Synthesis of triterpenoid saponins from Aesculus chinensis based on transcriptome data / 中国中药杂志

Aesculus chinensis belongs to Hippocastanaceae family,bears medicinal and ornamental values. The oleanane type triterpenoid saponin aescin is regarded as active ingredient and accumulated in seed. In order to understand its molecular basis of the triterpenoid biosynthesis,we used high-throughput sequencing under Illumina Hi Seq 2000 platform to obtain the transcriptome data of seed and flower from A. chinensis to further mine the genes involved in its metabolic pathway. Unigene's de novo splicing was performed using Trinity software; the transcriptome results were annotated with KEGG database to predict the specific pathways of the aescin triterpenoid metabolism. Terpenoid and triterpenoid pathways were found from transcriptome data,and forty seven and twenty seven corresponding genes were uncovered respectively. It was found that there are eight kinds of enzymes related to the terpenoid metabolism pathway precursors and three kinds of enzymes related to the triterpenoid metabolism pathway. In this study,five genes corresponding to triterpene cyclase were analyzed in A. chinensis for the first time,which may participate in the synthesis of triterpenoid. It' s revealed that there were thirty three differential genes associated with the ko00900 and ko00909 pathways by analysis on the difference in transcriptome expression between seeds and flowers; seventeen unigenes were up-regulated and sixteen unigenes were down-regulated in the seeds relative to flowers. In this study, qRT-PCR experiments were used to verify the expression of three key enzyme genes of SQE( Unigene25806),HMGS( Unigene36710),and β-AS( Unigene33291). The results of qRT-PCR were consistent with the transcriptome data. The candidate genes related to triterpenoid saponin aescin synthesis in A. chinensis found in this study can provide theoretical basis for the metabolism synthesis and regulation of aescin.

Serosurvey of Avian metapneumovirus, Orithobacterium rhinotracheale, and Chlamydia psittaci and Their Potential Association with Avian Airsacculitis / 生物医学与环境科学（英文）

Seasonal outbreaks of airsacculitis in China's poultry cause great economic losses annually. This study tried to unveil the potential role of Avian metapneumovirus (AMPV), Ornithobacterium rhinotracheale (ORT) and Chlamydia psittaci (CPS) in avian airsacculitis. A serological investigation of 673 breeder chickens and a case-controlled study of 430 birds were undertaken. Results showed that infection with AMPV, ORT, and CPS was highly associated with the disease. The correlation between AMPV and CPS were positively robust in both layers and broilers. Finally, we determined the co-infection with AMPV, ORT, and CPS was prevalent in the sampled poultry farms suffering from respiratory diseases and the outbreak of airsacculitis was closely related to simultaneous exposure to all three agents.

Development of a Novel PmpD-N ELISA for Chlamydia psittaci Infection / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Chlamydia psittaci is an avian respiratory pathogen and zoonotic agent. The wide prevalence of C. psittaci poses a threat to the poultry industry and its employees. However, few commercial kits are available for detecting avian antibodies excluding the in-house ELISA kit. In this study, we developed a novel ELISA kit for detecting antibodies against C. psittaci based on the N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) as the coating antigen.</p><p><b>METHODS</b>The antigen concentrations, primary antibody, and cut-off value were determined and optimized. The ELISA, designated PmpD-N ELISA, was assessed for sensitivity, specificity, and concordance using sera samples from 48 experimentally infected and 168 uninfected SPF chickens.</p><p><b>RESULTS</b>The sensitivity and specificity of PmpD-N ELISA were 97.9%, 100%, respectively, while the concordance was 98.1% as compared to that of MOMP-ELISA. No cross-reaction with positive sera for other avian pathogens was found. Using PmpD-N ELISA, 799/836 clinical samples were positive, including 93.0% and 98.1% positivity in layers and broilers, respectively.</p><p><b>CONCLUSION</b>These data indicate that indirect ELISA with PmpD-N as the antigen candidate is a promising approach for the surveillance of C. psittaci infection.</p>

Erratum: RETRACTED: Effect of overexpression of hypoxia-inducible factor-1α induced by hyperoxia in vivo in LNCaP tumors on tumor growth rate (APJTM (2015) 8(10) (813–820)(S1995764515001364)(10.1016/j.apjtm.2015.09.007))

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor. The authors have plagiarized part of a paper that had already appeared in Int. J. Biochem. Cell Biol., 51 (2014), 65-74. http://dx.doi.org/10.1016/j.biocel.2014.03.019. One of the conditions of submission of a paper for publication is that authors declare explicitly that their work is original and has not appeared in a publication elsewhere. Re-use of any data should be appropriately cited. As such this article represents a severe abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Research progress of gene recombinant mesenchymal stem cells as tumor targeting delivery vehicles / 药学学报

The applications of targeting gene delivery systems in tumor therapy have attracted extensive attention of researchers in recent years, as they can selectively deliver the therapeutic gene to tumor sites, improve the success rate of gene therapy and reduce the side effects. Therefore, design and development of novel gene delivery vehicles have been a hot area of current research. Recent studies have shown that mesenchymal stem cells (MSCs) have the ability to migrate towards and engraft into the tumor sites. Therefore, these properties make them a great hope for efficient targeted-delivery vehicles in cancer gene therapy. In this review, we examine the promising of utilization of MSCs as a targeted-delivery vehicle for cancer gene therapy, and summarize various challenges and concerns regarding this therapy.

Therapeutic effect of fibroblast growth factor 21 on NAFLD in MSG-iR mice and its mechanism / 药学学报

This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on NAFLD in MSG-IR mice and to provide mechanism insights into its therapeutic effect. The MSG-IR mice with insulin resistance were treated with high dose (0.1 micromol.kg-1d-1) and low dose (0.025 micromol.kg-1d-1) of FGF21 once a day for 5 weeks. Body weight was measured weekly. At the end of the experiment, serum lipids, insulin and aminotransferases were measured. Hepatic steatosis was observed. The expression of key genes regulating energy metabolism were detected by real-time PCR. The results showed that after 5 weeks treatment, both doses of FGF21 reduced body weight (P<0.01), corrected dyslipidemia (P<0.01), reversed steatosis and restored the liver morphology in the MSG model mice and significantly ameliorated insulin resistance. Additionally, real-time PCR showed that FGF21 significantly reduced transcription levels of fat synthetic genes, decreased fat synthesis and promoted lipolysis and energy metabolism by up-regulating key genes of lipolysis, thereby liver fat accumulation was reduced and liver function was restored to normal levels. In conclusion, FGF21 significantly reduces body weight of the MSG-IR mice, ameliorates insulin resistance, reverses hepatic steatosis. These findings provide a theoretical support for clinical application of FGF21 as a novel therapeutics for treatment of NAFLD.

Antitumor activity of the recombinant rClone30-CD/5-FC system / 药学学报

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.

Constitutive expression of human angiostatin in Pichia pastoris using glycerol as only carbon source / 生物工程学报

Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 (pGAP9K-AS)in shaking flask. The result shows that oleic acid is the best (163 mg/L) compared with glycerol (83mg/L), glucose (76 mg/L)and methanol (57 mg/L). Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 (pGAP9K-AS) in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P < 0.01).

Expression of human tumstatin in Pichia pastoris and its bioactivity / 生物工程学报

Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.

Expression of N domain of chromogranin A in Bacillus subtilis and its antifungal activity / 生物工程学报

Chromogranin A (CGA) is a soluble protein existed in most secreted cells and neurons. It was recently found that the bovine CGA N terminal region has vasoinhibitory, antibacterial and antifungal activities. Since the need for effective antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for fungal infections, it becomes imperative to find antifungal compounds with low toxicity toward mammalian cells. To study the antifungal activity of CGA N terminal region, the DNA fragment encoding for the N terminal 1-76 amino acid sequence (CGA1-76) of human CGA was amplified by PCR technique. After DNA sequence analysis, the amplified DNA fragment was cloned into the Bacillus subtilis inducible and expression vector pSBPTQ constructed in this study and the resultant plasmid pSVTQ was then transformed into triple-protease deficient Bacillus subtilis strain DB403 competent cells. The transformants was screened on LB plates containing 10 microg/mL kanamycin. The positive transformant DB403 (pSVTQ) was grown on kanmycin containing 2 x MSR medium and sucrose was added to 2% final concentration for induction after 2h cultivation. The culture supernatant was used to run SDS-PAGE. The result of SDS-PAGE showed that the CGA1-76 was expressed by sucrose induction and the expressed product secreted into the medium with a yield of 5 mg/L. The expressed product reacts specifically with mouse anti CGA47-68 monoclonal antibody. The antifungal activity of the expressed product was examined by adding the culture supernatant to the fungal spore or Candida albican suspensions at appropriate proportion and found that the recombinant human CGA1-76 produced in Bacillus subtilis inhibits the growth of Fusarium sp. Alternaria sp. and Candida albican at the concerntration of 4 micromol/L. These results demonstrate that human CGA1-76 has expressed in Bacillus subtilis and the expressed product is immunogenic and has the antifungal activity.

Cloning and expression of kringle 1-3 gene of human plasminogen and the purification and bioactivity of its expressed product / 生物工程学报

Kringle 1-3 domain is a recently found angiogenesis inhibitor with anti-angiogenesis and anti-tumor activity. The kringle 1-3 gene was amplified by PCR technique using angiostatin gene as template. After DNA sequencing, the PCR product was cloned into pPIC9K resulting in recombinant plasmid pPIC9K13 which was then transformed into Pichia pastoris GS115. The high copy integration transformants screened by PCR and G418 methods were cultivated in flasks. The K1-3 was expressed and secreted to the medium and has immunogenic activity as shown by SDS-PAGE and Western blotting. High cell density culture was carried out in 30-liter and 80-liter bioreactor, the biomass reaches 300 OD after methanol induction, and the expressed product is 200 mg/L. The fermentation supernatant was purified by Streamline SP and Phenyl Sepharose Chromatography, the product appears as a single band on SDS-PAGE, with a purity of 95%-96%. The purified product has anti-angiogenesis and anti-tumor activity.