Keizo Torii

Age-associated accumulation of 8-hydroxydeoxyguanosine in mitochondrial DNA of human diaphragm

This is the first report that age-associated accumulation of 8-hydroxydeoxyguanosine (8-OH-dG) does occur in human mitochondrial DNA (mtDNA) in muscle of diaphragm. We extracted mtDNA from human diaphragm muscles from differing age groups, and determined the amount of 8-OH-dG by ultramicro-high performance liquid chromatography/mass-spectrometry system. With the same specimen, multiple deletions of mtDNA were detected by electrophoresis after amplification by the polymerase chain reaction method. In subjects below age 55, the level of 8-OH-dG in mtDNA was below 0.02% of the total deoxyguanosine (dG), whereas, in subjects over age 65, the level of 8-OH-dG increased with age at a rate of ca. 0.25% per 10 years, reaching 0.51% at age 85. Moreover, a concomitant increase in multiple deletions was detected with the increase in age. These results suggest that, in younger diaphragms, replication of mtDNA dilutes out 8-OH-dG being not detectable. In the elderly subjects aged over 65, the replication rate might be slowed down leading to the accumulation of 8-OH-dG in mtDNA, which would accelerate the age-associated multiple deletions of mtDNA observed among the subjects.

Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates

ABSTRACT Tetracycline-resistant (Tetr) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tetr gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tetr genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tetr strains transferred Tetr genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tetr strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.

Characterization of Group C and G Streptococcal Strains That Cause Streptococcal Toxic Shock Syndrome

ABSTRACT Twelve strains (the largest number ever reported) of group C and G1 streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as Streptococcus dysgalactiae subsp. equisimilis, and one strain was identified as Streptococcus equi subsp. zooepidemicus. We found that it was the first reported case of STSS caused by S. equi subsp. zooepidemicus. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the S. dysgalactiae strains belonged to clusters I and II, both of which were closely related. The emm types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular S. dysgalactiae strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the spegg gene was detected in seven S. dysgalactiae strains, with none of the other superantigen genes being detected in any of the strains. However, the sagA gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.

Matrix metalloproteinases in the pathogenesis of asthma and COPD: implications for therapy.

While asthma is an inflammatory disorder of the airways involving mediators released from mast cells and eosinophils, inflammation alone is insufficient to explain the chronic nature of the disease. Recent progress in the understanding of disease pathogenesis has revealed that airway remodeling, which is at least in part due to an excess of extracellular matrix (ECM) deposition in the airway wall, plays a significant role in airflow obstruction. Matrix metalloproteinases (MMPs) have been suggested to be the major proteolytic enzymes to induce airway remodeling in asthma and COPD. It has been widely accepted that different inflammatory processes are involved in asthma and COPD with different inflammatory cells, mediators, and responses to treatments. Despite these different processes, airflow obstruction and airway remodeling characterize these two diseases. MMP-2 and -9 have been reported to be involved in the pathogenesis of airway remodeling in both diseases and MMP-12, in addition to these MMPs, in the pathogenesis of COPD.In this review, we discuss the current views on the role of MMPs in the pathogenesis of bronchial asthma and COPD. Anti-MMP therapy could theoretically be useful to prevent airway remodeling in asthma and COPD. However, to date no clinical data are available regarding the efficacy of anti-MMP therapies in the treatment of patients with asthma and COPD.

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid‐rich conditions. We found that the Shiga Toxin‐producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K‐12 strain in Luria–Bertani (LB)‐2‐morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini‐Tn5 mutagenesis and, from 50 000 colonies, five mutants that showed a clear acetic acid‐sensitive phenotype were isolated. The insertion of mini‐Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O‐polysaccharide, loss of both O‐polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O‐polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O‐polysaccharide and ECA, and normal growth was restored in organic acid‐rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O‐polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O‐polysaccharide and ECA as well as an acid resistance mechanism.

Effect of Antibiotics on Group A Streptococcus Exoprotein Production Analyzed by Two-Dimensional Gel Electrophoresis

ABSTRACT High-dose clindamycin (CLDM) and benzylpenicillin (PCG) are the recommended chemotherapeutic remedies for toxic shock-like syndrome caused by group A streptococci. One reason for this is that it has been shown that CLDM suppresses the expression of some exoproteins, e.g., SpeB, SpeA, and streptolysin O (Slo). We analyzed the effects of antibiotics on the production of whole exoproteins by two-dimensional gel electrophoresis. Unexpectedly, we found that the levels of several exoproteins, Slo, NAD+-glycohydrolase (Nga), M protein, and Sic, were increased by CLDM treatment, although we also confirmed previous findings that the levels of various exoproteins, including SpeB, were decreased. The increases in exoprotein levels were also detected by using other protein synthesis inhibitor antibiotics: erythromycin, kanamycin, tetracycline, chloramphenicol, and linezolid. Peptidoglycan synthesis inhibitors (such as PCG, cefazolin, and imipenem), DNA replication inhibitors (such as gatifloxacin), and an RNA polymerase inhibitor (rifampin) did not have significant effects on exoprotein production. The combination of CLDM and PCG had no advantageous effects with regard to exoprotein production compared to the effect achieved with CLDM alone. We also analyzed the transcriptional levels of slo and nga by reverse transcription-PCR and found that this change was also detected at the transcriptional level. Furthermore, the phenomenon was seen not only in strains of the M1 serotype but also in strains of the other M serotypes. Our study suggests that the clinical effectiveness of CLDM might be due to the inhibition of the production of a limited number of exoproteins.

A New Phylogenetic Cluster of Cereulide-Producing Bacillus cereus Strains

ABSTRACT Phenotypic and molecular studies have established that cereulide-producing strains of Bacillus cereus are a distinct and probably recently emerged clone within the Bacillus population. We analyzed a set of B. cereus strains, both cereulide producers and nonproducers, by multilocus sequence typing. Consistent with earlier reports, nonproducers demonstrated high heterogeneity. Most cereulide-producing strains and all flagellar antigen type H1 strains were allocated to the known sequence type of exclusively emetic B. cereus strains. Several cereulide-producing strains, however, were recovered at a new phylogenetic location, all of which were serotype H3 or H12. We hypothesize that the group of cereulide producers is diversifying progressively, probably by lateral transfer of the corresponding gene complex.

Matrix Metalloproteinases and Tissue lnhibitors of Matrix Metalloproteinases in Sputum from Patients with Bronchial Asthma

To examine a possibility that matrix metalloproteinases (MMPs) participate in the pathogenesis of asthma and/or the development of asthma attack, we measured the concentrations of MMP-2, MMP-9, and their respective tissue inhibitors of metalloproteinases (TIMP)-2 and TIMP-1, in induced sputa collected from 28 patients with moderate to severe bronchial asthma. Specimens were collected during both the attack and the remission from 15 age- and sex-matched healthy control subjects. The concentration of MMP-9 was significantly (p < 0.05) higher in the patients, even during the remission, as compared to that in healthy controls. The concentrations of MMP-9 (p < 0.05) and its specific inhibitor TIMP-1 (p < 0.01), and MMP-2 (p < 0.01) in these patients during the attack were significantly higher than those in healthy controls. In these patients, the MMP-9 concentration was significantly higher (p < 0.05) during the attack than during the remission. These results suggest that MMPs and TIMPs may be involved in the pathogenesis of bronchial asthma, and that the increased MMP-9 might be involved in the development of attack in patients with chronic asthma.

Phylogenetic Analysis of Bacillus cereus Isolates from Severe Systemic Infections Using Multilocus Sequence Typing Scheme

Bacillus cereus strains from cases of severe or lethal systemic infections, including respiratory symptoms cases, were analyzed using multilocus sequence typing scheme of B. cereus MLST database. The isolates were evenly distributed between the two main clades, and 60% of them had allele profiles new to the database. Half of the collections strains clustered in a lineage neighboring Bacillus anthracis phylogenetic origin. Strains from lethal cases with respiratory symptoms were allocated in both main clades. This is the first report of strains causing respiratory symptoms to be identified as genetically distant from B. anthracis. The phylogenetic location of the presented here strains was compared with all previously submitted to the database isolates from systemic infections, and were found to appear in the same clusters where clinical isolates from other studies had been assigned. It seems that the pathogenic strains are forming clusters on the phylogenetic tree.

Growth Phase-Dependent Effect of Clindamycin on Production of Exoproteins by Streptococcus pyogenes

ABSTRACT The administration of high-dose clindamycin plus benzylpenicillin has been recommended for the treatment of streptococcal toxic shock-like syndrome caused by Streptococcus pyogenes, and clindamycin has been found to be more effective than beta-lactams in retrospective analyses of human cases. Although therapeutic doses of clindamycin have also been shown to be effective against experimental infections and clindamycin has great efficacy against the production of bacterial exoproteins, we recently reported that the level of production of some exoproteins was unchanged or even increased by a subinhibitory dose of clindamycin when it is added upon the initiation of bacterial culture and the treated cultures were analyzed by two-dimensional gel electrophoresis. In this study we further examined the effect of clindamycin on the production of exoproteins by adding it to Streptococcus pyogenes cultures during various growth phases. We found that the levels of production of some proteins, NAD+ glycohydrolase, streptolysin O, and streptococcal inhibitor of complement, were increased when clindamycin was added at early-log-phase growth, which was the result that was seen when clindamycin was added at the beginning of culture. However, clindamycin inhibited the production of most types of proteins when it was administered to Streptococcus pyogenes cultures at mid-log-phase growth. In csrS- or mga-knockout bacterial strains, the increase in exoproteins seen in parental strains was considerably inhibited. Our study indicates that the in vitro effect of clindamycin on the production of exoproteins greatly depends on the growth phase of bacteria and some regulatory factors of Streptococcus pyogenes that are involved in this phenomenon.