Biological evaluation of Phellinus linteus-fermented broths as anti-inflammatory agents.

Phellinus linteus and its constituent hispolon induce potent anti-inflammatory activity in macrophages. Efficient production of the effective constituent and the biological function of P. linteus in the regulation of innate sensing have rarely been investigated. The aim of this study was to efficiently manufacture P. linteus-fermented broth containing the effective constituent, hispolon, and evaluate its immunoregulatory functions in macrophages. Four distinct fermented broths (PL1-4) and the medium dialyzate (MD) were prepared to screen suitable culture conditions for the mycelial growth of P. linteus. The P. linteus-fermented broth exhibited a dose-responsive inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production by murine macrophages. In addition, the P. linteus-fermented broths suppressed macrophage LPS-mediated nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α. Among the tested samples from P. linteus, PL4 contained vast amounts of hispolon and showed the greatest anti-inflammatory activity in both the RAW264.7 cells and murine primary peritoneal exudate macrophages (PEMs). This study demonstrates that the purification of the effective constituent from P. linteus-fermented broth may enable the production of a potent therapeutic agent for anti-inflammation in macrophages.

Helicobacter pylori CagA-mediated IL-8 induction in gastric epithelial cells is cholesterol-dependent and requires the C-terminal tyrosine phosphorylation-containing domain.

Upon infection of the gastric epithelial cells, the Helicobacter pylori cytotoxin-associated gene A (CagA) virulence protein is injected into the epithelial cells via the type IV secretion system (TFSS), which is dependent on cholesterol. Translocated CagA is targeted by the membrane-recruited c-Src family kinases in which a tyrosine residue in the Glu-Pro-Ile-Tyr-Ala (EPIYA)-repeat region, which can be phosphorylated, induces cellular responses, including interleukin-8 (IL-8) secretion and hummingbird phenotype formation. In this study, we explored the role of EPIYA-containing C-terminal domain (CTD) in CagA tethering to the membrane lipid rafts and in IL-8 activity. We found that disruption of the lipid rafts reduced the level of CagA translocation/phosphorylation as well as CagA-mediated IL-8 secretion. By CagA truncated mutagenesis, we identified that the CTD, rather than the N-terminal domain, was responsible for CagA tethering to the plasma membrane and association with detergent-resistant membranes, leading to CagA-induced IL-8 promoter activity. Our results suggest that CagA CTD-containing EPIYAs directly interact with cholesterol-rich microdomains that induce efficient IL-8 secretion in the epithelial cells.

Cholesterol depletion reduces entry of Campylobacter jejuni cytolethal distending toxin and attenuates intoxication of host cells.

Campylobacter jejuni is a common cause of pediatric diarrhea worldwide. Cytolethal distending toxin, produced by Campylobacter jejuni, is a putative virulence factor that induces cell cycle arrest and apoptosis in eukaryotic cells. Cellular cholesterol, a major component of lipid rafts, has a pivotal role in regulating signaling transduction and protein trafficking as well as pathogen internalization. In this study, we demonstrated that cell intoxication by Campylobacter jejuni cytolethal distending toxin is through the association of cytolethal distending toxin subunits and membrane cholesterol-rich microdomains. Cytolethal distending toxin subunits cofractionated with detergent-resistant membranes, while the distribution reduced upon the depletion of cholesterol, suggesting that cytolethal distending toxin subunits are associated with lipid rafts. The disruption of cholesterol using methyl-ß-cyclodextrin not only reduced the binding activity of cytolethal distending toxin subunits on the cell membrane but also impaired their delivery and attenuated toxin-induced cell cycle arrest. Accordingly, cell intoxication by cytolethal distending toxin was restored by cholesterol replenishment. These findings suggest that membrane cholesterol plays a critical role in the Campylobacter jejuni cytolethal distending toxin-induced pathogenesis of host cells.

Visual experience-independent functional expression of NMDA receptors in the developing rabbit retina.

PURPOSE: Activation of the NMDA glutamate receptors is critical for the initiation of synaptic plasticity. In the developing rat retina, NMDA receptor function has been associated with visual experience, though the light-dependent regulation of the subunit composition of the NMDA receptors is controversial. In the present study, the functional expression of NMDA receptors in the developing rabbit retina was characterized and the impact of light deprivation on how the subunit composition of NMDA receptors is regulated was examined. METHODS: Antibodies against NR1 and NR2A/B were used to examine neonatal expression patterns of the NMDA receptor subunits. Furthermore, the functional NMDA receptors were mapped using the agmatine (AGB) activation assay. RESULTS: Although NR1 and NR2A/B subunit immunoreactivity was prominently detectable only immediately after birth, AGB activation assay showed that functional NMDA receptors could be identified as early as embryonic day 21. No significant difference was observed between normal- and dark-reared animals in terms of their NR1 and NR2A/B expression. In addition, a comparison of AGB permeation between normal- and dark-reared animals showed no difference in functional expression of NMDA receptors. CONCLUSIONS: These results indicate that NMDA receptors participate in the synaptic maturation of retinal circuits during the early stages of development but that the functional NMDA receptors, including their subunit composition, in the developing rabbit retina are independent of the rabbit's visual experience.

Prevalence of antimicrobial resistance in Helicobacter pylori isolates in Taiwan in relation to consumption of antimicrobial agents.

During 1998-2004, a total of 218 Helicobacter pylori isolates were obtained from patients who were randomised to receive one of the following regimens in a medical centre in Taiwan: lansoprazole, amoxicillin and clarithromycin (LAC) therapy; or lansoprazole, metronidazole and clarithromycin (LMC) therapy. In the LMC group, resistance rates for metronidazole and clarithromycin reduced from 48.6% (1998-2000) to 20.4% (2001-2004) (P<0.05) and from 13.5% to 6.3% (P<0.05), respectively. Analysis of annual antimicrobial consumption found that metronidazole use was slowly decreased both in the total population and in gastrointestinal disease patients. The per-protocol analysis revealed a higher eradication rate for patients using LMC therapy in 2001-2004 (82.6% vs. 75.0%), whilst there was similar efficacy for LAC therapy (84.8% vs. 84.2%). This observation suggests an effective programme to control H. pylori antibiotic resistance and hence elevate its cure rate.

Nosocomial outbreak of infection with multidrug-resistant Acinetobacter baumannii in a medical center in Taiwan.

OBJECTIVE: To investigate a nosocomial outbreak of infection with multidrug-resistant (MDR) Acinetobacter baumannii in the intensive care units at China Medical University Hospital in Taiwan. DESIGN: Prospective outbreak investigation. SETTING: Three intensive care units in a 2,000-bed university hospital in Taichung, Taiwan. METHODS: Thirty-eight stable patients in 3 intensive care units, all of whom had undergone an invasive procedure, were enrolled in our study. Ninety-four A. baumannii strains were isolated from the patients or the environment in the 3 intensive care units, during the period from January 1 through December 31, 2006. We characterized A. baumannii isolates by use of repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) and random amplified polymorphic DNA (RAPD) fingerprinting. The clinical characteristics of the source patients and the environment were noted. RESULTS: All of the clinical isolates were determined to belong to the same epidemic strain of MDR A. baumannii by the use of antimicrobial susceptibility tests, REP-PCR, and RAPD fingerprinting. All patients involved in the infection outbreak had undergone an invasive procedure. The outbreak strain was also isolated from the environment and the equipment in the intensive care units. Moreover, an environmental survey of one of the intensive care units found that both the patients and the environment harbored the same outbreak strain. CONCLUSION: The outbreak strain of A. baumannii might have been transmitted among medical staff and administration equipment. Routine and aggressive environmental and equipment disinfection is essential for preventing recurrent outbreaks of nosocomial infection with MDR A. baumannii.

Localization and functional mapping of AMPA receptor subunits in the developing rabbit retina.

PURPOSE: Glutamate has been suggested to regulate the development of retinal neurons, but ontogenic expression of ionotropic glutamate receptors has only recently been characterized in the rat retina. The purpose of this study was to examine the expression patterns of AMPA receptors and to functionally map glutamatergic drive in the developing rabbit retina. METHODS: The retinas from New Zealand White rabbits of different developmental stages (embryonic days [E]21 and 26, postnatal days [P]0-10, and adult) were isolated and cryosectioned into vertical slices. Antibodies against GluR1, -R2/3, and -R4 were used to examine the postnatal expression patterns of the AMPA receptor subunits. To further map the glutamatergic drive in the developing rabbit retina, an agmatine (AGB)-activation assay was also used. RESULTS: All AMPA receptor subunits, including GluR1, -R2/3, and -R4, were expressed in the inner plexiform layer as early as E26 and were convincingly labeled in the outer plexiform layer at P2. These AMPA subunits showed different spatial distribution and temporal expression patterns across the postnatal stages examined. The immunoreactivity of the AMPA subunits was weak at P0 to P2 and then showed a striking increase at P4 to P6. The AGB activation assay revealed that some amacrine and ganglion cells were activated with 2 microM AMPA as early as E26 and, in the presence of an increased concentration of AMPA (20 microM), some potential horizontal cells were activated at the same stage. CONCLUSIONS: AMPA glutamate receptors express and function during the early stages of the developing rabbit retina, indicating that AMPA receptors are functional before synapse formation. The period of increasing expression pattern of AMPA subunits also coincides with the switch of the glutamatergic drive of the retinal wave and thus may contribute to the synaptic maturation in the retinal circuits.

Cholesterol depletion reduces Helicobacter pylori CagA translocation and CagA-induced responses in AGS cells.

Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-beta-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.

18beta-glycyrrhetinic acid promotes src interaction with connexin43 in rat cardiomyocytes.

The mechanism by which 18beta-glycyrrhetinic acid regulates gap junction intercellular communication (GJIC) remains poorly understood. In this study, treatment of cultured rat neonatal cardiomyocytes with 18beta-glycyrrhetinic acid resulted in dose-dependent inhibition of GJIC as assessed by fluorescent dye transfer analysis. 18beta-Glycyrrhetinic acid induced time-dependent serine/threonine dephosphorylation and redistribution of connexin43 (Cx43) in cardiomyocytes and the induced Cx43 dephosphorylation was prevented by the protein phosphatase inhibitor, calyculin A. However, functional analyses showed that the inhibitory effect of 18beta-glycyrrhetinic acid on dye spreading among cardiomyocytes was not blocked by calyculin A, but was blocked by the Src-selective tyrosine kinase inhibitor, PP2. 18beta-Glycyrrhetinic acid also induced an increase in the levels of phosphorylated Src, and this effect was prevented by PP2. Immunoprecipitation using anti-Cx43 and anti-p-Src antibodies showed that 18beta-glycyrrhetinic acid increased the association between p-Src and Cx43 and induced tyrosine phosphorylation of Cx43. We conclude that the inhibitory effect of 18beta-glycyrrhetinic acid on GJIC in cardiomyocytes involves Src-mediated tyrosine phosphorylation of Cx43.