Aldehyde oxidase-dependent species difference in hepatic metabolism of fasudil to hydroxyfasudil

<p>1. An investigation on the metabolic mechanism of fasudil to hydroxyfasudil was conducted <i>in vitro</i> using liver subcellular fractions of different species. Hydroxyfasudil was generated in large amounts by rat liver S9 and to a similar extent by human liver S9 but was not detected in dog liver S9 incubations.</p> <p>2. Studies with various molybdenum hydroxylase inhibitors demonstrated that aldehyde oxidase (AO), but not xanthine oxidase (XO), selectively catalyzed fasudil to hydroxyfasudil in both rat and human liver cytosol. In addition, the oxygen atom incorporated into hydroxyfasudil was derived from water rather than atmospheric oxygen, which further corroborated AO involvement.</p> <p>3. Enzyme kinetics experiments revealed that fasudil had a higher affinity to human hepatic AO than to rat hepatic AO. Besides, significantly different <i>in vivo</i> pharmacokinetic parameters observed between male and female rats indicated that the AO activity in rats was gender-dependent.</p> <p>4. The present study provided first evidences that AO causes differences in fasudil metabolism between species.</p

Pre-vaccination (Day 1) and post-vaccination (Day 22) geometric mean titre against a) A/H1N1, b) A/H3N2 and c) B viral strains in subjects without pre-vaccination immunoprotection

<p><b>Copyright information:</b></p><p>Taken from "Safety and immunogenicity of an MF59™-adjuvanted subunit influenza vaccine in elderly Chinese subjects"</p><p>http://www.immunityageing.com/content/5/1/2</p><p>Immunity & ageing : I & A 2008;5():2-2.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2291031.</p><p></p

Geometric mean titre ratios (Day22:Day1) in all subjects in the Phase II/III trial

<p><b>Copyright information:</b></p><p>Taken from "Safety and immunogenicity of an MF59™-adjuvanted subunit influenza vaccine in elderly Chinese subjects"</p><p>http://www.immunityageing.com/content/5/1/2</p><p>Immunity & ageing : I & A 2008;5():2-2.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2291031.</p><p></p

DataSheet_1_Effects of the epiphytic patterns on endophytes and metabolites of Dendrobium nobile Lindl.docx

IntroductionDendrobium is an epiphytic herb plant with neuroprotective, gastroprotective, anti-inflammatory, and immunomodulatory effects. It is often found attached to tree trunks or rocks. With the development of the dendrobium industry, numerous epiphytic patterns exist, such as crushed stone, stump, and sawdust. The study of metabolites and endophytes of D. nobile under different epiphytic patterns, which revealed the effects of epiphytic patterns on D. nobile from the perspectives of metabolomics and microbiology, is of great significance for the healthy development of D. nobile.MethodsIn the study, the D. nobile under five epiphytic patterns grown in the same environment were selected. The metabolites were investigated by widely targeted metabolomics, and the endophytes were sequenced using high-throughput sequencing methods. Then, a correlation analysis between the different metabolites and endophytes was performed.ResultsA total of 1,032 metabolites were annotated in D. nobile. There are more flavonoids and phenolic acids accumulated on the epiphytic pattern of Danxia stone, whereas the accumulation of lipids on the other epiphytic patterns and 16 differential metabolites was screened out. The endophyte composition of D. nobile was dominated by Proteobacteria, Actinomycetes, unidentified bacteria, Firmicutes, and Cyanobacteria. For endophytic fungi, Basidiomycota and Ascomycota were the dominant phyla of D. nobile. The relative abundance of Spirosoma, Nocardioides, and Arrhenia in the Danxia stone was significantly higher than that of other epiphytic patterns. According to correlation analysis, we found a significant correlation between differential metabolites and Spirosoma, Nocardioides, and Arrheni.DiscussionThis study confirmed that Dendrobium quality was affected by its epiphytic patterns and revealed its possible causes from a microbiological point of view.</p

JNK/mTOR regulates GRP78 induction through ATF4 in human CCA cells.

<p>(A) After treated with SP600125 (SP, 20 µM) for 48 h, ATF4 and phosphorylated eIF2α in QBC939, RBE and HCCC-9810 cells were analyzed using western blot. (B) After treated with rapamycin (Rap, 20 nM) for 48 h, phosphorylated eIF2α and phosphorylated p70S6K in QBC939, RBE and HCCC-9810 cells were analyzed using western blot. (C) After treated with salubrinal (Sal, 25 µM) for 30 h with or without SP600125 (SP, 20 µM) and rapamycin (Rap, 20 nM) preincubation for 1 h, ATF4 and phosphorylated eIF2α were analyzed using western blot in HepG2 cells. (D) After treated with PF-4708671 (PF, 10 µM) and 4EGI-1 (50 µM) for 48 h, GRP78 and ATF4 in QBC939, RBE and HCCC-9810 cells were analyzed using western blot. (E) QBC939, RBE and HCCC-9810 cells were treated with rapamycin (Rap, 20 nM) for 12 h, and ATF4 and GRP78 mRNA levels were analyzed using real-time RT-PCR. Values are means±S.D. Columns, mean of three individual experiments; bars, SD. *Significantly different from control value.</p

JNK promotes the activity of mTOR in human CCA cells.

<p>(A) After treated with SP600125 (SP, 20 µM) for indicated time periods, phosphorylated p70S6K in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. Rapamycin (Rap, 20 nM)-treated cholangiocarcinoma cells were used as positive control. (B) After transfected with siJNK for 60 h, phosphorylated p70S6K in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. (C) After treated with SP600125 (SP, 20 µM) for indicated time periods, phosphorylated mTOR in QBC939, RBE and HCCC-9810 cells was analyzed using western blot. (D) After treated with SP600125 (SP, 20 µM) for indicated time periods, phosphorylated Raptor and phosphorylated 4E-BP1 in QBC939, RBE and HCCC-9810 cells were analyzed using western blot.</p

Expression of GRP78 and phosphorylated JNK in human CCA.

<p>(A) GRP78 and phosphorylated JNK in human CCA were analyzed using immunohistochemistry. (B) Summary of experimental findings. In human CCA cells, the phosphorylation of eIF2α initiates ATF4 expression, which then induces GRP78 accumulation. GRP78 plays an important role in promoting human CCA cells proliferation and invasion. Suppression of mTOR inhibits eIF2α-induced ATF4 expression, which leads to a decrease in GRP78 levels. JNK blocking decreases GRP78 levels through inhibiting the activity of mTOR. Similarly, PI3K/Akt blocking decreases GRP78 levels through inhibiting the activity of mTOR.</p

eIF2α/ATF4 induces GRP78 accumulation in human CCA cells.

<p>(A) Western blot analysis of GRP78 in human cholangiocarcinoma cells. DMSO- and tunicamycin (Tun, 2.0 µg/ml)-treated HepG2 cells were used as negative and positive control, respectively. (B) RT-PCR analysis of spliced XBP1 mRNA in human CCA cells. Tunicamycin (Tun, 2.0 µg/ml)-treated QBC939 cells were used as positive control. (C) Western blot analysis of phosphorylated eIF2α and ATF4 in human CCA cells. Salubrinal (Sal, 25 µM)-treated HepG2 cells were used as positive control. (D) After transfected with ATF4 siRNA for 60 h, QBC939, RBE and HCCC-9810 cells were subjected to western blot analysis.</p

JNK Contributes to the Tumorigenic Potential of Human Cholangiocarcinoma Cells through the mTOR Pathway Regulated GRP78 Induction

<div><p>Less is known about the roles of c-Jun N-terminal kinase (JNK) in cholangiocarcinoma (CCA). Here, we report that JNK exerts its oncogenic action in human CCA cells, partially due to the mammalian target of rapamycin (mTOR) pathway regulated glucose-regulated protein 78 (GRP78) induction. In human CCA cells, the phosphorylation of eukaryotic initiation factor alpha (eIF2α) results in the accumulation of activating transcription factor 4 (ATF4) and GRP78 independent of unfolded protein response (UPR). Suppression of GRP78 expression decreases the proliferation and invasion of human CCA cells. It's notable that mTOR is required for eIF2α phosphorylation-induced ATF4 and GRP78 expression. Importantly, JNK promotes eIF2α/ATF4-mediated GRP78 induction through regulating the activity of mTOR. Thus, our study implicates JNK/mTOR signaling plays an important role in cholangiocarcinogenesis, partially through promoting the eIF2α/ATF4/GRP78 pathway.</p></div

GRP78 promotes human CCA cells proliferation and invasion.

<p>(A) GRP78 suppression inhibits human CCA cells proliferation. QBC939, RBE and HCCC-9810 cells were transfected with siGRP78 for indicated time periods. Cell viability was determined by CCK8 assay. (B and C) GRP78 suppression inhibits human CCA cells migration and invasion. To knockdown GRP78, human CCA cells were transfected with siGRP78 for 36 h before transferring to 24-well transwell chambers. The migration (B) and invasion (C) of QBC939, RBE and HCCC-9810 cells with or without siGRP78 treatment were analyzed using transwell assay. (D) The effects of siGRP78 on GRP78 suppression were measured using western blot. Values are means±S.D. Columns, mean of three individual experiments; bars, SD. *Significantly different from control value.</p