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BACKGROUND Baicalin, one of the main bioactive components extracted from the traditional Chinese medicine baical Skullcap root, has an anti-tumor activity which had been studied in several cancers. However, its role in human mesothelioma remains unknown. In this study, we investigated the anti-tumor mechanisms of baicalin in the mesothelioma cell line MESO924. MATERIAL AND METHODS Effects of baicalin on mesothelioma were assessed by measuring cell viability, apoptosis, migration, invasion, inactivation of signaling intermediates, and cell-cycle alterations. RESULTS Baicalin inhibited the proliferation, migration, and invasion of human mesothelioma cells and increased their apoptosis, all in a dose-dependent manner. Specifically, baicalin decreased the expression of p-EGFR, p-AKT, p-MAPK, p-S6, Bcl-2, and VEGF and increased the expression of Bax in mesothelioma cells. The suppressed mesothelioma cellular proliferation is due to the arrest of the S cell cycle by baicalin. Inhibition of the PI3K/AKT/mTOR signaling pathway by a PI3K/AKT/mTOR inhibitor augmented the anti-proliferation effects induced by baicalin. In addition, baicalin increased the sensitivity of MESO924 to the chemotherapeutic drugs doxorubicin, cisplatin, and pemetrexed. CONCLUSIONS These results highlight the roles of baicalin in inhibiting cell growth, migration, and invasion of mesothelioma cells while increasing apoptosis and sensitizing cells to chemotherapeutic agents through the PI3K/AKT/mTOR signaling pathway, which indicates that baicalin could be a useful drug for mesothelioma therapy.

Protein kinase CK2 alpha (CK2α) is involved in the development of multiple malignancies. Overexpression of Y-box binding protein 1 (YBX1) is related to tumor proliferation, drug resistance, and poor prognosis. Studies have demonstrated that both CK2 and YBX1 could regulate the PI3K/AKT pathway. In addition, we predicted that CK2 might be the upstream kinase of YBX1 through the Human Protein Reference Database (HPRD). Herein, we hypothesize that CK2 may interact with YBX1 and they regulate the PI3K/AKT signaling pathway together. Expressions of CK2α and YBX1 in cancer cell lines were evaluated by immunoblotting. The results showed that CK2α could regulate the expression of YBX1 at the transcriptional level, which is dependent on its enzymatic activity. Synergistic effects of PI3K/AKT pathway inactivation could be observed through combined inhibition of CK2α and YBX1, and YBX1 was required for CK2α-induced PI3K/AKT pathway activation. Further results demonstrated that CK2α could interact with YBX1 and PI3K/AKT antagonist decreased cell resistance to doxorubicin induced by co-activation of CK2α and YBX1. These results indicated that combined inhibition of CK2α and YBX1 showed synergistic effects in inactivating the PI3K/AKT signaling pathway and may be one of the mechanisms involved in tumor growth and migration.

Phosphatase of Regenerating Liver-3 (PRL-3) is a newly identified colorectal cancer metastasis-related protein, which is a 22 kDa non-classical protein tyrosine phosphatase with a C-terminal prenylation motif. In this study, the inhibition kinetics of protein tyrosine phosphatases (PTPs) by a fluorescent substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) was evaluated. PRL-3 exhibits classical Michaelis-Menten kinetics with a v max value of 11.3 μmol · L −1 · min −1 . Analysis of PRL-3 by a Michaelis-Menten plot and a double-reciprocal plot indicated that the inhibitor magnolol can cause K m to increase, but does not alter the v max value, which suggests the competitive inhibition of PRL-3. At the same time, it was found that DiFMUP is a more sensitive substrate for PRL-3 than para-nitro-phenyl phosphate (pNPP) that is more frequently used at present. Furthermore, the method of screening for PTPs by the use of DiFMUP was developed, which studied the acceptance of DiFMUP by other PTPs.

A tetranuclear copper(II) complex [Cu4(NSI)4] · 2C2H5OH · 2H2O (NSI=hydroxethylsalicydeneimine) has been synthesized and characterized by X-ray diffraction analysis. The compound crystallizes in the monoclinic system, space group P2(1), a=9.494(3) A, b=18.687(5) A, c=13.149(4) A, β=110.162(5)°, Z=2, R1=0.0482 and wR2=0.0978. The crystal structure contains a tetranuclear pseudo-cubane core based on an approximately cubane array of alternating copper and oxygen atoms. Each copper atom resides in a distorted square planar coordination environment with one nitrogen and three oxygen atoms from two NSI ligands. The tetranuclear units are linked in the crystal by O–H⋯O hydrogen bonds and weak Cu⋯O co-ordination bonds into one-dimensional structure. Variable temperature (5–300 K) magnetic measurements indicate the existence of ferromagnetic interactions among copper atoms. The IR and ESR spectra have also been investigated.

The inhibition of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) is considered able to decrease serum cholesterol levels and dramatically reduce the risk for cardiovascular and cerebrovascular diseases. The statins, competitive inhibitors of HMGCR, have been employed to control hypercholesterolemia. But their side effects, especially their safety of long-term administration have attracted great attention. Therefore, there is still an urgent requirement for the development of safer inhibitors of HMGCR with less serious side effects. In this study, we cloned and purified the catalytic domain of human HMGCR (△HMGCR), and applied the method of Ultra Performance Liquid Chromatography (UPLC) to assay △HMGCR activity and screen its inhibitors from natural products. The results indicated that EGCG can inhibit △HMGCR in the presence of some glycerol in vitro and can decrease cellular total cholesterol in HepG2 cells. As a consequence, it is promising to put EGCG into the development of hypolipidemic health product.

Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that regulates multiple developmental processes throughout the metazoans. It is a dual function protein, working as a transcription factor in the nucleus and as a tyrosine phosphatase in the cytoplasm. In this study, we isolated EYA-1 of Caenorhabditis elegans, the only homolog of Eyes absent, and set up an effective feeding-based RNAi (RNA interference) against the gene. We found that knockdown of EYA-1 decreased heat and oxidative stress tolerance and accelerated the onset of paralysis mediated by Aβ1-42 proteotoxicity and polyQ. Under heat stress (35°C), EYA-1 knockdown shortened the mean lifespan by 16.8%, which could be attributed to decrease in heat shock protein-16.2 (hsp-16.2) expression. Under oxidative stress, EYA-1 knockdown could shorten the mean lifespan by 18.7%, which could be attributed to intracellular ROS accumulation and the decrease of superoxide dismutase-3 (sod-3) protein expression. Moreover, EYA-1 knockdown animals also showed increased lipofuscin accumulation under oxidative stress. Further studies demonstrated that EYA-1 knockdown could not inhibit daf-16 nuclear accumulation in wild-type worms in response to stress. On the other hand, EYA-1 deficiency did not further reduce stress resistance of daf-16 mutants, which are stress sensitive. Quantitative real-time PCR results also showed that the expression of two daf-16 target genes, hsp-12.3 and sod-3, was downregulated in EYA-1 RNAi-treated worms under stress. All this evidence indicates EYA-1 is required for stress resistance of worms, and it might act downstream of daf-16 to regulate expression of stress resistance-associated genes.

To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis.Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K(+) efflux and Na(+) influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively.Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 μg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K(+) efflux and Na(+) influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect.Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections.

Four kinds of supports were used to prepare immobilized papain using different methods: simple absorption on Celite, ionic absorption on CM-cellulose and QAE-Sephadex, and covalent cross-linking on egg white protein. The direct effects of the support on the catalytic properties of the enzyme were examined by studying the dependence of water content, pH, ionic strength, and reaction temperature on the yields of a model dipeptide Boc-Phe-ValOMe in ethyl acetate under thermodynamic control. It was found that the supports with low hydrophilicity demonstrated much higher catalytic activity by the enzyme than those with high hydrophilicity. Egg white protein was the best support with a good yield (94.5%) of the dipeptide. Papain and α-chymotrypsin immobilized on egg white protein were used to synthesize a number of dipeptides with Boc-Phe-XOMe showing different substrate specificity. Alcalase was used in the synthesis of some dipeptides containing hydrophilic amino acids and d -amino acids with reasonable yields.

A novel iron(III)–nickel(II) heterodinuclear complex containing bidentate bridging and terminal monodentate coordinated diphenylphosphate ligands has been synthesized, and its structure and properties have been clarified.

Environmental Protection Agency and the World Health Organization determined that bromate is a potential human carcinogen, even at low µg/l levels in drinking water. In order to decrease the chemical risk of bromate, the monitor parameters have increased and maximum contaminant levels of the parameters have been controlled strictly. A simple technique for the determination of trace level of bromate in drinking water with ion chromatography is presented. In the selected concentration and velocity of the sodium hydroxide eluent, bromate and other anions can be separated with the Dionex DX-120 IC machine and the IonPac As16 anion-exchange column in linear gradient. Bromate can be certified by retention time and fixed the quantity by peak area. Prior to ion chromatography, the drinking water sample was treated with electrolyzing argentine electrode to minimize the disturbance of superfluous chloride and then concentrated in a microwave oven in 10 minutes. The detection limit of bromate was decreased to 0.5µg/l. The method was applicable for the detection of bromate in real drinking water.

Aerobic granular sludge was cultured successively and stably existed in the membrane bioreactors, inoculated by floccular activated sludge and fed with glucose as organic substrate by means of special alternating anaerobic-aerobic operation mode. Microscopic observation of the granular showed that the morphology of granules was nearly spherical or elliptical with dense structure and its mean diameter was 1000 mum. Nitrogen and carbon components in ammonium nitrogen wastewater were efficiently removed by using an aerobic granular sludge membrane bioreactor where biofilm was fixed on a hollow-fiber membrane. The experimental results showed that the effluent ammonia nitrogen could be constant at less than 10 mg/L and COD was averaged at 46.2 mg/L under the conditions of HRT 8 h, the volume load of COD 600 mg/L, and the concentration of ammonia nitrogen 40 mg/L. The removal rates of nitrate nitrogen and nitrite nitrogen were approximately 90%. In addition, the introduction of aerobic granules into the MBR system was beneficial to controlling membrane fouling.

Spatial variability and distribution of soil arsenic in Zibo maize producing area, Shandong Province were studied using geostatistics and geographic information system (GIS) techniques. Positive skewed distribution was observed; Logarithm transformation was applied in order to achieve normality in the data set and to reduce the affect of outliers. The results show that experimental semi-variogram exists distinct anisotropic and has the strongest continuity in the direction of northeastern 70 degrees. Exponential model was used to fit experimental semi-variogram. Normal kriging was used to predict the spatial distribution of soil arsenic in Zibo maize producing area. It can be concluded that there is clear special heterogeneity in soil arsenic and wastewater irrigation is the main reason affected spatial variability. In fact, the concentrations of soil arsenic in Zibo maize producing area decrease in the pass 20 years.

A novel method was described for detection carbamate pesticides residue in cucumber by immobilizing acetylcholinesterase enzyme in a micro-screen plate with 96 wells based on GA-BSA-gelatin gels. The concentrations of BSA, GA and gelatin in enzyme immobilization solution were optimized and the best concentrations were 5%, 0.5% and 0.1%, respectively. To analyze the pesticides residue 5g cucumber sample was homogenized with 10 mL acetone then an aliquot of 5mL extract was collected in a 10 mL test tube with a cap, into which 2g sodium chloride and 2mL dichloromethane were added in sequence. After shaking, 1 mL of the supernatant aliquot was evaporated by a blower in a small beaker and dissolved by 20% methanol-water solution then 50 microL was piped to a sample well. After incubation 10 minutes the absorbance was detected. The proposed method offered a rapid, simple and inexpensive means to in screen of batch samples. The minimum detection limit of this method was in a range of 0.1-0.2 mg/kg for cucumber samples.

The lysin gene of Bacillus anthracis-diagnosing bacteriophage, obtained by PCR amplification,was cloned into the Escherichia coli exepression vector pET22b which has been cleaved by EcoR I and Nde I. The recombinant vector pET22b-gamma lysin was verified to be correctly constructed by PCR, sequencing and enzyme digestion, and highly expressed in E. coli BL21 (DE3), which accounted for about 40 percent of total protein in E. coli BL21 (DE3), while in the 5L fermentor the expression level reached 15g/L. After expression, disruption and purification with three-step chromatography, Streamline SP, SP HP and Sephacryl S-100, the recombinant gamma lysin was finally obtained with purity of higher than 95 percent as determined by gel scan. The final yield following SP HP was 19.1 percent, with a greater-than-350-fold increase in specific activity. The pure enzyme has been shown active to Bacillus anthracis, and not to E. coli, Bacillus subtilis and Bacillus cereus. Its specific activity was about 1400 u/mg.

The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.

A high-performance liquid chromatograph equipped with an evaporative light scattering detector (ELSD) (HPLC–ELSD) was used to assay the ceramides in yeast cells. The HPLC–ELSD method employed a cyanopropyl bonded column (CN column) that effectively separated the main interfering substance ergosterol without any derivatization process; most other interfering substances were also removed. The method can be applied for routine assay of ceramide content in yeast.