Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris

[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.

Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

AIM： To investigate the expression and methylation status of the secreted frizzled-related protein 2 （SFRP2） in esophageal squamous cell carcinoma （ESCC） and ex- plore its role in ESCC carcinogenesis.METHODS： Seven ESCC cell lines （KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1） and one immortalized human esophageal epithelial cell line （Het- 1A）, 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction （RT-PCR） was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expres- sion and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2＇-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we es-tablished stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.RESULTS： SFRP2 mRNA was expressed in the im- mortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2＇-deoxycytidine could restore the expres- sion of SFRP2 mRNA in the three ESCC cell lines lack- ing SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue （0.939 ± 0.398 vs 1.51 ± 0.399, P 〈 0.01）. SFRP2 methylation was higher in tumor tissue than in paired normal tissue （95% vs 65%, P 〈 0.05）. The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by in- troducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro （47.17% 4± 15.61% vs 17% ：1： 3.6%, P = 0.031） and tumor growth in nude mice （917.86：1：249.35 mm3 vs 337.23 ± 124.43 mm3, P 〈 0.05）. Using flow cytom- etry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells （P = 0.025）. The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts. CONCLUSION： Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.

Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer

AIM： To identify the methylation of secreted frizzled-related protein 1 （SFRP1） in gastric cancer and to investigate the aberrant expression of SFRP1 and its correlation with the clinical pathological features of patients. METHODS： We determined SFRP1 methylation and SFRP1 mRNA expression in 3 gastric cancer cell lines SGC-7901, BGC-823, HGC-27, from 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens by methylation-specific （MSP） PCR and RT-PCR respectively. Fisher＇s exact test was used to analyze the statistical association between clinical pathological data and aberrant expression of SFRP1. RESULTS： In 3 cancer cell lines, BGC-823 and HGC-27 had methylated SFRP1 and lost SFRP1 mRNA expression. After treatment of BGC-823 and HGC-27 with the demethylating agent, 5-aza-2′-deoxycytidine, SFRP1 was re-expressed. In 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens, hypermethylation of SFRP1 was detected in 23 （44%） and 8 （15%） specimens respectively （x^2= 10.34, P 〈 0.01）. Loss of SFRP1 expression was detected in 17（33%） and 6 （12%） specimens respectively （x^2= 6.75, P 〈 0.01）. There was a significant correlation between SFRP1 hypermethylation and SFRP1 expression loss. SFRP1 expression was also correlated significantly with tumor stage and lymph node status, but not with patient sex, age and histological type. CONCLUSION： SFRP1 inactivation is a common and early event caused mainly by hypermethylation in gastric cancer. SFRP1 expression loss may be correlated with tumor metastasis in primary gastric cancer.

戊型肝炎病毒多拷贝分泌表达载体的构建及在毕赤酵母中的表达

目的:将胞内表达载体p AO815改建成分泌型表达载体,并体外构建戊型肝炎病毒ORF2128-660多拷贝重组表达质粒,进一步比较不同转化子在毕赤酵母中的表达水平,以得到高表达重组菌株。方法:利用重叠PCR技术将α-factor信号肽基因与目的基因ORF2128-660拼接后插入去磷酸化的p AO815载体,得到分泌型表达重组质粒α-ORF2128-660/p AO815(单拷贝),然后将Bam HⅠ和BglⅡ双酶切获得的目的基因表达盒(AOX-α-ORF2128-660)插入到去磷酸化的重组质粒α-ORF2128-660/p AO815中,得到2(AOX-α-ORF2128-660)/p AO815(2拷贝),电转化毕赤酵母GS115,甲醇诱导,SDS-PAGE分析不同拷贝数转化子的表达产量,ELISA检测表达产物的生物活性。结果:α-factor信号肽基因与目的基因ORF2128-660已克隆入表达载体中,目的蛋白分泌至诱导液上清中,相对分子质量约为59 000,与阴性对照比,单拷贝、2拷贝转化子表达的产物均有生物活性但表达水平无区别。结论:成功改建成分泌型表达载体,构建了多拷贝重组表达质粒,并且成功表达了目的蛋白,为利用改造的p AO815载体表达其他蛋白奠定了基础。

Understanding the Safety, Health and Environmental （SHE） Challenges of Xenobiotics and Their Remedial Approaches

With the use of over 100,000 industrially produced chemicals, there have been several concerns on human health and environment. Most of these chemicals are exposed into the natural environment during the life cycles of their production, transportation, storage, consumption, and as by-products and wastes. The rising rates of cancer, obesity, and infertility suggests that there are compounds recently introduced to the environment that have altered the chemistry of the human body, and it is only with the monitoring of xenobiotics such as Bisphenol A （BPA）, nonyphenols, estrogen （natural and synthetic） and other endocrine-disrupting compounds （EDCs） that patterns and links could be drawn. This paper investigates the safety, environmental and health （SHE） impacts caused by BPA, nonyphenols and estrogens. Derived from petroleum, bisphenol A is used in manufacturing plastic consumer products, including certain water bottles, in dental sealants for children＇s teeth, and in resins used to line tin cans. Nonyphenol is one of the by-products of alkylphenolpolyethoxilates which is widely used as nonionic surfactants. Synthetic estrogen used for birth control pills as well as natural estrogen excreted by women through urine enters the domestic wastewater streams. These compounds are considered to be EDCs and have severe SHE concerns. In this paper, the challenges of entry of these compounds （xenobiotics） into nature, health and environmental issues and their remediation have been reviewed in detail.

Establishment of a transgenic mouse model with liver-specific expression of secretory immunoglobulin D

Mutation of mevalonate kinase （MVK） is thought to account for most cases of hyperimmunoglobulinemia D syndrome （HIDS） with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D （IgD） and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD （cslgD） specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgeues did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS.