Molecular Transportation Conversion of Membrane Tension Using a Mechanosensitive Channel in Asymmetric Lipid-Protein Vesicles.

Giant lipid vesicles composed of a lipid bilayer form complex membrane structures and enzyme network reactions that can be used to construct well-defined artificial cell models based on microfluidic technologies and synthetic biology. As a different approach to cell-mimicking systems, we formed an asymmetric lipid-amphiphilic protein (oleosin) vesicle containing a lipid and an oleosin monolayer in the outer and inner leaflets, respectively. These asymmetric vesicles enabled the reconstitution and function of ß-barrel types of membrane proteins (OmpG) and the fission of vesicles stimulated by lysophospholipids. These applications combine the advantages of the high stability of lipids and oleosin leaflets in asymmetric lipid-oleosin vesicles. In this study, to evaluate the versatility of this asymmetric lipid-oleosin vesicle, the molecular transport of the mechanosensitive channel of large conductance (MscL) with an α-helix was evaluated by changing the tension of the asymmetric vesicle membrane with lysophospholipid. A nanopore of MscL assembled as a pentamer of MscLs transports small molecules of less than 10 kDa by sensing physical stress at the lipid bilayer. The amount and maximum size of the small molecules transported via MscL in the asymmetric lipid-oleosin vesicles were compared to those in the lipid vesicles. We revealed the existence of the C- and N-terminal regions (cytoplasmic side) of MscL on the inner leaflet of the asymmetric lipid-oleosin vesicles using an insertion direction assay. Furthermore, the change in the tension of the lipid-oleosin membrane activated the proteins in these vesicles, inducing their transportation through MscL nanopores. Therefore, asymmetric lipid-oleosin vesicles containing MscL can be used as substrates to study the external environment response of complex artificial cell models.

Quantitative analysis of vibration waves based on Fourier transform in magnetic resonance elastography.

We developed a novel magnetic resonance elastography (MRE) analysis method based on Fourier transform to assess the responsive characteristics for different tissue stiffness and degree of transmission of the vibration wave emanating from a passive driver during MRE. A phantom tissue study was conducted with an MRE sequence and vibration wave system using a clinical MR scanner. The phantom tissue consisted of two layers of agar: 0.75 wt% and 1.0 wt%. Phase-unwrapped images derived from acquired MRE phase images were used to generate a phase profile curve, with a line plotted for the phase-unwrapped images. Fourier transform was performed, and the peak value of the power spectrum was derived. The damping rate/ratio was calculated using the Hilbert transform of the phase profile. We found that the mean shear stiffness value of 1.0 wt% agar was higher than that of 0.75 wt% agar. The responsive frequency of the 0.75 wt% agar layer showed a wider range and the damping rate of the signal showed a higher value than the respective values of the 1.0 wt% agar layer. In conclusion, Fourier transform analysis of MRE enabled us to obtain more detailed information of the tissue characteristics and vibration-wave conditions.

Presence of Staphylococcus aureus ST398 and ST9 in Swine in Japan.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is mainly associated with swine and is capable of causing zoonotic infections. The methicillin-resistant S. aureus (MRSA) multilocus sequence type (ST) 398 of swine origin is predominant in Europe and North America, whereas ST9 is predominant in Asia. To evaluate the possible emergence of MRSA in swine, we examined the ST and spa type of 15 methicillin-susceptible S. aureus (MSSA) isolates obtained from swine in 8 different prefectures from north to south Japan between 2003 and 2009. Sequence analyses revealed that 6 porcine MSSA isolates belonged to ST398; 6 to ST9; and 1 each to ST5, ST97, and ST705. Of the 6 MSSA ST398 strains, 4 were classified as spa type t034. This study illustrated that there is a reservoir in Japanese swine of livestock-associated MSSA types.

Prevalence and mechanism of antimicrobial resistance in Staphylococcus aureus isolates from diseased cattle, swine and chickens in Japan.

Antimicrobial administration is essential for the control and treatment of diseases in animals, but the emergence and prevalence of antimicrobial-resistant Staphylococcus aureus is a significant concern during animal production. Here we investigated the antimicrobial susceptibility of S. aureus from diseased food-producing animals and molecularly characterized the methicillin-resistant and fluoroquinolone-resistant isolates. A total of 290 S. aureus isolates obtained from cattle (n=246), swine (n=16), and chickens (n=28) between 2003 and 2009 were examined for antimicrobial susceptibility against 9 antimicrobials using an agar dilution method. Resistance to penicillin (PC) was most frequently found (24.8%), followed by oxytetracycline (OTC, 10.0%), dihydrostreptomycin (4.1%), erythromycin (EM, 3.1%), enrofloxacin (ERFX, 2.1%), and kanamycin (1.7%). The PC resistance rate was significantly higher in swine than in cattle (P<0.01) and chickens (P<0.01). The resistance rates to OTC, EM and ERFX were significantly higher in swine and chickens than in cattle (P<0.05). Methicillin-resistant S. aureus (MRSA) was recovered from milk derived from a cow with mastitis in 2003; sequence type 8, SCCmec type IV and spa type t024. In the six ERFX-resistant strains isolated after 2003, amino acid substitutions in ParC with/without GyrA were detected. As the prevalence of MRSA and FQ-resistant S. aureus in the animals should be noticed, continuous monitoring is necessary to control resistance to clinically important antimicrobials in S. aureus from food-producing animals.

Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan.

A total of 318 Escherichia coli isolates obtained from different food-producing animals affected with colibacillosis between 2001 and 2006 were subjected to phylogenetic analysis: 72 bovine isolates, 89 poultry isolates and 157 porcine isolates. Overall, the phylogenetic group A was predominant in isolates from cattle (36/72, 50%) and pigs (101/157, 64.3%) whereas groups A (44/89, 49.4%) and D (40/89, 44.9%) were predominant in isolates from poultry. In addition, group B2 was not found among diseased food-producing animals except for a poultry isolate. Thus, the phylogenetic group distribution of E. coli from diseased animals was different by animal species. Among the 318 isolates, cefazolin resistance (minimum inhibitory concentrations: ≥32 µg/ml) was found in six bovine isolates, 29 poultry isolates and three porcine isolates. Of them, 11 isolates (nine from poultry and two from cattle) produced extended spectrum ß-lactamase (ESBL). The two bovine isolates produced bla(CTX-M-2), while the nine poultry isolates produced bla(CTX-M-25) (4), bla(SHV-2) (3), bla(CTX-M-15) (1) and bla(CTX-M-2) (1). Thus, our results showed that several types of ESBL were identified and three types of ß-lactamase (SHV-2, CTX-M-25 and CTX-M-15) were observed for the first time in E. coli from diseased animals in Japan.

A proof of the DBRF-MEGN method, an algorithm for deducing minimum equivalent gene networks.

BACKGROUND: We previously developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method, which deduces the most parsimonious signed directed graphs (SDGs) consistent with expression profiles of single-gene deletion mutants. However, until the present study, we have not presented the details of the method's algorithm or a proof of the algorithm. RESULTS: We describe in detail the algorithm of the DBRF-MEGN method and prove that the algorithm deduces all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants. CONCLUSIONS: The DBRF-MEGN method provides all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.

Contribution of enhanced efflux to reduced susceptibility of Salmonella enterica serovar Choleraesuis to fluoroquinolone and other antimicrobials.

We examined antimicrobial susceptibility and efflux systems in laboratory-derived mutants of Salmonella enterica serovar Choleraesuis selected by culture on fluoroquinolone-containing plates. The mutants exhibited decreased susceptibilities to quinolones and several other antimicrobials. Mutations in the gyrA gene were not always found in the mutants. Accumulation assays revealed that intracellular enrofloxacin concentrations were significantly lower in the mutants compared with parent isolates. Increased expression of acrB mRNA can explain the decreased susceptibilities to several antimicrobials but not in the case of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Decreased susceptibility to CCCP may result from the increased expression of emrA mRNA. These results suggest that the enhancement of multiple efflux pumps is responsible for decreased susceptibilities to several antimicrobials in the laboratory-derived mutants.

Isolation of meticillin-resistant Staphylococcus aureus (MRSA) from swine in Japan.

Meticillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 398 is widely prevalent in swine in Europe and North America. To determine the prevalence of MRSA, and specifically ST398, in Japanese swine, a total of 115 nasal swabs and 115 faecal samples from swine reared at 23 farms located in eastern Japan were investigated. MRSA was isolated from a nasal sample (0.9%) but not from any faecal samples. The strain of MRSA was classified as ST221 by multilocus sequence typing and as t002 by spa typing. The MRSA isolate exhibited resistance to ampicillin, meticillin and dihydrostreptomycin. Interestingly, it remained susceptible to cefazolin, ceftiofur, imipenem, gentamicin, kanamycin, chloramphenicol, oxytetracycline, erythromycin, azithromycin, tylosin, vancomycin, enrofloxacin and trimethoprim. The prevalence of MRSA amongst swine was low and MRSA ST398 was not recovered in the present study.

Comparison of in vitro activities and pharmacokinetics/pharmacodynamics estimations of veterinary fluoroquinolones against avian pathogenic Escherichia coli isolates.

We analyzed in vitro activities and pharmacokinetics/pharmacodynamics (PK/PD) parameters of veterinary fluoroquinolones against avian pathogenic Escherichia coli (APEC) strains from cases of avian colibacillosis. The median of minimum inhibitory concentration (MIC(50)) values against APEC strains for enrofloxacin (ERFX) and danofloxacin (DNFX) were 0.25 µg/ml and for norfloxacin (NFLX) and ofloxacin (OFLX) were 0.5 µg/ml. The percentage of resistant strains for ERFX, DNFX, NFLX, and OFLX were 24.4%, 23.6%, 22.8%, and 23.6%, respectively. Scattergrams of the MICs of ERFX compared to DNFX, NFLX, and OFLX for 127 strains demonstrate a clear correlation between the MIC of ERFX and that of other fluoroquinolones. The differences in amino acid substitution in GyrA may play a role in the variation of MIC values for fluoroquinolones. The ratios of peak serum concentration to MIC (C(max):MIC) and ratios of area under the curve to MIC (AUC:MIC) were relatively high in ERFX and OFLX compared to other fluoroquinolones. These results indicate that although the in vitro activities of these fluoroquinolones against APEC isolates are slightly different, the PK/PD values vary with PK parameters. Therefore, we need to consider the PK/PD parameters in the choice of fluoroquinolones during treatment of avian colibacillosis.

Molecular typing of avian pathogenic Escherichia coli O78 strains in Japan by using multilocus sequence typing and pulsed-field gel electrophoresis.

Avian pathogenic Escherichia coli (APEC) serogroup O78 isolates in Japan were characterized using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Thirty-four of the serotype O78 isolates were clustered into 11 PFGE types with 80% similarity and were classified into 5 sequence types (STs), ST23, ST117, ST155, ST369 and 1 novel ST (ST1645). The most dominant ST was ST23 (54.3%). Fluoroquinolone-resistant strains belonged to ST23 (9 strains), ST155 (3 strains) and ST117 (1 strain). The combined findings of MLST and PFGE suggested that 1 genotype belonging to ST23 was the genotype specific to fluoroquinolone-resistant strains. These results indicate that the combination of MLST and PFGE is useful for conducting epidemiological studies on APEC O78 strains.

[Regional differences in the prevalence of Japanese cedar-pollen allergy].

PURPOSE: According to the 2008 survey conducted by Baba et al., the average prevalence of Japanese cedar-pollen allergy is 26.5% in Japan, although there are great regional differences in the prevalence of this disease. In this study, we investigated the causes of its regional differences. METHODS: Regional differences in the 2008 prevalence of cedar-pollen allergy in 47 prefectures, obtained by Baba et al., were examined in terms of the correlations with the following factors: mean cedar pollen count in each region, the pollen dispersal period, relative humidity in February and March, and the concentrations of SPM, NOx and Ox. The pollen counts of cedar and cypress and the sum of these pollen counts were also measured. RESULTS: The prevalence of cedar-pollen allergy had the highest correlation with the pollen dispersal period, followed by the correlation with the pollen count and relative humidity in the order. There was no statistically significant correlation between the prevalence of cedar-pollen allergy and air pollutants such as SPM.

The fickle mutation of a cytoplasmic tyrosine kinase effects sensitization but not dishabituation in Drosophila melanogaster.

fickle is a P-element mutation identified from a screen for defects in courtship behavior and disrupts the fly homolog of Bruton's tyrosine kinase (Btk) gene (Baba et al., 1999). Here, we show that habituation of the olfactory jump reflex also is defective in fickle. Unlike, the prototypical memory mutants, rutabaga and dunce, which habituate more slowly than normal, fickle flies habituate faster than normal. fickle's faster-than-normal response decrement did not appear to be due to sensorimotor fatigue, and dishabituation of the jump response was normal. Based on a long-standing "two opponent process" theory of habituation, these data suggested that behavioral sensitization might be defective in fickle. To test this hypothesis, we designed a olfactory sensitization procedure, using the same stimuli to habituate (odor) and dishabituate (vortexing) flies. Mutant flies failed to show any sensitization with this procedure. Our study reveals a "genetic dissection" of sensitization and dishabituation and, for the first time, provides a biological confirmation of the two opponent process theory of habituation.

A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran insects.

A second-generation linkage map was constructed for the silkworm, Bombyx mori, focusing on mapping Bombyx sequences appearing in public nucleotide databases and bacterial artificial chromosome (BAC) contigs. A total of 874 BAC contigs containing 5067 clones (22% of the library) were constructed by PCR-based screening with sequence-tagged sites (STSs) derived from whole-genome shotgun (WGS) sequences. A total of 523 BAC contigs, including 342 independent genes registered in public databases and 85 expressed sequence tags (ESTs), were placed onto the linkage map. We found significant synteny and conserved gene order between B. mori and a nymphalid butterfly, Heliconius melpomene, in four linkage groups (LGs), strongly suggesting that using B. mori as a reference for comparative genomics in Lepidotera is highly feasible.

DBRF-MEGN method: an algorithm for deducing minimum equivalent gene networks from large-scale gene expression profiles of gene deletion mutants.

MOTIVATION: Large-scale gene expression profiles measured in gene deletion mutants are invaluable sources for identifying gene regulatory networks. Signed directed graph (SDG) is the most common representation of gene networks in genetics and cell biology. However, no practical procedure that deduces SDGs consistent with such profiles has been developed. RESULTS: We developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method in which an algorithm deduces the most parsimonious SDGs consistent with expression profiles of gene deletion mutants. Positive (or negative) directed edges representing positive (or negative) gene regulations are deduced by comparing the gene expression level between the wild-type and mutant. The most parsimonious SDGs are deduced using graph theoretical procedures. Compensation for excess removal of edges by restoring a minimum number of edges makes the method applicable to cyclic gene networks. Use of independent groups of edges greatly reduces the computational cost, thus making the method applicable to large-scale expression profiles. We confirmed the applicability of our method by applying it to the gene expression profiles of 265 Saccharomyces cerevisiae deletion mutants, and we confirmed our method's validity by comparing the pheromone response pathway, general amino acid control system, and copper and iron homeostasis system deduced by our method with those reported in the literature. Interpretation of the gene network deduced from the S. cerevisiae expression profiles by using our method led to the prediction of 132 transcriptional targets and modulators of transcriptional activity of 18 transcriptional regulators. AVAILABILITY: The software is available on request.

lingerer, a Drosophila gene involved in initiation and termination of copulation, encodes a set of novel cytoplasmic proteins.

In an effort to uncover genetic components underlying the courtship behavior of Drosophila melanogaster, we have characterized a novel gene, lingerer (lig), mutations of which result in abnormal copulation. Males carrying a hypomorphic mutation in lig fail to withdraw their genitalia upon termination of copulation, but display no overt abnormalities in their genitalia. A severe reduction in the dosage of the lig gene causes repeated attempted copulations but no successful copulations. Complete loss of lig function results in lethality during early pupal stages. lig is localized to polytene segment 44A on the second chromosome and encodes three alternatively spliced transcripts that generate two types of 150-kD proteins, Lig-A and Lig-B, differing only at the C terminus. Lig proteins show no similarity to known proteins. However, a set of homologous proteins in mammals suggest that Drosophila Lig belongs to a family of proteins that share five highly conserved domains. Lig is a cytoplasmic protein expressed in the central nervous system (CNS), imaginal discs, and gonads. Lig-A expression is selectively reduced in lig mutants and the ubiquitous supply of this protein at the beginning of metamorphosis restores the copulatory defects of the lig mutant. We propose that lig may act in the nervous system to mediate the control of copulatory organs during courtship.