Biomimetic flavin-catalysed reactions for organic synthesis.

Using simple riboflavin related compounds as biomimetic catalysts, catalytic oxidation of various substrates with hydrogen peroxide or molecular oxygen can be performed selectively under mild conditions. The principle of these reactions is fundamental and will provide a wide scope for environmentally benign future practical methods.

Factors associated with acute otitis media in children due to penicillin intermediately resistant Streptococcus pneumoniae.

OBJECTIVES: This study was conducted to analyze the association between recent antimicrobial use and acute otitis media (AOM) due to Streptococcus pneumoniae intermediately resistant to penicillin (PISP). The influence of drug resistance on the clinical course of AOM was also assessed. METHODS: A retrospective cohort study was conducted in infants at Japanese Red Cross Medical Center, Tokyo. Children included in the study were under 24 months of age and diagnosed with AOM due to infection with S. pneumoniae between April 2002 and March 2007. Crude risk ratios (cRR) of PISP infection in cases with recent antibiotic use and other factors were obtained. The Mantel-Haenszel estimate was applied for the adjustment of cRR. RESULTS: Of 35 children, 13 had AOM due to penicillin-susceptible S. pneumoniae (PSSP) and 22 had AOM due to PISP. The adjusted risk ratio (aRR) of penicillin antibiotic use within 1 month was 1.99 (95% confidence interval (CI): 1.36-2.92), and the aRR of penicillin antibiotic use within 1 week was 1.93 (95% CI: 1.33-2.67). Recent use of penicillin antibiotics was an associated factor for AOM due to PISP. The clinical course was not clearly different between cases infected with PSSP and those with PISP. CONCLUSION: Recent use of penicillin antibiotics might be a selective pressure for PISP.

TAK1 represses transcription of the human telomerase reverse transcriptase gene.

In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, namely, the human telomerase reverse transcriptase (hTERT). However, the molecular mechanisms involved in the regulation of hTERT expression have not been completely clarified. We have previously reported that transforming growth factor beta (TGF-beta) represses the expression of the hTERT gene. In the present study, we demonstrated that TGF-beta-activated kinase 1 (TAK1), originally identified as a mitogen-activated kinase kinase kinase, represses the hTERT core promoter activity in an E-box-independent manner, and it also represses the transcription of the hTERT gene in human lung adenocarcinoma cell line, A549 cells. This TAK1-induced repression was found to be caused by the recruitment of histone deacetylase to Sp1 at the hTERT promoter and a consequent reduction in the amount of acetylated histone H4 at the hTERT promoter. Finally, we demonstrated that TAK1 induces cellular senescence programs in normal human diploid cells. Thus, we assume that TAK1 triggers the repression mechanisms of the hTERT gene as a result of evoking cellular senescence programs. Considered together, TAK1 is thought to play a causative role in the determination of a finite replicative lifespan of normal and cancer cells.

Mycoplasma bovis-associated suppurative otitis media and pneumonia in bull calves.

Outbreaks of Mycoplasma bovis-associated otitis media and pneumonia occurred on four beef cattle farms in Hokkaido, Japan between 2000 and 2001. The morbidity and mortality were estimated at 8-40 and 30-100%, respectively. Eight calves with bilateral ear droop and exudative otitis media were examined bacteriologically and histopathologically. M. bovis was isolated post mortem from nasal swabs and from the ears, lungs, lymph nodes (cranial and pulmonary), brain and heart of all calves. At necropsy, suppurative exudates were observed in the tympanic bullae of all cases. Numerous abscesses were also found in the petrous portion of the temporal bone and lungs in seven cases. Histopathologically, the exudates within the tympanic bullae consisted of a mixture of neutrophils, necrotic cell debris and fibrin, and the tympanic mucosa was thickened with neutrophil and macrophage infiltration and proliferation of fibrous connective tissue. Pulmonary lesions included extensive foci of coagulative necrosis surrounded by numerous neutrophils. Hepatocytes or renal tubular epithelial cells were enlarged with hyaline cytoplasmic inclusions in four calves. Immunohistochemical labelling confirmed the presence of M. bovis antigen in the cytoplasm of the inflammatory cells in the middle ear, temporal bone and lungs, and was also demonstrated within the cytoplasmic inclusions of the hepatocytes and renal tubular epithelial cells. Ultrastructurally, mycoplasma-like organisms, 200-500 microm in diameter, were found within not only hepatocytes and renal tubular epithelia but also within axons of the facial nerves. The present results show that M. bovis spreads to multiple organs and is capable of invading various kinds of host cell. The intracellular localization may be favourable for evading host immune responses.

Mucosal immunoadjuvant activity of the low toxic recombinant Escherichia coli heat-labile enterotoxin produced by Bacillus brevis for the bacterial subunit or component vaccine in pigs and cattle.

A gene encoding the mature Escherichia coli heat-labile enterotoxin (LT) lacking the nick site in the A subunit by deleting tripeptides was introduced in a vector pNH301 and expressed extracellularly as mutant molecule of holotoxin at high levels in Bacilus brevis HPD31-S5 of the host bacterium. The mucosal adjuvant activities of the produced mutant LT (mLT) preparation were studied in pigs and cattle. Intranasal immunization of pigs with the recombinant subunit vaccine of Erysipelothrix rhusiopathiae or the component vaccine of Bordetella bronchiseptica mixed with the mLT resulted in a substantial enhancement of both mucosal and serum-specific antibody levels. The immunized pigs were also protected when challenge-exposed intradermally with a highly virulent E. rhusiopathiae strain or challenge-exposed intranasally with a highly virulent strain of B. bronchiseptica. The mLT intranasally administered with recombinant intimin (an outer membrane adhesin) of E. coli O157:H7 also induced an elevation of IgA-specific antibody in the nasal secretion and saliva of calves as well as an elevation of IgG1-specific antibody level against the intimin in the sera and colostrum of cows. The three kinds tested protein antigens were poorly immunogenic when antigen administered intranasally alone. The mLT intranasally administered at a higher effective dose did not induce local adverse reactions or diarrhea in pigs and cattle. The present study demonstrates that the recombinant mLT produced using the B. brevis expression system might represent promising immunoadjuvants for the potential application of intranasal vaccines directed against infectious diseases in pigs and cattle.

Truncated surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae serotype 1a elicits protection against challenge with serotypes 1a and 2b in pigs.

Erysipelothrix rhusiopathiae is a causal agent of swine erysipelas, which is of economic importance in the swine industry by virtue of causing acute septicemia, chronic arthritis, and endocarditis. However, little is known about the genetic properties of its protective antigens. Recently, a surface protective antigen (SpaA) gene was identified from serotype 2 in a mouse model. We cloned spaA from virulent strain Fujisawa (serotype 1a) and determined that the N-terminal 342 amino acids without C-terminal repeats of 20 amino acids have the ability to elicit protection in mice. Fusions of 342 amino acids of Fujisawa SpaA and histidine hexamer (HisSpa1.0) protected pigs against challenge with both serotype 1 and serotype 2, the most important serotypes in the swine industry. Pigs immunized with HisSpa1.0 reacted well with both HisSpa1.0 and intact SpaA by enzyme-linked immunosorbent assay and immunoblotting. Serum collected at the time of challenge from a pig immunized with HisSpa1. 0 markedly enhanced the in vitro phagocytic and killing activity of pig neutrophils against the bacteria. DNA sequences of protective regions of spaA genes from five strains of serotypes 1 and 2 were almost identical. The full DNA sequences also seemed to be conserved among strains of all 12 serotype reference strains harboring the spaA gene by restriction fragment length polymorphism analysis of PCR products. These results indicates that SpaA is a common protective antigen of serotypes 1 and 2 of E. rhusiopathiae in swine and will be a useful tool for development of new types of vaccines and diagnostic tools for effective control of the disease.

Serologic evidence that streptococcal superantigens are not involved in the pathogenesis of Kawasaki disease.

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology and is associated with marked activation of T cells and monocyte macrophages, leading to the assumption that superantigens are involved in its pathogenesis. To determine if an association exists between streptococcal superantigens and KD, we examined serum antibody responses to superantigens in sera from 50 paired acute and convalescent KD patients using purified recombinant streptococcal superantigens, such as SPEA, SPEC, SSA and MF. We found a very low frequency of detection of anti-superantigen antibodies by ELISA and no marked IgG seroconversion to each superantigen, indicating the absence of a serological relationship between toxin-producing streptococcal infection and the onset of KD.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody against Streptococcus suis type 2 in infected pigs.

An ELISA test for the detection of antibody against S. suis type 2 in pigs was developed and applied to field sera. The best sensitivity and specificity of ELISA were obtained when a purified polysaccharide antigen was used. It showed no cross reaction with sera against other serotypes of S. suis and other pathogenic bacteria. A total of 264 sera were collected from 20 pig farms and examined with the antibody against S. suis type 2. In the affected farms, 17.0% of pigs tested were positive, 9.8% in the adjacent farms, but only 3.4% in the free farms. The difference of the positive percentages between the affected and the free farms was statistically significant (P < 0.05).

Recombinant 46-kilodalton surface antigen (P46) of Mycoplasma hyopneumoniae expressed in Escherichia coli can be used for early specific diagnosis of mycoplasmal pneumonia of swine by enzyme-linked immunosorbent assay.

The 46-kDa surface antigen (P46) is the early and species-specific immunogenic protein of Mycoplasma hyopneumoniae. Three TGA codons encoding tryptophan in the P46 gene were replaced with TGG by an in vitro mutagenesis technique. The mutated P46 gene was expressed in Escherichia coli by using the chelating peptide tag system. The purified recombinant P46 was successfully used in an enzyme-linked immunosorbent assay for detection of antibodies against M. hyopneumoniae in swine serum. It did not cross-react with sera from swine infected with Mycoplasma flocculate, Mycoplasma hyorhinis, or Mycoplasma hyosynoviae. With this method, mycoplasmal pneumonia of swine was detectable within 2 weeks after infection.

Biologically active extracellular products of oral viridans streptococci and the aetiology of Kawasaki disease.

A bacteriological study of isolates from the oral cavity of patients with Kawasaki disease (KD), age-matched non-KD patients and healthy children, showed that over half the KD and control isolates had gram-positive, catalase-negative cocci. About 50% of these organisms were identified as viridans streptococci by means of an API Strep 20 kit. Further identification by fluorometric DNA-DNA hybridisation demonstrated that the predominant species were S. oralis and S. mitis, each of which accounted for 25% of the isolates of viridans streptococci; 40% of viridans strains were unidentifiable; and S. sanguis and S. parasanguis were minor components. Studies in vivo showed that insertion of culture supernates of most of the viridans streptococci increased capillary permeability and induced redness with swelling and occasional bleeding in rabbit skin. One-third of S. mitis strains and one-fifth of the unidentified strains caused aggregation of human blood platelets, whereas S. oralis and other strains had no such effect. The distribution of extracellular lipoteichoic acids and glucan produced in the presence of sucrose was also examined. There were no significant differences in the recovery rate of viridans streptococci forming these biologically active extracellular products between KD and control groups.

Ureaplasma canigenitalium sp. nov., isolated from dogs.

Ureaplasma strains isolated from dogs (Canis familiaris) were characterized and compared with the type strains of five previously described species of the genus Ureaplasma, Ureaplasma urealyticum (isolated from humans), Ureaplasma diversum (isolated from cattle), Ureaplasma gallorale (isolated from chickens), Ureaplasma cati (isolated from cats), and Ureaplasma felinum (isolated from cats). The canine strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked a cell wall, passed through 450-nm-pore-size membrane filters, required cholesterol for growth, and formed minute colonies (diameter, 20 to 140 microns) on agar medium. These canine ureaplasma strains have been reported to be members of four serovars. The four serovars of canine strains fell into a single group on the basis of their genomic properties, as determined by DNA-DNA hybridization. On the basis of these findings, we propose that ureaplasmas with these characteristics belong to a new species, Ureaplasma canigenitalium, with strain D6P-C (= ATCC 51252) as the type strain.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analyses of ureaplasmas isolated from dogs.

To define the antigenic relatedness among unspeciated ureaplasma strains isolated from dogs, four canine ureaplasma isolates representing four serotypes were compared with the five type strains of the established species in Genus Ureaplasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Although all the strains showed distinct electrophoretic patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the Western blotting patterns were much more distinct. By Western blotting, the five type strains of established species reacted strongly with homologous antisera and showed slight cross reactions with heterologous antisera. However canine strains which showed little cross reactions with the established Ureaplasma species showed a variety of cross reactions among the four canine serotypes used.

Cardiac sequelae of Kawasaki disease in Japan: statistical analysis.

The proportions of Kawasaki disease patients with cardiac sequelae in Japan were analyzed using nationwide survey data from the 6 1/2-year period July 1982 through December 1988. Of 46,864 cases of Kawasaki disease reported in the surveys, 7637 or 16.3% had cardiac sequelae such as dilation or stenosis of coronary arteries, myocardial infarction, and valvar lesions 1 month or more after onset. The prevalence of cardiac sequelae was particularly high in males, infants younger than 1 year, and children older than 5 years of age. In sequential observation, there was no correlation between the prevalence of cardiac sequelae and periods of high or low incidence of the disease. The prevalence of cardiac sequelae overall declined steadily over the observation period, perhaps as a consequence of increasing use of intravenous gamma globulin. However, children older than the age of 5 years manifested increasing prevalence of cardiac sequelae over the observation period, probably as a result of lower rates of intravenous gamma globulin administration.

Ureaplasma felinum sp. nov. and Ureaplasma cati sp. nov. isolated from the oral cavities of cats.

Seven ureaplasma strains isolated from the oral cavities of domestic cats (Felis domestica) were characterized and compared with the type strains of the three previously established species of this genus, Ureaplasma urealyticum (humans), Ureaplasma diversum (cattle), and Ureaplasma gallorale (chickens). The feline strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked cell walls, passed through 0.45-micron membrane filters, required cholesterol for growth, and formed minute (15- to 140-microns) colonies on agar medium. The seven feline strains fell into two distinct groups based on (i) their antigenic properties (determined by using the metabolism and growth inhibition and indirect immunoperoxidase procedures), (ii) their genomic properties (determined by using DNA-DNA hybridization and DNA cleavage pattern procedures), and (iii) their polypeptide profiles (determined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses). Based on these properties, the two feline groups were unrelated to each other or to the three previously established species, and each group represents a distinct Ureaplasma species. Thus, we propose that ureaplasmas with these phylogenetic and genomic properties be given taxonomic status as Ureaplasma felinum and Ureaplasma cati, with strain FT2-B (= ATCC 49229 = NCTC 11709) and strain F2 (= ATCC 49228 = NCTC 11710) as the type strains, respectively.

Steroid hormone production in testis, ovary, and adrenal gland of immature rats irradiated in utero with 60Co.

Pregnant rats received whole-body irradiation at 20 days of gestation with 2.6 Gy lambda rays from a 60Co source. Endocrinological effects before maturation were studied using testes and adrenal glands obtained from male offspring and ovaries from female offspring irradiated in utero. Seminiferous tubules of the irradiated male offspring were remarkably atrophied with free germinal epithelium and containing only Sertoli cells. Female offspring also had atrophied ovaries. Testicular tissue obtained from intact and 60Co-irradiated rats was incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione as a substrate. Intermediates for androgen production and catabolic metabolites were isolated after the incubation. The amounts of these metabolites produced by the irradiated testes were low in comparison with the control. The activities of delta 5-3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17,20-lyase, and delta 4-5 alpha-reductase in the irradiated testes were 30-40% of those in nonirradiated testes. Also, the activities of 17 beta- and 20 alpha-hydroxysteroid dehydrogenases were 72 and 52% of the control, respectively. In adrenal glands, the 21-hydroxylase activity of the irradiated animals was 38% of the control, but the delta 5-3 beta-hydroxysteroid dehydrogenase activity was comparable to that of the control. On the other hand, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase of the irradiated ovary was only 19% of the control. These results suggest that 60Co irradiation of the fetus in utero markedly affects the production of steroid hormones in testes, ovaries, and adrenal glands after birth.

A nationwide incidence survey of Kawasaki disease in 1985-1986 in Japan.

Nationwide epidemiological surveys of Kawasaki disease have been conducted nine times in Japan since 1970. By the end of 1986, 83,857 (male:female ratio, 1.4) cases were reported. We summarize the results of these surveys, especially the latest survey of cases from January 1985 to December 1986. There were three epidemic years - 1979, 1982, and 1986. The ratios of the number of patients diagnosed in each of those years to the number in the preceding year were 2.0, 2.4, and 1.7, respectively. The last epidemic started in a metropolitan area of Tokyo in December 1985 and propagated northwards and southwards to involve almost all of the country in six months. The age-specific incidence curve showed a unimodal peak at nine to 11 months of age. The proportion of sibling cases was approximately 2%. The epidemiological pictures suggested that the disease was caused by an unknown biologic agent that is common in the community and that spreads easily among very young children.