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1.  A Comparison between Revised NCEP ATP III and IDF Definitions in Diagnosing Metabolic Syndrome in an Urban Sri Lankan Population: The Ragama Health Study 
ISRN Endocrinology  2013;2013:320176.
Background. The prevalence of metabolic syndrome (MetS) within individual cohorts varies with the definition used. The aim of this study was to compare the prevalence of MetS between IDF and revised NCEP ATP III criteria in an urban Sri Lankan population and to investigate the characteristics of discrepant cases. Methods. 2985 individuals, aged 35–65 years, were recruited to the study. Anthropometric and blood pressure measurements and laboratory investigations were carried out following standard protocols. Results. Age and sex-adjusted prevalences of MetS were 46.1% and 38.9% by revised NCEP and IDF definitions, respectively. IDF criteria failed to identify 21% of men and 7% of women identified by the revised NCEP criteria. The discrepant group had more adverse metabolic profiles despite having a lower waist circumference than those diagnosed by both criteria. Conclusion. MetS is common in this urban Sri Lankan cohort regardless of the definition used. The revised NCEP definition was more appropriate in identifying the metabolically abnormal but nonobese individuals, especially among the males predisposed to type 2 diabetes or cardiovascular disease. Further research is needed to determine the suitability of the currently accepted Asian-specific cut-offs for waist circumference in Sri Lankan adults.
doi:10.1155/2013/320176
PMCID: PMC3600172  PMID: 23533799
2.  Meta-analysis of genome-wide association studies for panic disorder in the Japanese population 
Translational Psychiatry  2012;2(11):e186-.
Panic disorder (PD) is a moderately heritable anxiety disorder whose pathogenesis is not well understood. Due to the lack of power in previous association studies, genes that are truly associated with PD might not be detected. In this study, we conducted a genome-wide association study (GWAS) in two independent data sets using the Affymetrix Mapping 500K Array or Genome-Wide Human SNP Array 6.0. We obtained imputed genotypes for each GWAS and performed a meta-analysis of two GWAS data sets (718 cases and 1717 controls). For follow-up, 12 single-nucleotide polymorphisms (SNPs) were tested in 329 cases and 861 controls. Gene ontology enrichment and candidate gene analyses were conducted using the GWAS or meta-analysis results. We also applied the polygenic score analysis to our two GWAS samples to test the hypothesis of polygenic components contributing to PD. Although genome-wide significant SNPs were not detected in either of the GWAS nor the meta-analysis, suggestive associations were observed in several loci such as BDKRB2 (P=1.3 × 10−5, odds ratio=1.31). Among previous candidate genes, supportive evidence for association of NPY5R with PD was obtained (gene-wise corrected P=6.4 × 10−4). Polygenic scores calculated from weakly associated SNPs (P<0.3 and 0.4) in the discovery sample were significantly associated with PD status in the target sample in both directions (sample I to sample II and vice versa) (P<0.05). Our findings suggest that large sets of common variants of small effects collectively account for risk of PD.
doi:10.1038/tp.2012.89
PMCID: PMC3565774  PMID: 23149450
BDKRB2; gene ontology; GWAS; NPY5R; panic disorder; polygenic score
3.  Absence of age-related prefrontal NAA change in adults with autism spectrum disorders 
Translational Psychiatry  2012;2(10):e178-.
Atypical trajectory of brain growth in autism spectrum disorders (ASDs) has been recognized as a potential etiology of an atypical course of behavioral development. Numerous neuroimaging studies have focused on childhood to investigate atypical age-related change of brain structure and function, because it is a period of neuron and synapse maturation. Recent studies, however, have shown that the atypical age-related structural change of autistic brain expands beyond childhood and constitutes neural underpinnings for lifelong difficulty to behavioral adaptation. Thus, we examined effects of aging on neurochemical aspects of brain maturation using 3-T proton magnetic resonance spectroscopy (1H-MRS) with single voxel in the medial prefrontal cortex (PFC) in 24 adult men with non-medicated high-functioning ASDs and 25 age-, IQ- and parental-socioeconomic-background-matched men with typical development (TD). Multivariate analyses of covariance demonstrated significantly high N-acetylaspartate (NAA) level in the ASD subjects compared with the TD subjects (F=4.83, P=0.033). The low NAA level showed a significant positive correlation with advanced age in the TD group (r=−0.618, P=0.001), but was not evident among the ASD individuals (r=0.258, P=0.223). Fisher's r-to-z transformation showed a significant difference in the correlations between the ASD and TD groups (Z=−3.23, P=0.001), which indicated that the age–NAA relationship was significantly specific to people with TD. The current 1H-MRS study provided new evidence that atypical age-related change of neurochemical aspects of brain maturation in ASD individuals expands beyond childhood and persists during adulthood.
doi:10.1038/tp.2012.108
PMCID: PMC3565815  PMID: 23092982
anterior cingulate; Asperger syndrome; autistic disorder; case–control; human; pervasive developmental disorder
4.  Video Capsule Retention in a Zenker Diverticulum 
Case Reports in Gastroenterology  2011;5(2):361-365.
We report the case of a video capsule endoscope lodged within a Zenker diverticulum. The system that was equipped with a real-time viewer showed an unchanging image unlike esophageal or gastric mucosa, suggesting that the capsule was elsewhere. The presence of cervical discomfort suggested video capsule retention in a Zenker diverticulum. The capsule was removed endoscopically and reinserted using a hood-assisted endoscope and the procedure was completed.
doi:10.1159/000329882
PMCID: PMC3134059  PMID: 21769288
Video capsule; Retention; Zenker diverticulum
5.  Disseminated keratoconjunctival epitheliopathy in a patient with systemic lupus erythematosus 
The British Journal of Ophthalmology  2006;90(8):1068-1070.
doi:10.1136/bjo.2006.090498
PMCID: PMC1857182  PMID: 16854840
systemic lupus erythematosus; keratoconjunctival epitheliopathy; inflammation; impression cytology; transmission electron microscopy
6.  Efficacy of Carotid Artery Stenting 
Interventional Neuroradiology  2007;13(Suppl 1):94-99.
Summary
Carotid Artery Stenting(CAS) was performed for 51 lesions in 46 patients for almost clinically symptomatic stenotic (>70%)lesions of cervical carotid arteries. The lesions involved the contralateral occlusion cases in eight cases, the bilateral stenotic cases in six cases and the ipsilateral internal carotid artery stenotic cases in two cases. In all cases, endovascular technique was performed from a transfemoral approach under local anesthesia primarily. Under systemic heparinization, CAS was performed using a self-expanding stent system. For the pre-stenting and post-stenting dilatation, percutaneous transluminal angioplasty (PTA) balloon catheters were used. The balloon was inflated up to the pressure of six to ten atoms for 20 to 30 seconds. After CAS, stenotic lesion dilated successfully in all cases (0-20% residual stenosis; mean, 5.5%) even if in the case of the contra-lateral occlusion cases, more than 90% severe stenotic cases, and the tortuous artery cases. The cerebral protection system was always used, mainly distal blocking balloon type. Only one symptomatic complication occurred after CAS. Follow-up ultrasonic carotid echogram was performed in 30 cases. No cases showed restenosis (more than 50% restenosis). Clinical follow up was performed in all cases for one to 41 months (mean, 15.2 months) and no clinical deterioration such as TIA or stroke occurred. CAS is technically feasible and can be performed with relatively low morbidity even if complicated stenotic cases. It may be useful, but the increase the number of patients and the long-term follow-up are necessary to evaluate the safety and usefulness of this method.
PMCID: PMC3345473  PMID: 20566084
carotid artery stenting, carotid artery stenosis, endovascular technique
7.  Inflammatory cells in brush cytology samples correlate with the severity of corneal lesions in atopic keratoconjunctivitis 
The British Journal of Ophthalmology  2004;88(12):1504-1505.
Background: Inflammatory cells infiltrating to the tarsal conjunctiva are thought to be involved in the pathogenesis of corneal lesions in severe allergic conjunctival diseases. The relation between such cells and the severity of corneal lesions was studied.
Methods: Six patients with atopic keratoconjunctivitis (AKC) were enrolled in this study. Tarsal brush cytology findings and the severity of corneal damage at that point were recorded and analysed for correlation.
Results: Four out of six patients exhibited correlation between eosinophils and corneal damage. Three out of six patients exhibited correlation between neutrophils and corneal damage. Two out of six patients exhibited correlation between both eosinophils and neutrophils and corneal damage. Analysis of all data from all patients taken together revealed that both eosinophils and neutrophils in brush cytology samples significantly correlated with corneal damage.
Conclusions: Inflammatory cells in brush cytology samples correlated with corneal damage. Evaluation of the relative percentages of inflammatory cells in brush cytology samples is a useful method of assessing disease activity in allergic conjunctival disease.
doi:10.1136/bjo.2004.047167
PMCID: PMC1772414  PMID: 15548799
atopic keratoconjunctivitis; brush cytology; eosinophils; neutrophils; allergy
8.  Endovascular Treatment of Ruptured Anterior Communicating Artery Aneurysms 
Interventional Neuroradiology  2006;12(Suppl 1):61-66.
Summary
The aim of this paper is to provide a review of our experience in using the endovascular treatment of ruptured anterior communicating artery (ACoA) aneurysms. Between March 1997 and May 2004, 211 ruptured aneurysms were treated with Guglielmi detachable coil (GDC) system in Mito Medical Center, 73 were located at the ACoA. Two cases were incomplete embolization, and performed microsurgical clipping. In the initial embolization for the 71 aneurysms, complete occlusion was achieved in 44 aneurysms, neck remnant in 11 aneurysms and body filling in 16 aneurysms. Intra-operative complication was occurred in six cases (8.2%).
Aneurysm perforation was occurred in three cases (4.1%), thromboembolic complication was occurred in three cases (4.1%). Acute rebleeding were observed in two cases (2.7%). Endovascular treatment is an effective technique for treating ACoA aneurysms, and 3D-rotational angiography is important diagnostic tool for evaluating the ACoA complex.
PMCID: PMC3387970  PMID: 20569604
anterior communicating artery aneurysm, Guglielmi Detachable Coil, subarachnoid haemorrhage
10.  Structure of cag pathogenicity island in Japanese Helicobacter pylori isolates 
Gut  1999;44(3):336-341.
BACKGROUND—cag pathogenicity island (PAI) is reported to be a major virulence factor of Helicobacter pylori. 
AIM—To characterise cagA and the cag PAI in Japanese H pylori strains. 
METHODS—H pylori isolates from Japanese patients were evaluated for CagA by immunoblot, for cagA transcription by northern blot, and for cagA and 13 other cag PAI genes by Southern blot. cagA negative strains from Western countries were also studied. Induction of interleukin-8 secretion from gastric epithelial cells was also investigated. 
RESULTS—All Japanese strains retained cagA. Fifty nine of 63 (94%) strains had all the cag PAI genes. In the remaining four, cag PAI was partially deleted, lacking cagA transcripts and not producing CagA protein. Details of the PAI of these strains were checked; three lacked cagB to cagQ (cagI) and continuously cagS to cag13 (cagII), and the remaining one lacked cagB to cag8. Western cagA negative strains completely lacked cag PAI including cagA. Nucleotide sequence analysis in one strain in which the cag PAI was partially deleted showed that the partial deletion contained 25 kb of cag PAI and the cagA promoter. Interleukin-8 induction was lower with the cag PAI partial deletion strains than with the intact ones. All Japanese cag PAI deleted strains were derived from patients with non-ulcer dyspepsia, whereas 41of 59 (70%) CagA-producing strains were from patients with peptic ulcers or gastric cancer (p<0.05). 
CONCLUSIONS—Most Japanese H pylori strains had the intact cag PAI. However, some lacked most of the cag PAI in spite of the presence of cagA. Thus the presence of the cagA gene is not an invariable marker of cag PAI related virulence in Japanese strains. 


Keywords: Helicobacter pylori; pathogenicity island; Japanese
PMCID: PMC1727424  PMID: 10026317
11.  Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan 
Gut  1998;42(3):338-343.
Background—VacA and CagA proteins have been reported to be major virulence factors of Helicobacter pylori. However, antibodies against these proteins are frequently found in the sera of Japanese patients regardless of their gastroduodenal status. 
Aim—To evaluate the expression of VacA and CagA proteins by H pylori strains isolated in Japan. 
Methods—By using specific antibodies raised against recombinant VacA and CagA proteins, the expression of VacA and CagA was evaluated in 68 H pylori strains isolated from Japanese patients; a vacuolating assay and genotyping of the vacA gene were also used in the evaluation. The results were analysed in relation to the gastroduodenal diseases of the hosts. 
Results—VacA and CagA proteins were expressed in 59/68 (87%) and in 61/68 (90%) isolates respectively. The vacuolating assay was positive in 57/68 (84%) isolates, indicating that most immunologically VacA positive strains produced active cytotoxin. The prevalence of infection with strains expressing CagA and positive for vacuolating activity (Type I) was very high, 54/68 (79%), irrespective of the gastroduodenal status of the host. 
Conclusion—Most H pylori isolates in Japan are positive for vacuolating cytotoxin and CagA, and thus these virulence factors cannot be used as markers to discern the risk of developing serious gastroduodenal pathologies in the hosts. However, the high prevalence of infection with strains positive for vacuolating cytotoxin and CagA may contribute to the characteristics of H pylori infection in Japan. 


Keywords: VacA; Helicobacter pylori; CagA; ulcer; non-ulcer dyspepsia
PMCID: PMC1727030  PMID: 9577338
12.  Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta-lactamase genes. 
Antimicrobial Agents and Chemotherapy  1997;41(12):2606-2611.
Escherichia coli HKY56, which demonstrated resistance to various beta-lactams except carbapenems, was isolated from the throat swab of an inpatient in 1994. Conjugal transfer of cephem resistance from HKY56 to E. coli CSH2 was not successful. Three cefotaxime-resistant E. coli clones harboring plasmid pMRE001, pMRE002, or pMRE003, each of which carried a 3.4-, 5.8-, or 6.2-kb EcoRI fragment insert, respectively, were obtained from HKY56. Although restriction analysis suggested their different origins, these clones showed similar profiles of resistance to various beta-lactams. The sequence of 10 amino acid residues at the N terminus of beta-lactamase purified from E. coli HB101(pMRE001) was identical to that of Toho-1. This Toho-1-like beta-lactamase-1 (TLB-1) was able to hydrolyze cefoperazone and cefotaxime efficiently, but it failed to hydrolyze cephamycins. A Toho-1-specific DNA probe was hybridized with three distinct EcoRI fragments derived from the chromosomal DNA of strain HKY56, and these fragments corresponded to DNA inserts carried by pMRE001, pMRE002, and pMRE003, respectively. PCR and Southern hybridization analysis suggested that all six cephem-resistant E. coli strains, strains HKY273, HKY285, HKY288, HKY305, HKY316, and HKY335, which were isolated in 1996 at the same hospital where strain HKY56 had been isolated, also possessed multiple Toho-1-like beta-lactamase (TLB) genes, and the hybridization patterns obtained with the Toho-1-specific probe were quite similar among these six isolates. The DNA fingerprinting patterns observed by pulsed-field gel electrophoresis revealed that among the E. coli isolates tested, all isolates except HKY56 possessed a similar genetic background. These findings suggested that E. coli strains that carry chromosomally multiplied TLB genes may have been proliferating and transmitted among patients in the same hospital.
PMCID: PMC164177  PMID: 9420027
13.  Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator. 
Journal of Virology  1997;71(11):8856-8859.
The hepatitis C virus (HCV) nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids and fused to the DNA-binding domain of GAL4, strongly activates transcription in yeast and human hepatoma cells. Transcriptional activation by the HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis.
PMCID: PMC192353  PMID: 9343247
14.  In vitro activities of 10 antimicrobial agents against bacterial vaginosis-associated anaerobic isolates from pregnant Japanese and Thai women. 
Antimicrobial Agents and Chemotherapy  1997;41(10):2297-2299.
The in vitro activities of 10 antimicrobial agents against 159 bacterial vaginosis-associated anaerobic isolates from pregnant Japanese and Thai women were determined. Clindamycin, imipenem, cefmetazole, amoxicillin, amoxicillin-clavulanate, and metronidazole were highly active against all anaerobic isolates except Prevotella bivia and Mobiluncus species, which were resistant to amoxicillin and metronidazole, respectively. Cefotiam, ceftazidime, and ofloxacin were variably effective, while cefaclor was the least effective agent.
PMCID: PMC164113  PMID: 9333068
15.  Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline. 
Journal of Bacteriology  1997;179(14):4480-4485.
The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.
PMCID: PMC179282  PMID: 9226256
16.  In vivo selection of Klebsiella pneumoniae strains with enhanced quinolone resistance during fluoroquinolone treatment of urinary tract infections. 
We report two cases of failure of fluoroquinolone treatment of urinary tract infections with Klebsiella pneumoniae strains harboring quinolone resistance-associated alterations in GyrA and ParC and in vivo selection of posttreatment isolates with enhanced fluoroquinolone resistance. Active efflux leading to decreased accumulation of a drug enhanced fluoroquinolone resistance in one posttreatment isolate, and an additional mutation in parC resulting in an additional amino acid change in ParC was associated with increased resistance in the other.
PMCID: PMC163971  PMID: 9210697
17.  Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts. 
Methyl formate synthase, which catalyzes methyl formate formation during the growth of methylotrophic yeasts, was purified to homogeneity from methanol-grown Candida boidinii and Pichia methanolica cells. Both purified enzymes were tetrameric, with identical subunits with molecular masses of 42 to 45 kDa, containing two atoms of zinc per subunit. The enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl group of the hemiacetal adduct [CH2(OH)OCH3] of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate. Although neither methanol nor formaldehyde alone acted as a substrate for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogenase activity toward aliphatic long-chain alcohols such as octanol, showing that they belong to the class III alcohol dehydrogenase family. The methyl formate synthase activity of C. boidinii was found in the mitochondrial fraction in subcellular fractionation experiments, suggesting that methyl formate synthase is a homolog of Saccharomyces cerevisiae Adh3p. These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in methylotrophic yeasts. The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed.
PMCID: PMC168467  PMID: 9143107
18.  PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams. 
Journal of Clinical Microbiology  1996;34(12):2909-2913.
We applied PCR to the rapid detection of the metallo-beta-lactamase gene, blaIMP, in clinically isolated gram-negative rods. A total of 54 high-level ceftazidime-resistant strains (MICs, > 128 micrograms/ml) were subjected to PCR analyses with the blaIMP-specific primers, since the blaIMP-bearing clinical isolates tested in our previous study always demonstrated high-level resistance to ceftazidime. Twenty-two blaIMP-positive strains including 9 Pseudomonas aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae strains were newly identified from 18 different hospitals in Japan. These strains were mostly isolated from urine samples and showed high-level resistance to almost every cephem, while their levels of resistance to carbapenems were diverse. The PCR analyses with novel integrase gene-specific (intI3) and acc(6')-Ib gene-specific primers suggested that the integron structure found in a large plasmid harbored by S. marcescens AK9373 was also well conserved among blaIMP-positive strains. These results imply that the blaIMP gene cassettes have been dispersing into various gram-negative rods with the help of the newly identified integron element. Thus, the PCR-aided rapid detection will be helpful for the early recognition of emerging blaIMP-positive clinical isolates which demonstrate consistent resistance to beta-lactams.
PMCID: PMC229432  PMID: 8940421
19.  Effects of Clostridium difficile toxin A and toxin B on phospholipase D activation in human promyelocytic leukemic HL60 cells. 
Infection and Immunity  1996;64(11):4433-4437.
The possible involvement of Rho family GTP-binding proteins in the regulation of phospholipase D (PLD) activity has recently been demonstrated. In the present study, to further examine the role of Rho family proteins in PLD activation of human promyelocytic leukemic HL60 cells, we used toxin A and toxin B from the anaerobic bacterium Clostridium difficile, which was shown to glucosylate Rho family proteins and inhibit their interaction with effectors. Pretreatment of [3H]oleic acid-labeled HL60 cell lysates with either one of the toxins resulted in a remarkable inhibition of membrane PLD activity stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The magnitude of inhibition of PLD activity was correlated well with the extent of toxin A- or B-induced glucosylation of 22-kDa RhoA in HL60 cells, toxin B being more effective than toxin A. GTPgammaS-stimulated PLD activation measured with the exogenous substrate containing phosphatidylinositol 4,5-bisphosphate was also inhibited by toxin B. Toxin B had no effect on GTP-gammaS-induced translocation of RhoA from cytosol to membranes. Furthermore, the toxin B pretreatment also suppressed PLD activation induced by 4beta-phorbol 12-myristate 13-acetate in HL60 cell lysates. Thus, it was indicated that Rho family proteins play a key role in GTPgammaS- and 40-phorbol 12-myristate 13-acetate-induced PLD activity in HL60 cells. In addition, the results obtained here indicate that C. difficile toxins are a useful tool for researching the regulation of the Rho family protein-mediated PLD activation and also provide a clue toward understanding the pathogenic background of pseudomembranous colitis from the viewpoint of signal transduction.
PMCID: PMC174394  PMID: 8890188
20.  Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus type 1-infected cell line, MT-2. 
Journal of Virology  1996;70(10):7219-7223.
We recently found that a human T-cell leukemia virus type 1-infected cell line, MT-2, could support the replication of hepatitis C virus (HCV) (N. Kato, T. Nakazawa, T. Mizutani, and K. Shimotohno, Biochem. Biophys. Res. Commun. 206:863-869, 1995). In order to develop a culture system in which HCV replicates more efficiently, we examined the efficiency of HCV replication in cloned MT-2 cell lines by the limiting dilution method. Consequently, we obtained five clones in which intracellular positive-stranded HCV RNA could be detected until at least 21 days postinoculation (p.i.), as opposed to 15 days p.i. in uncloned MT-2 cells. MT-2C, one of the five clones which supported HCV replication up to 30 days p.i., was used for further characterization of HCV replication. Semiquantitative analysis of HCV by PCR revealed that RNA synthesis in infected cells increased after inoculation, reached a maximum level at 4 days p.i., and maintained this level until at least 11 days p.i. The 5' untranslated region of negative-stranded HCV RNA was also detected in the infected cells by two different methods with strand specificity. These results suggest that HCV replicated and multiplied in the MT-2C cells. HCV-infected MT-2C cells that were treated with antibiotics, such as G418 and hygromycin B, sustained HCV RNA for a longer period than did untreated cells. We demonstrated inhibitory effects on HCV replication by an antisense oligonucleotide complementary to the HCV core encoding region and by interferon-alpha. Furthermore, cell-free viral transmission was demonstrated by this culture system. These results suggest that our cell culture system will be useful for studying the mechanism of HCV replication, for screening antiviral agents, and for developing HCV vaccines.
PMCID: PMC190776  PMID: 8794370
21.  Molecular aspects of high-level resistance to sulbactam-cefoperazone in Klebsiella oxytoca clinical isolates. 
Nine Klebsiella oxytoca strains which demonstrated resistance to the combination of sulbactam and cefoperazone were isolated from geographically separate hospitals in Japan in 1995. Among them, K. oxytoca SB23 showed high-level resistance to sulbactam-cefoperazone (MIC > 128 micrograms/ml) and aztreonam (MIC, 128 micrograms/ml). The sulbactam-cefoperazone resistance was not transferred from strain SB23 to Escherichia coli CSH2 by conjugation, beta-Lactamase RbiA, produced by strain SB23, was purified, and the molecular mass was estimated to be 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic parameters for RbiA revealed that cefoperazone and aztreonam were hydrolyzed efficiently by this enzyme. Moreover, ceftazidime and imipenem were also hydrolyzed weakly by RbiA, although strain SB23 did not show any resistance to these agents. Clavulanate, sulbactam, and tazobactam failed to block the hydrolysis of cefoperazone by RbiA. The structural gene of RbiA (blaRBI) was cloned and sequenced, and the deduced amino acid sequence of RbiA demonstrated high-level similarities to those of the beta-lactamases found in K. oxytoca D488, E23004, and plasmid-mediated MEN-1, which have been classified into Bush functional group 2be. Although RbiA demonstrates high-level molecular similarity to the enzymes in group 2be, from an enzymological point of view, this enzyme might be differentiated from the enzymes in that group. Hybridization analysis revealed that beta-lactamase genes highly similar to blaRBI were generally encoded on the chromosome of the sulbactam-cefoperazone-resistant clinical isolates of K. oxytoca tested in the study, despite their different derivations. This observation suggests that sulbactam-cefoperazone-resistant A. oxytoca strains which produce RbiA-type beta-lactamases have been proliferating in many hospitals in Japan.
PMCID: PMC163460  PMID: 8878568
22.  Cloning of the aldehyde reductase gene from a red yeast, Sporobolomyces salmonicolor, and characterization of the gene and its product. 
An NADPH-dependent aldehyde reductase (ALR) isolated from a red yeast, Sporobolomyces salmonicolor, catalyzes the reduction of a variety of carbonyl compounds. To investigate its primary structure, we cloned and sequenced the cDNA coding for ALR. The aldehyde reductase gene (ALR) comprises 969 bp and encodes a polypeptide of 35,232 Da. The deduced amino acid sequence showed a high degree of similarity to other members of the aldo-keto reductase superfamily. Analysis of the genomic DNA sequence indicated that the ALR gene was interrupted by six introns (two in the 5' noncoding region and four in the coding region). Southern hybridization analysis of the genomic DNA from S. salmonicolor indicated that there was one copy of the gene. The ALR gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified to homogeneity and showed the same catalytic properties as did the enzyme from S. salmonicolor.
PMCID: PMC168011  PMID: 8779568
23.  Isolation and characterization of a novel oxygenase that catalyzes the first step of n-alkane oxidation in Acinetobacter sp. strain M-1. 
Journal of Bacteriology  1996;178(13):3695-3700.
In the Finnerty pathway for n-alkane, oxidation in Acinetobacter sp., n-alkanes are postulated to be attacked by a dioxygenase and the product, n-alkyl hydroperoxide, is further metabolized to the corresponding aldehyde via the peroxy acid [W. R. Finnerty, P. 184-188, in A. H. Applewhite (ed.), Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry, 1988]. However, no biochemical evidence regarding the first-step reaction is available. In this study, we found a novel n-alkane-oxidizing enzyme that requires only molecular oxygen, i.e., not NAD(P)H, in our isolate, Acinetobacter sp. strain M-1, and purified it to apparent homogeneity by gel electrophoresis. The purified enzyme is a homodimeric protein with a molecular mass of 134 kDa, contains 1 mol of flavin adenine dinucleotide per mol of subunit, and requires CU2+ for its activity. The enzyme uses n-alkanes ranging in length from 10 to 30 carbon atoms and is also active toward n-alkenes (C12 to C20) and some aromatic compounds with substituted alkyl groups but not toward a branched alkane, alcohol, or aldehyde. Transient accumulation of n-alkyl hydroperoxide was detected in the course of the reaction, and no oxygen radical scavengers affected the enzyme activity. From these properties, the enzyme is most probably a dioxygenase that catalyzes the introduction of two atoms of oxygen to the substrate, leading to the formation of the corresponding n-alkyl hydroperoxide. The enzymatic evidence strongly supports the existence of an n-alkane oxidation pathway, which is initiated by a dioxygenase reaction, in Acinetobacter spp.
PMCID: PMC178149  PMID: 8682768
24.  Bioconversion of 7-Aminocephalosporanic Acid by Intact Rhodotorula glutinis Cells 
When 7-aminocephalosporanic acid (7-ACA) was used as a single carbon source in the enrichment culture medium for screening 7-ACA-degrading microorganisms, pink yeast colonies appeared frequently, and these were identified as Rhodotorula glutinis. These intact R. glutinis cells converted (i) 7-ACA to deacetyl-7-ACA (7-ADACA) and (ii) monochloroacetyl-7-ACA to monochloroacetyl-7-ADACA at sufficiently high levels to be of commercial interest. Acetylation of 7-ADACA to 7-ACA, the reverse reaction of hydrolysis in an organic medium with methyl acetate as an acetyl donor, was also demonstrated.
PMCID: PMC1388908  PMID: 16535370
25.  Structure of the 3' terminus of the hepatitis C virus genome. 
Journal of Virology  1996;70(5):3307-3312.
Hepatitis C virus (HCV), a positive-strand RNA virus, has been considered to have a poly(U) stretch at the 3' terminus of the genome. We previously found a novel 98-nucleotide sequence downstream from the poly(U) stretch on the HCV genome by primer extension analysis of the 5' end of the antigenomic-strand RNA in infected liver (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215: 744-749, 1995). Here, we show that the novel sequence is a highly conserved 3' tail of the HCV genome. We repeated primer extension analyses with four HCV-infected liver samples and found the 98-nucleotide sequence in all the samples. Furthermore, experiments in which RNA oligonucleotide was ligated to the 3' end of the HCV genome existing in infectious serum revealed nearly identical 3' termini with no extra sequence downstream from the 98-nucleotide sequence, suggesting that this sequence is the tail of the HCV genome. This tail sequence was highly conserved among individuals and even between the two most genetically distant HCV types, II/1b and III/2a. Computer modeling predicted that the tail sequence can form a conserved stem-and-loop structure. These results suggest that the novel 3' tail is a common structure of the HCV genome that plays an important role in initiation of genomic replication.
PMCID: PMC190199  PMID: 8627816

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