We compared the in-vitro antimycobacterial activities of rifabutin and KRM-1648, two rifamycin derivatives, with that of rifampicin against 163 strains of Mycobacterium tuberculosis. We also evaluated the correlation between the level of resistance to rifampicin, rifabutin and KRM-1648 and genetic alterations in the rpoB gene. All 82 strains susceptible to rifampicin or resistant to rifampicin with MICs < or = 16 mg/L were susceptible to rifabutin and KRM-1648 with MICs < or = 1 mg/L. Seventy-six of 81 strains resistant to rifampicin with MICs > or = 32 mg/L were resistant to both rifabutin and KRM-1648, but with lower MICs than those of rifampicin. KRM-1648 showed more potent antimycobacterial activity than rifabutin against organisms with low MICs (< or = 1 mg/L), while rifabutin was more active than KRM-1648 against organisms with high MICs (> or = 2 mg/L). A total of 96 genetic alterations around the 69 bp core region of the rpoB gene were detected in 92 strains. Alterations at codons 515, 521 and 533 in the rpoB gene did not influence the susceptibility to rifampicin, rifabutin and KRM-1648. Point mutations at codons 516 and 529, deletion at codon 518 and insertion at codon 514 influenced the susceptibility to rifampicin but not that to rifabutin or KRM-1648. With the exception of one strain, all alterations at codon 513 and 531 correlated with resistance to the three test drugs. The resistant phenotype of strains with an alteration at codon 526 depended on the type of amino acid substitution. Our results suggest that analysis of genetic alterations in the rpoB gene might be useful not only for predicting rifampicin susceptibility, but also for deciding when to use rifabutin for treating tuberculosis. Further studies may be required to determine the usefulness of KRM-1648.

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ, a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.Oxygenic photosynthesis provides us with food, oxygen, and fuel and is therefore vital to life on the earth. The first reaction occurring in oxygenic photosynthesis is the splitting of water into electrons, protons, and molecular oxygen, among which electrons and protons are utilized for the synthesis of NADPH and ATP, whereas oxygen is supplied to the atmosphere for maintaining aerobic life forms. The water splitting reaction is catalyzed by photosystem II (PSII), 5 an extremely large membrane-protein complex located in thylakoid membranes from prokaryotic cyanobacteria to higher plants. In the case of cyanobacteria, the crystal structure of PSII has been solved with its resolution gradually increased to an atomic level of 1.9 Å (1-6), which showed that PSII contains 17 transmembrane subunits and 3 peripheral, hydrophilic subunits with a total molecular mass of 700 kDa for a dimer.The first oxygenic photosynthetic organism is believed to be the ancestor of cyanobacteria some 2.7 billion years ago (7). Although the subunit compositions of PSII from cyanobacteria to higher plants we see today are rather conserved, some apparent differences exist in both the transmembrane and peripheral subunits among cyanobacteria, various algae, and higher plants (8). One of the remarkable differences is found in the composition and function of extrinsic proteins associated in the lumenal side and required for maintaining the optimal function of the water-splitting reaction. In cyanobacteria, three extrinsic proteins of PsbO (33 kDa), Ps...

Coinfections of bacteria and influenza are a major cause of excessive mortality during influenza epidemics. However, the mechanism of the synergy between influenza virus and bacteria are poorly understood.In this study, mice were inoculated with influenza virus, followed 2 days later by inoculation with Streptococcus pneumoniae. The kinetics of viral titres, bacterial numbers and the immune response (cytokine and chemokine production) were also analysed.Short-term survival correlated with pathological changes in the lungs of infected mice. Influenza virus or S. pneumoniae infection alone induced moderate pneumonia; however, severe bronchopneumonia with massive haemorrhage in coinfected mice, which caused death of these mice y2 days after inoculation with S. pneumoniae, was noted. Intrapulmonary levels of inflammatory cytokines/chemokines, type-1 T-helper cell cytokines and Toll-like receptors, and the related mitogen-activated protein kinase signalling molecules (phosphorylated extracellular signal-regulated kinase -1 and -2, p38 and c-Jun N-terminal kinase), were increased in coinfected mice.These results suggest that immune mediators, including cytokines and chemokines, through Toll-like receptors/mitogen-activated protein kinase pathways, play important roles in the pathology of coinfection caused by influenza virus and Streptococcus pneumoniae.

We analyzed the relationship between rifampin MICs and rpoB mutations of 40 clinical isolates of Mycobacterium tuberculosis. A point mutation in either codon 516, 526, or 531 was found in 13 strains requiring MICs of > or = 64 micrograms/ml, while 21 strains requiring MICs of < or = 1 microgram/ml showed no alteration in these codons. However, 3 of these 21 strains contained a point mutation in either codon 515 or 533. Of the other six strains requiring MICs between 2 and 32 micrograms/ml, three contained a point mutation in codon 516 or 526, while no alteration was detected in the other three. Our results indicate that the sequencing analysis of a 69-bp fragment in the rpoB gene is useful in predicting rifampin-resistant phenotypes.

b This is the first report of a detailed relationship between triazole treatment history and triazole MICs for 154 Aspergillus fumigatus clinical isolates. The duration of itraconazole dosage increased as the itraconazole MIC increased, and a positive correlation was observed (r ‫؍‬ 0.5700, P < 0.0001). The number of itraconazole-naïve isolates dramatically decreased as the itraconazole MIC increased, particularly for MICs exceeding 2 g/ml (0.5 g/ml versus 2 g/ml, P ‫؍‬ 0.03). We also examined the relationship between cumulative itraconazole usage and the MICs of other azoles. A positive correlation existed between itraconazole dosage period and posaconazole MIC (r ‫؍‬ 0.5237, P < 0.0001). The number of itraconazole-naïve isolates also decreased as the posaconazole MIC increased, particularly for MICs exceeding 0.5 g/ml (0.25 g/ml versus 0.5 g/ml, P ‫؍‬ 0.004). Conversely, the correlation coefficient obtained from the scattergram of itraconazole usage and voriconazole MICs was small (r ‫؍‬ ؊0.2627, P ‫؍‬ 0.001). Susceptibility to three triazole agents did not change as the duration of voriconazole exposure changed. In addition, we carried out detailed analysis, including microsatellite genotyping, for isolates obtained from patients infected with azole-resistant A. fumigatus. We confirmed the presence of acquired resistance to itraconazole and posaconazole due to a G54 substitution in the cyp51A gene for a patient with chronic pulmonary aspergillosis after oral itraconazole therapy. We should consider the possible appearance of azole-resistant A. fumigatus if itraconazole is used for extended periods.

In a newly established murine model of chronic Pseudomonas aeruginosa respiratory infection mimicking diffuse panbronchiolitis (DPB), we investigated the effect of oral administration of clarithromycin on lymphocyte accumulation in the lung. Infection was produced by placement of a plastic tube precoated with P. aeruginosa in the bronchus. The number of bacteria on the tube was 6.25 +/- 0.22 log10 colony-forming units (cfu)/ml. Viable bacteria were constantly isolated at 10(5) to 10(6) cfu/specimen from the lungs for more than 1 yr. The histopathologic features resembled those of DPB consisting of massive accumulation of lymphocytes in the lung. The total number of pulmonary lymphocytes started to increase on Day 7, reaching a peak level within 12 d of intratracheal challenge. The number remained steady at that level for up to 120 d. There was also a steady fall in the CD4+/CD8+ ratio in the lungs, commencing on Day 7 and persisting to Day 120. A 10-d course of oral clarithromycin (10 mg/kg/d) from Day 7 resulted in a reduction of lymphocyte numbers to baseline level, although the dose did not influence the number of bacteria in the lungs. Treatment also increased the CD4+/CD8+ ratio to the baseline level from Day 7 to 17. Our results were similar to those detected in bronchoalveolar lavage fluid of patients with DPB and suggest that the therapeutic benefits of clarithromycin are due to its anti-inflammatory properties rather than antimicrobial effect.

It is crucial to consider the presence of UDs and meningoencephalitis for the choice of antifungals and treatment duration for cryptococcosis in non-HIV patients. Three- and six months-administration of azoles for pulmonary cryptococcosis with or without UDs, respectively is reasonable.

Diagnosing chronic pulmonary aspergillosis (CPA) is complicated, and there are limited data available regarding the identification of galactomannan (GM) in clinical specimens to assist the detection of this infection. The purpose of this study was to evaluate the detection of GM in bronchoalveolar lavage fluid (BALF) and serum and to assess its utility for diagnosing CPA. We retrospectively reviewed the diagnostic and clinical characteristics of 144 patients, with and without CPA, in Nagasaki University Hospital, Japan, whose BAL and serum specimens were examined for the presence of GM. The Platelia Aspergillus enzyme immunoassay (PA EIA) was performed according to the manufacturer's instructions. The mean values of BALF GM antigen were 4.535 (range, 0.062-14.120) and 0.430 (range, 0.062-9.285) in CPA (18) and non-CPA (126) patients, respectively. The mean values of serum GM antigen were 1.557 (range, 0.232-5.397) and 0.864 (range, 0.028-8.956) in CPA and non-CPA patients, respectively. PA EIA of BALF is superior to the test with serum, with the optimal cut-off values for BALF and serum of 0.4 and 0.7, respectively. The sensitivity and specificity of PA EIA in BALF at a cut-off of 0.4 were 77.2% and 77.0%, respectively, whereas with serum at a cut-off of 0.7, they were 66.7% and 63.5%, respectively. GM testing using BALF showed reasonable sensitivity and specificity as compared to that using serum. Thus, assessing GM levels in BALF may enhance the accuracy of diagnosing CPA.