Ken-ichiro Iida

The Expression of Two Splice Variants of Metabotropic Glutamate Receptor Subtype 5 in the Rat Brain and Neuronal Cells During Development

Abstract: We previously reported that a variant with extra amino acid residues exists in the metabotropic glutamate receptor subtype 5 (mGluR5). Either of the two isoforms, named mGluR5b and mGluR5a for the isoforms with and without the inserted sequence, respectively, generated Ca2+‐activated Cl− current when expressed in Xenopus oocytes. We herein report that these two isoforms are produced by the alternative splicing of the exon skipping type. When examined during the course of postnatal development, the major mGluR5 isotype mRNA was observed to switch from mGluR5a to mGluR5b in the rat hippocampus and the cerebral cortex. We also investigated two cell lines that could be differentiated into neuron‐like cells in vitro. Whereas the mGluR5b mRNA was hardly detectable in either undifferentiated or differentiated NG108‐15 cells, the relative amounts of the two variant mRNAs changed after the induction of differentiation in the P19 cells. An extracellular application of trans‐d,l‐1‐amino‐1,3‐cyclopentanedicarboxylate on the neuron‐like P19 cells induced intracellular Ca2+ mobilization, thus suggesting that the cells could express functional mGluR(s) coupled to phospholipase C and other components that could mediate the signal transduction pathway. This cell line may thus provide a model system for studying both mGluR5 expression and other mGluR‐induced phenomena at the molecular level.

Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria

Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 μg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 μg/ml) led to breaks of double‐stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single‐stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 μg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.

Hydrogen Peroxide Production in Streptococcus pyogenes: Involvement of Lactate Oxidase and Coupling with Aerobic Utilization of Lactate

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H2O2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147:2469-2477, 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H2O2-producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H2O2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H2O2 production in S. pyogenes. Of the other two potential H2O2-producing enzymes of this bacterium, NADH and alpha-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H2O2-scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H2O2. Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H2O2-producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.

Method To Detect Only Live Bacteria during PCR Amplification

ABSTRACT Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 108 heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 108 heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 103 to 107 antibiotic-treated L. monocytogenes cells but could detect 102 live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 103 to 107 heat-treated cells but could detect 101 live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.

Release of Shiga Toxin by Membrane Vesicles in Shigella dysenteriae Serotype 1 Strains and In Vitro Effects of Antimicrobials on Toxin Production and Release

Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.

Alteration in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in Quinolone-resistant Shigella dysenteriae serotype 1 clinical isolates from Kolkata, India.

After the emergence of multidrug-resistant Shigella strains (8, 11), fluoroquinolones, such as ciprofloxacin and norfloxacin, were used in India to treat shigellosis. However, recently, genetically clonal ciprofloxacin-resistant Shigella dysenteriae serotype 1 strains have been isolated from sporadic and epidemic cases of dysentery in southern Asia, including eastern India (1, 9, 12; S. Dutta, A. Ghosh, K. Ghosh, D. Dutta, S. K. Bhattacharya, G. B. Nair, and S.-I. Yoshida, Letter, J. Clin. Microbiol. 41:5833-5834, 2003). Quinolone resistance is linked mainly to mutations located in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) (3, 4, 5). Replacement of residues Ser 80 and Glu 84 in parC commonly supplements gyrA mutations at residues Ser 83 and Asp 87 in members of the Enterobacteriaceae family to develop high fluoroquinolone resistance (13, 14). Besides topoisomerase mutations, overexpression of the energy-dependent multidrug efflux pump AcrAB due to mutations within repressor (AcrR) and multiple target and nontarget gene changes also contribute to fluoroquinolone resistance phenotypes in bacteria (2, 7, 15). In this report, we demonstrate the mutations in the QRDRs of gyrA, gyrB, parC, and parE genes of fluoroquinolone-resistant S. dysenteriae serotype 1 strains isolated in Kolkata, India. The type strain of S. dysenteriae serotype 1 (GTC 786) was procured from the culture collection of the Graduate School of Medicine, Gifu University, Gifu, Japan. Among 12 wild strains of S. dysenteriae serotype 1 included in this study, eight DS strains from sporadic cases (1) and one SKN outbreak strain (9) of dysentery showed resistance to fluoroquinolones, such as nalidixic acid, ciprofloxacin, and norfloxacin. The other three IBM strains were susceptible to all fluoroquinolones (8). These two groups of strains were compared to the type strain to determine any mutations. The QRDRs of the gyrA (648-bp), gyrB (309-bp), parC (249-bp), and parE (290-bp) genes for all study strains were amplified with primer pairs designed from the genome sequence of S. flexneri 2457T, which is available at the DBGET database. For gyrA, the forward primer 5′ TAC ACC GGT CAA CAT TGA GG 3′ (nucleotide [nt] 24 to 43) and the reverse primer 5′ TTA ATG ATT GCC GCC GTC GG 3′ (nt 652 to 671) were used. For gyrB, the forward primer 5′ TGA AAT GAC CCG CCG TAA AGG 3′ (nt 1170 to 1190) and the reverse primer 5′ GCT GTG ATA ACG CAG TTT GTC CGG G 3′ (nt 1455 to 1479) were used. For parC, the forward primer 5′ GTC TGA ACT GGG CCT GAA TGC 3′ (nt 147 to 167) and the reverse primer 5′ AGC AGC TCG GAA TAT TTC GAC AA 3′ (nt 373 to 395) were used. For parE, the forward primer 5′ ATG CGT GCG GCT AAA AAA GTG 3′ (nt 1066 to 1086) and the reverse primer 5′ TCG TCG CTG TCA GGA TCG ATA C 3′ (nt 1334 to 1355) were used. The amplification was performed in a DNA thermal cycler as follows: (i) 30 cycles, with 1 cycle consisting of 1 min at 92°C, 1 min at 64°C, and 2 min at 74°C; and (ii) a final extension step of 10 min at 74°C. The purified PCR products were used directly as templates for sequencing, which was performed with CEQ dye terminator cycle sequencing using the Quickstart kit (Beckman Coulter, Inc.) in an automated sequencer (CEQ 2000 XL DNA analysis system; Beckman Coulter). The amino acid sequences of QRDRs were determined, and the homology of sequences was performed using the DNASIS program (Hitachi Software, Tokyo, Japan). The MICs of nalidixic acid, ciprofloxacin, and norfloxacin were determined for each strain by using the Etest kit (AB Biodisk, Solna, Sweden), and the readings were interpreted using the National Committee for Clinical Laboratory Standards (NCCLS) breakpoint criteria (6). In Table ​Table1,1, two groups (groups A and B) were discerned on the basis of their fluoroquinolone resistance profiles. None of the strains possessed any mutation in the gyrB or parE gene. Mutations in amino acid sequences were detected in GyrA and ParC regions of all (100%) fluoroquinolone-resistant strains (group A). However, no single parC mutation was found without the concomitant presence of a gyrA mutation. TABLE 1. MICs of antimicrobial agents and amino acid changes in the GyrA and ParC subunits of fluoroquinolone-resistant S. dysenteriae serotype 1 All fluoroquinolone-resistant strains showed two mutations, one mutation in gyrA at codon 83 (C→T transition), resulting in the replacement of serine (TCG) by leucine (TTG), and one mutation at codon 87 (G→A or A→G transition), resulting in the replacement of aspartic acid (GAC) by asparagine (AAC) or glycine (GGC). Another mutation at codon 80 (G→T transition) of parC resulted in the replacement of serine (AGC) by isoleucine (ATC), but no change was found at codon 84 (GAA; glutamic acid) of the gene. Since only the QRDRs of the genes were sequenced and the wild-type susceptibility was not knocked in, we do not rule out the possibility of mutations, although unlikely, present outside the region sequenced. Our results corroborated earlier findings of mutations in the gyrA gene of quinolone-resistant S. dysenteriae serotype 1strains (10, 12). A difference in the amino acid substitution at codon 87 of the GyrA subunit of the sporadic strain (this study) and outbreak strain (12) is noteworthy. However, in addition to the gyrA mutation, the present study also reports another substitution at codon 80 of the parC gene of fluoroquinolone-resistant S. dysenteriae serotype 1 strains.

Concerted Action of Lactate Oxidase and Pyruvate Oxidase in Aerobic Growth of Streptococcus pneumoniae : Role of Lactate as an Energy Source

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H(2)O(2)-forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.

Leptospira idonii sp. nov., isolated from environmental water.

Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira, but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6 ± 1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1(T) grew in Korthofs medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1(T) within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira. The strain was not pathogenic to hamsters. Strain Eri-1(T) exhibited low levels (11.2-12.6 %) of similarity by DNA-DNA hybridization to the three most closely related species of the genus Leptospira. The DNA G+C content of the genome of strain Eri-1(T) was 42.5 ± 0.1 mol%. These results suggest that strain Eri-1(T) represents a novel species of the genus Leptospira, for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1(T) ( = DSM 26084(T) = JCM 18486(T)).

A Novel Agar Medium to Detect Hydrogen Peroxide-Producing Bacteria Based on the Prussian Blue-Forming Reaction

The classic method for H2O2 detection involving Prussian blue formation was adapted to formulate a novel agar medium that makes possible in situ detection of H2O2 produced by bacteria. Using this medium, colonies of H2O2‐producing species including Streptococcus pyogenes were easily identified by the appearance of blue halos. The utility of the medium was further illustrated by its successful application to the isolation of H2O2‐producing mutants from a non‐H2O2‐producing stain of S. pyogenes.

Type 1 fimbriation and its phase switching in diarrheagenic Escherichia coli strains.

ABSTRACT Type 1 fimbriae can be expressed by most Escherichia coli strains and mediate mannose-sensitive (MS) adherence to mammalian epithelial cells. However, the role of type 1 fimbriae in enteric pathogenesis has been unclear. Expression of type 1 fimbriae inE. coli is phase variable and is associated with the inversion of a short DNA element (fim switch). Forty-six strains of diarrheagenic E. coli were examined for the expression of type 1 fimbriae. Only four of these strains were originally type 1 fimbriated. Seventeen strains, originally nonfimbriated, expressed type 1 fimbriae in association with off-to-on inversion of the fim switch, after serial passages in static culture. The switching frequencies of these strains, from fimbriate to nonfimbriate, were greater than that of the laboratory strain E. coli K-12. None of the 16 strains of serovar O157:H7 or O157:H− expressed type 1 fimbriae after serial passages in static culture. The nucleotide sequence analysis of thefim switch region revealed that all of the O157:H7 and O157:H− strains had a 16-bp deletion in the invertible element, and the fim switch was locked in the “off” orientation. The results suggest that expression of type 1 fimbriae may be regulated differently in different E. coli pathogens causing enteric infections.