IFN-gamma-induced iNOS mRNA expression is inhibited by rebamipide in murine macrophage RAW264.7 cells.

To investigate the effects of rebamipide, an antigastritis and anti-gastric ulcer drug, on inducible nitric oxide synthase (iNOS), murine macrophage RAW264.7 cells were treated with interferon-gamma (IFN-gamma) in the presence of rebamipide. NO production was stimulated by IFN-gamma, and the level was attenuated by rebamipide in a dose-dependent manner. Therefore, we investigated the possibility that either rebamipide directly inhibited iNOS enzyme activity or that it reduced iNOS mRNA expression. In a cell-free system, rebamipide did not affect iNOS enzyme activity; however, rebamipide inhibited iNOS mRNA and protein expression induced by IFN-gamma. Thus, we concluded that rebamipide inhibited IFN-gamma-induced NO production as a result of its inhibitory action on iNOS mRNA expression.

Delayed gastric emptying by Helicobacter pylori lipopolysaccharide in conscious rats.

The present study was carried out to investigate the possibility that lipopolysaccharide deprived from Helicobacter pylori may alter gastric motility. To address the question, we examined the effect of H. pylori lipopolysaccharide on gastric emptying in conscious rats. Gastric emptying was evaluated by the phenol red method. Time-course and dose-related effects of intraperitoneal administration of H. pylori lipopolysaccharide were investigated. Intraperitoneal injection of H. pylori lipopolysaccharide significantly suppressed gastric emptying of a liquid meal in a dose-dependent manner. The inhibitory action of H. pylori lipopolysaccharide was observed 2, 4, 8, or 12 hr after the injection. These results suggest for the first time that H. pylori lipopolysaccharide may suppress gastric emptying in a long-lasting fashion. It is also suggested that H. pylori may influence gastric function through its cell wall structure named lipopolysaccharide.

Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45.

Accumulating evidence suggests an important role of interleukin-8 (IL-8) in Helicobacter pylori infection-associated chronic atrophic gastritis and peptic ulcer. We observed in this study that a gastric cancer-derived cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H. pylori but not with killed H. pylori, H. pylori culture supernatants, or live H. pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria. Moreover, the tyrosine kinase inhibitor herbimycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 cells cocultured with live bacteria, suggesting the involvement of a tyrosine kinase(s) in H. pylori-induced IL-8 production. In addition, coculture of H. pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5'-flanking region of the IL-8 gene (bp -133 to +44), indicating that the induction of IL-8 production occurred at the transcriptional level. This region contain three cis elements important for induction of IL-8 gene expression: AP-1 (-126 to -120 bp), NF-IL6 (-94 to -81 bp), and NF-kappaB (-80 to -70 bp) binding sites. Mutation of the NF-kappaB binding site abrogated completely the induction of luciferase activity, whereas that of the AP-1 site partially reduced the induction. However, mutation of the NF-IL6 binding site resulted in no decrease in the induction of luciferase activity. Moreover, specific NF-kappaB complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H. pylori. Collectively, these results suggest that H. pylori induced the activation of NF-kappaB as well as AP-1, leading to IL-8 gene transcription.

Helicobacter pylori lipopolysaccharide inhibits acid secretion in pylorus-ligated conscious rats.

To examine the effect of Helicobacter pylori lipopolysaccharide on gastric secretion, the present study was carried out using pylorus ligated conscious rats. Intraperitoneal administration of Helicobacter pylori lipopolysaccharide significantly inhibited gastric acid secretion (4 hr) in a dose-dependent manner (0.033-1.0 mg/rat). The Helicobacter pylori lipopolysaccharide (1 mg/rat)-induced acid inhibition was still observed 8 hr after injection. Gastric acid secretion (4 hr) was compared in the rats that had received intraperitoneal administration of 1 mg/rat dose of Helicobacter pylori lipopolysaccharide or saline alone 24 hr before. There was no significant difference in gastric acid secretion between the saline- and H. pylori lipopolysaccharide-treated rats. These results suggest for the first time that H. pylori lipopolysaccharide may inhibit acid production, and this acid inhibition may be long-lasting. It is also demonstrated that this anti-secretory action of H. pylori lipopolysaccharide has a reversible effect on gastric secretion. All these results suggest that H. pylori lipopolysaccharide might be involved in the low acid secretory function seen in patients with acute H. pylori infection.

Evaluation of OPC-17116 against important pathogens that cause respiratory tract infections.

The antibacterial activity of OPC-17116, a new fluoroquinolone antibacterial agent, against important pathogens that cause respiratory tract infections was evaluated in vitro and in vivo and compared with those of ciprofloxacin, ofloxacin, and norfloxacin. The pharmacokinetic profiles of OPC-17116 were studied in both mice and rats given the drug orally at doses of 50 and 40 mg/kg of body weight, respectively. OPC-17116 showed a high degree of distribution in the lung tissues of both species, with maximum concentrations of 29.6 and 32.0 micrograms/g, respectively. Furthermore, the drug concentrations in lung tissue were about 10 to 15 times greater than the concentrations in plasma. OPC-17116 showed potent antibacterial activity against such pathogens as Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, and Moraxella catarrhalis. The MICs of this compound for 90% of these organisms except methicillin-resistant S. aureus and P. aeruginosa ranged from < or = 0.006 to 0.78 microgram/ml. The in vitro antibacterial activity of OPC-17116 was reflected by the efficacy of a single oral dose against systemic bacterial infections in mice. OPC-17116 showed a superior effect against gram-positive bacteria, H. influenzae, and M. catarrhalis. In comparison with the other reference compounds, the efficacy of OPC-17116 was less than that of ciprofloxacin against K. pneumoniae and P. aeruginosa. OPC-17116 showed a greater therapeutic effect than the other drugs against experimental acute pneumonia caused by these organisms in mice or rats. This excellent therapeutic effect against respiratory tract infections may be a result of its high level of distribution in lung tissue.

Comparative in vitro activities of a new quinolone, OPC-17116, possessing potent activity against gram-positive bacteria.

The in vitro antibacterial activity of OPC-17116, a new fluoroquinolone, against a wide variety of clinical isolates was evaluated and compared with those of ciprofloxacin, ofloxacin, and norfloxacin. OPC-17116 showed potent broad-spectrum activity against gram-positive and -negative bacteria. The activity of this compound against gram-positive bacteria was higher than those of other quinolones, and its activity against gram-negative and anaerobic bacteria was roughly comparable to those of other quinolones. OPC-17116 had potent activity against important pathogens of respiratory tract infections such as Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, and Branhamella catarrhalis. The MICs of this compound against 90% of these organisms, except for methicillin-resistant S. aureus, ranged from less than or equal to 0.006 to 3.13 micrograms/ml. OPC-17116 at more than one-half the MICs was bactericidal against clinical isolates of S. aureus, Escherichia coli, K. pneumoniae, and P. aeruginosa. The activity of OPC-17116 was decreased by several culture conditions such as acidic pH, high concentration of Mg2+ ions, and inoculum size of 10(7) CFU/ml. OPC-17116 inhibited the supercoiling activity of DNA gyrases from E. coli KL-16 and S. aureus SA113 (50% inhibitory concentrations, 0.19 and 23.0 micrograms/ml, respectively). The amount of OPC-17116 accumulation was higher than that of other quinolones in S. aureus.

Mechanisms of clinical resistance to fluoroquinolones in Staphylococcus aureus.

Mechanisms of Staphylococcus aureus resistance to fluoroquinolones were characterized. Subunit A and B proteins of DNA gyrase were partially purified from fluoroquinolone-susceptible strain SA113 and resistant isolate MS16405, which was 250- to 1,000-fold less susceptible to fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, temafloxacin, and sparfloxacin than SA113 was. The supercoiling activity of the gyrase from SA113 was inhibited by the fluoroquinolones, and the 50% inhibitory concentrations of the drugs correlated well with their MICs. In contrast, the gyrase from MS16405 was insensitive to inhibition of supercoiling by all of the quinolones tested, even at 800 micrograms/ml. Combinations of heterologous gyrase subunits showed that subunit A from MS16405 conferred fluoroquinolone resistance, suggesting that an alteration in gyrase subunit A is a cause of the fluoroquinolone resistance in MS16405. Uptake of hydrophilic fluoroquinolones such as ciprofloxacin and norfloxacin by MS16405 was significantly lower than that by SA113. Furthermore, this difference was abolished by the addition of an energy inhibitor, carbonyl cyanide m-chlorophenylhydrazone, suggesting that an alteration in an energy-dependent process, such as an active efflux of hydrophilic quinolones, may lead to decreased drug uptake and hence to increased resistance to fluoroquinolones in MS16405. These findings suggest that the fluoroquinolone resistance in MS16405 is due mainly to an alteration in subunit A of DNA gyrase and may also be associated with an alteration in the drug uptake process.

Mechanisms of clinical resistance to fluoroquinolones in Enterococcus faecalis.

About 10% of 100 clinical isolates of Enterococcus faecalis were resistant to greater than or equal to 25 micrograms of norfloxacin, ofloxacin, ciprofloxacin, and temafloxacin per ml. In this study, the DNA gyrase of E. faecalis was purified from a fluoroquinolone-susceptible strain (ATCC 19433) and two resistant isolates, MS16968 and MS16996. Strains MS16968 and MS16996 were 64- to 128-fold and 16- to 32-fold less susceptible, respectively, to fluoroquinolones than was ATCC 19433; MICs of nonquinolone antibacterial agents for these strains were almost equal. The DNA gyrase from ATCC 19433 had two subunits, designated A and B, with properties similar to those of DNA gyrase from other gram-positive bacteria such as Bacillus subtilis and Micrococcus luteus. Inhibition of the supercoiling activity of the enzyme from ATCC 19433 by the fluoroquinolones correlated with their antibacterial activities. In contrast, preparations of DNA gyrase from MS16968 and MS16996 were at least 30-fold less sensitive to inhibition of supercoiling by the fluoroquinolones than the gyrase from ATCC 19433 was. Experiments that combined heterologous gyrase subunits showed that the A subunit from either of the resistant isolates conferred resistance to fluoroquinolones. These findings indicate that an alteration in the gyrase A subunit is the major contributor to fluoroquinolone resistance in E. faecalis clinical isolates. A difference in drug uptake may also contribute to the level of fluoroquinolone resistance in these isolates.

Ultrastructural aspects of fragility of Pseudomonas aeruginosa outer membrane devoid of protein F.

The protein F-deficient cells of Pseudomonas aeruginosa were previously found to be more susceptible to osmotic shock than the sufficient cells (Gotoh et al., J. Bacteriol., in press). The protein F-deficient cells were observed by the thin-section method of electron microscopy to determine the effects of osmotic shock. The osmotic shock induced breakage of the protein F-deficient outer membrane, while it had no effect on the protein F-sufficient outer membrane. These results suggested that the cells lost their viability by the osmotic shock caused by fragility of the outer membrane.

Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane.

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane. Determination of the diffusion rates of saccharides and beta-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.