(A) receptor-linked currents in the central nervous system, the mechanism of action remains unclear. We therefore examined the effects of local anesthetics on gamma-aminobutyric acid (GABA)-induced currents using recombinant GABA(A) receptors with specific combinations of subunits. Murine GABA(A) receptors were expressed by injection of cRNAs encoding each subunit into Xenopus oocytes. The effects of local anesthetics (lidocaine, bupivacaine, procaine and tetracaine) on GABA-induced currents of receptors expressing different subunit combinations (alpha1beta2, alpha1beta2gamma2s, alpha4beta2gamma2s and beta2) were examined via the two electrode voltage clamp method. At alpha1beta2, alpha1beta2gamma2s and alpha4beta2gamma2s GABA(A) receptors, all local anesthetics inhibited GABA-induced currents in a dose-dependent manner. The presence of the gamma2s subunit resulted in a greater inhibition by all local anesthetics, but the presence of the alpha4 subunit resulted in less inhibition. At beta2 homomeric receptors, local anesthetics directly induced an outward current similar to that of picrotoxin. These data indicated that (1) the alpha and gamma subunits of GABA(A) receptors modulated the inhibitory effects of local anesthetics on GABA(A) function, and (2) local anesthetics can activate the beta2 subunit and may block the GABA(A) receptor channel pore.

Developmental changes in the protein and mRNA content of a p115/transcytosis-associated protein in the bovine mammary gland.

We measured the amounts of a vesicular transport factor, p115/transcytosis-associated protein (p115/TAP) and its mRNA, in mammary glands from cows in which lactation was induced hormonally. The highest level of p115/TAP mRNA, determined by Northern blotting, was detected in the developing stage. In contrast to the mRNA level, the amount of protein, determined by immunoblot analysis using anti-p115/TAP antibodies raised against a p115/TAP-derived recombinant fusion protein, was higher during the lactating stages than at other times. Immunohistochemical study showed that p115/TAP was predominantly localized in mammary epithelial cells. The p115/TAP was also detected in tissues other than the mammary gland but, in contrast to the situation in the mammary gland, the protein and its mRNA levels in those tissues were independent of the stage of lactation. The increased level of p115/TAP mRNA during the developing stage and the maintenance of p115/TAP protein during lactation suggest that the synthesis of p115/TAP is regulated during mammary development and differentiation, and also that the protein is involved in a function related to lactation.