Enhanced Bacterial Growth and Gene Expression of D-Amino Acid Dehydrogenase With D-Glutamate as the Sole Carbon Source

In a search for life-supporting, not life-assisting, D-amino acid metabolism, an environmental strain that grows better with D-glutamate as the sole carbon source was isolated from an ordinary river. The strain, designated as A25, exhibited a faster growth rate and greater cell yield with D-glutamate than with L-glutamate. Conversely, the D/L ratio of total cellular glutamate was as low as 4/96, which suggests that D-glutamate is more likely catabolized than anabolized. Strain A25 was phylogenetically most closely related to the gamma-proteobacterial species Raoultella ornithinolytica, with a 16S rRNA gene sequence similarity of 100%. A standard strain, R. ornithinolytica JCM 6096T, also showed similarly enhanced growth with D-glutamate, which was proven for the first time. Gene expression of the enzymes involved in D-amino acid metabolism was assayed by reverse-transcription quantitative PCR (RT-qPCR) using specifically designed primers. The targets were the genes encoding D-amino acid dehydrogenase (DAD; EC 1.4.99.1), glutamate racemase (EC 5.1.1.3), D-glutamate oxidase (EC 1.4.3.7 or EC 1.4.3.15), and UDP-N-acetyl-α-D-muramoyl-L-alanyl-D-glutamate ligase (EC 6.3.2.9). As a result, the growth of strains A25 and R. ornithinolytica JCM 6096T on D-glutamate was conspicuously associated with the enhanced expression of the DAD gene (dadA) in the exponential phase compared with the other enzyme genes. Pseudomonas aeruginosa is also known to grow on D-glutamate as the sole carbon source but to a lesser degree than with L-glutamate. A standard strain of P. aeruginosa, JCM 5962T, was tested for gene expression of the relevant enzymes by RT-qPCR and also showed enhanced dadA expression, but in the stationary phase. Reduction of ferricyanide with D-glutamate was detected in cell extracts of the tested strains, implying probable involvement of DAD in the D-glutamate catabolizing activity. DAD-mediated catalysis may have advantages in the one-step production of α-keto acids and non-production of H2O2 over other enzymes such as racemase and D-amino acid oxidase. The physiological and biochemical importance of DAD in D-amino acid metabolism is discussed

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam.

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic

Cardioprotective effects of chloroquine pretreatment on ischemic and reperfusion injury via activation of ERK1/2 in isolated rat hearts

Purpose Several therapeutic agents have been found to prevent myocardial ischemic and reperfusion (I/R) injury after cardiac surgery; however, no drug is routinely used to afford cardioprotective benefits in clinical settings. Herein, we aimed to determine whether chloroquine (CQ) pretreatment attenuates I/R injury after global ischemia in isolated rat hearts and elucidate mechanisms underlying the effects of CQ. Methods Isolated rat hearts were subjected to 30-min global ischemia, followed by 60-min reperfusion with Krebs-Henseleit buffer (KHB). Immediately before ischemia, 10 mL of pretreatment solutions (KHB, n = 4 or KHB + CQ [100 mu M], n = 4) were injected through the aortic root. Cardiac function was examined based on the rate pressure product (RPP). Myocardial apoptosis was evaluated using TUNEL staining. To assess the reperfusion ischemia salvage kinase pathway, protein expression levels of AKT and extracellular signal-regulated kinase (ERK1/2) were determined using western blotting. To investigate the role of ERK1/2, an ERK1/2 selective inhibitor was used in eight additional rats. Results The recovery rate of the RPP was higher in the KHB + CQ group than in the KHB group 60 min after I/R (KHB, 44 +/- 3% vs. KHB + CQ, 69 +/- 7%; P = 0.019, d = 2.2). CQ pretreatment reduced apoptosis and enhanced the phosphorylation of ERK1/2; however, AKT phosphorylation was unaltered. In addition, the ERK1/2 inhibitor abolished CQ-mediated cardioprotective effects. Conclusions CQ pretreatment showed protective effects on cardiac function after I/R by activating ERK1/2

Cardioprotective effects of chloroquine pretreatment on ischemic and reperfusion injury via activation of ERK1/2 in isolated rat hearts

Purpose Several therapeutic agents have been found to prevent myocardial ischemic and reperfusion (I/R) injury after cardiac surgery; however, no drug is routinely used to afford cardioprotective benefits in clinical settings. Herein, we aimed to determine whether chloroquine (CQ) pretreatment attenuates I/R injury after global ischemia in isolated rat hearts and elucidate mechanisms underlying the effects of CQ. Methods Isolated rat hearts were subjected to 30-min global ischemia, followed by 60-min reperfusion with Krebs-Henseleit buffer (KHB). Immediately before ischemia, 10 mL of pretreatment solutions (KHB, n = 4 or KHB + CQ [100 mu M], n = 4) were injected through the aortic root. Cardiac function was examined based on the rate pressure product (RPP). Myocardial apoptosis was evaluated using TUNEL staining. To assess the reperfusion ischemia salvage kinase pathway, protein expression levels of AKT and extracellular signal-regulated kinase (ERK1/2) were determined using western blotting. To investigate the role of ERK1/2, an ERK1/2 selective inhibitor was used in eight additional rats. Results The recovery rate of the RPP was higher in the KHB + CQ group than in the KHB group 60 min after I/R (KHB, 44 +/- 3% vs. KHB + CQ, 69 +/- 7%; P = 0.019, d = 2.2). CQ pretreatment reduced apoptosis and enhanced the phosphorylation of ERK1/2; however, AKT phosphorylation was unaltered. In addition, the ERK1/2 inhibitor abolished CQ-mediated cardioprotective effects. Conclusions CQ pretreatment showed protective effects on cardiac function after I/R by activating ERK1/2

The anti-inflammatory effects of flavanol-rich lychee fruit extract in rat hepatocytes.

Flavanol (flavan-3-ol)-rich lychee fruit extract (FRLFE) is a mixture of oligomerized polyphenols primarily derived from lychee fruit and is rich in flavanol monomers, dimers, and trimers. Supplementation with this functional food has been shown to suppress inflammation and tissue damage caused by high-intensity exercise training. However, it is unclear whether FRLFE has in vitro anti-inflammatory effects, such as suppressing the production of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and the proinflammatory mediator nitric oxide (NO), which is synthesized by inducible nitric oxide synthase (iNOS). Here, we analyzed the effects of FRLFE and its constituents on the expression of inflammatory genes in interleukin 1β (IL-1β)-treated rat hepatocytes. FRLFE decreased the mRNA and protein expression of the iNOS gene, leading to the suppression of IL-1β-induced NO production. FRLFE also decreased the levels of the iNOS antisense transcript, which stabilizes iNOS mRNA. By contrast, unprocessed lychee fruit extract, which is rich in flavanol polymers, and flavanol monomers had little effect on NO production. When a construct harboring the iNOS promoter fused to the firefly luciferase gene was used, FRLFE decreased the luciferase activity in the presence of IL-1β, suggesting that FRLFE suppresses the promoter activity of the iNOS gene at the transcriptional level. Electrophoretic mobility shift assays indicated that FRLFE reduced the nuclear transport of a key regulator, nuclear factor κB (NF-κB). Furthermore, FRLFE inhibited the phosphorylation of NF-κB inhibitor α (IκB-α). FRLFE also reduced the mRNA levels of NF-κB target genes encoding cytokines and chemokines, such as TNF-α. Therefore, FRLFE inhibited NF-κB activation and nuclear translocation to suppress the expression of these inflammatory genes. Our results suggest that flavanols may be responsible for the anti-inflammatory and hepatoprotective effects of FRLFE and may be used to treat inflammatory diseases

Inhibition of nitric oxide production by the flavanol monomers.

<p>When IL-1β increased nitric oxide (NO) in the medium, the NO level in the presence of IL-1β was set to 100%, whereas the NO level in the absence of IL-1β was set to 0%. Gallic acid was used as a positive control to monitor the suppression of IL-1β-indcuced NO production.</p><p>IC₅₀, half-maximal (50%) inhibitory concentration of NO production in IL-1β-treated hepatocytes.</p><p>NA, not applied because 50% suppression of NO production was not observed.</p><p>ND, not determined because NO levels increased.</p

FRLFE suppresses iNOS induction by the IL-1β signaling pathway.

<p>A pathway to activate the <i>iNOS</i> gene and the action of FRLFE are schematically depicted. The bold arrows indicate the decreases caused by FRLFE in this study. The proinflammatory cytokine IL-1β binds to its receptor (type I IL-1 receptor, IL1R1) to activate NF-κB through the IκB kinase (IKK) signaling pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Takimoto1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Lawrence1" target="_blank">[25]</a>. Activated IKK phosphorylates IκB-α, resulting in the degradation of IκB-α. A circled P denotes protein phosphorylation. Active NF-κB enters into the nucleus, binds to the <i>iNOS</i> gene promoter (κB sites), and activates transcription. FRLFE inhibits the phosphorylation of IκB-α and the nuclear translocation of NF-κB, resulting in a decrease in the nuclear levels of NF-κB. Because NF-κB also regulates the transcription of the iNOS asRNA, FRLFE reduces the level of iNOS asRNA, which interacts with and stabilizes the iNOS mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Matsui1" target="_blank">[17]</a>. Furthermore, FRLFE may interfere with the iNOS mRNA–asRNA interaction at a posttranscriptional level. Therefore, FRLFE significantly decreases the level of iNOS mRNA.</p