利用非经典分泌蛋白质实现脂肪酶A的分泌表达

【目的】以枯草芽孢杆菌脂肪酶A(Lipase A)为报告蛋白,尝试利用4种非经典分泌蛋白质及其前50个氨基酸作为分泌信号以实现其分泌表达。【方法】我们扩增了脂肪酶A的编码基因和非经典分泌蛋白质的编码序列,构建了8种针对脂肪酶A的分泌表达载体,并转化至枯草芽孢杆菌WB800菌株,通过测定重组菌株的酶活、利用蛋白质电泳和免疫印迹等技术检测脂肪酶A的分泌情况【结果】以Pdh A的氨基酸序列和Sod A、Eno的前50氨基酸序列作为分泌信号的重组菌株较好的实现了脂肪酶A的分泌表达。【结论】部分非经分泌蛋白质的编码基因或其前50个氨基酸序列能够引导脂肪酶A分泌至细胞外。     

The role of acyl-CoA thioesterase ACOT8I in mediating intracellular lipid metabolism in oleaginous fungus <Emphasis Type="Italic">Mortierella alpina</Emphasis>

Thioesterases (TEs) play an essential role in the metabolism of fatty acids (FAs). To explore the role of TEs in mediating intracellular lipid metabolism in the oleaginous fungus Mortierella alpina, the acyl-CoA thioesterase ACOT8I was overexpressed. The contents of total fatty acids (TFAs) were the same in the recombinant strains as in the wild-type M. alpina, whilst the production of free fatty acids (FFAs) was enhanced from about 0.9% (wild-type) to 2.8% (recombinant), a roughly threefold increase. Linoleic acid content in FFA form constituted about 9% of the TFAs in the FFA fraction in the recombinant strains but only about 1.3% in the wild-type M. alpina. The gamma-linolenic acid and arachidonic acid contents in FFA form accounted for about 4 and 25%, respectively, of the TFAs in the FFA fraction in the recombinant strains, whilst neither of them in FFA form were detected in the wild-type M. alpina. Overexpression of the TE ACOT8I in the oleaginous fungus M. alpina reinforced the flux from acyl-CoAs to FFAs, improved the production of FFAs and tailored the FA profiles of the lipid species.     

Recent advances in the research and development of human defensins

Human defensins are a family of cationic antimicrobial peptides with molecular weights of 4-5 kDa, containing a conserved six disulphide-linked cysteine motif. During the last two decades a series of endogenous alpha- and beta-human defensins were discovered. They exhibit a broad range of antimicrobial properties and are thought to be ideal therapeutic agents because of their potential ability to circumvent the problems of acquired resistance often observed with other antimicrobial therapies. Because of their appealing medical and pharmaceutical potential there has been an emphasis on human defensins in medical and molecular biology research in recent years. This paper aims to present a comprehensive review of recent advances in the study of human defensins including their discovery, classification, molecular properties, expression, mechanisms of action and potential medical applications. In addition, the advances in producing human defensins via genetic engineered cells are summarized from research works in our group (besides host cells including E. coli, B. subtilis and yeast systems, the cell-free protein synthesis system was also employed to express human beta-defensin-2) along with other related published works. The present challenges and prospects for the potential application of human defensins are also discussed.     

T-2 toxin induces apoptosis in differentiated murine embryonic stem cells through reactive oxygen species-mediated mitochondrial pathway

T-2 toxin, a member of the trichothecene mycotoxin family produced by the Fusarium fungi, has been shown to exert a variety of toxic effects on multiple targets in vivo. However, the embryonic toxicity of T-2 toxin in vitro remains unclear. In the present study, two permanent cell lines, embryonic stem cells (ES cells D3) and fibroblast 3T3 cells, were used to evaluate T-2 toxin toxicity. Differentiated mouse ES cells were cultivated as embryoid bodies along with T-2 toxin at different concentrations (0.5, 1, and 2 ng/ml) for 24 h. The increases in cellular reactive oxygen species (ROS), lipid and DNA oxidative damage, and loss of mitochondrial transmembrane potential were observed at 1 and 2 ng/ml concentrations. Flow cytometry showed that T-2 toxin induced cell cycle arrest and apoptosis. Furthermore, T-2 toxin opened the mitochondrial permeability transition pore, caused the release of cytochrome c from mitochondria and induced the upregulation of p53, caspase-9, caspase-3 expression and increased the ratio of Bax/Bcl-2. However, T-2 toxin-induced oxidative damage and apoptosis in differentiated ES cells decreased significantly in the presence of the antioxidant Trolox. Taken together, these results demonstrate that T-2 toxin induces oxidative stress and apoptosis in differentiated murine ES cells, and ROS-mediated mitochondrial pathway plays an important role in T-2 toxin induced apoptosis.     

SGT1 regulates Akt signaling by promoting beta-TrCP-dependent PHLPP1 degradation in gastric cancer cells

SGT1 (suppressor of G2 allele of Skp1) plays a role in various cellular processes including kinetochore assembly and protein ubiquitination by interacting with Skp1, a component of SCF E3 ligase complex. However, the function of SGT1 in cancer is largely unknown. Here, we showed that SGT1 was over-expressed in gastric cancer tissues and silencing of SGT1 by siRNAs significantly inhibited the growth and colony formation of gastric cancer cells. We further showed that SGT1 could regulate Akt signaling pathway by modulating Akt ser473 phosphorylation status. Moreover, we found that SGT1 was able to regulate the stability of PHLPP1, which is the direct phosphatase for Akt ser473 phosphorylation. Immunoprecipitation assay revealed that SGT1 could enhance the binding between PHLPP1 and beta-TrCP which has been documented to be able to target PHLPP1 for destruction. Decreased PHLPP1 in SGT1 over-expressed gastric cancer cells failed to dephosphorylate Akt and resulted in increased Akt ser473 phosphorylation and amplified downstream Akt signaling. Thus, our data revealed a previously uncovered role of SGT1 in gastric cancer development, and suggested that SGT1 could be a promising anti-cancer target to against gastric cancer.     

Expression and Purification of Integral Membrane Fatty Acid Desaturases

Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases.     

蝴蝶兰F3′5′H基因转化OT杂种百合Robina的研究

为了定向育种获得蓝色百合,该研究以百合Robina为蓝色基因最佳受体,以其花丝诱导产生的胚性愈伤组织和再生小植株小鳞片作为转化材料,利用农杆菌介导法,将蝴蝶兰F3′5′H基因导入百合Robina中。结果表明:以小鳞片为转化材料,预培养3d,OD_(600)为0.8,侵染10min,共培养3d,加入100μmol/L AS稳定转化率最高为12.78%;而以胚性愈伤为转化材料,预培养2d,OD_(600)为0.8,侵染10min,共培养3d,加入100μmol/L AS稳定转化率最高为12.22%。2种转化材料的最适潮霉素筛选浓度均为20mg/L。对抗性植株分别进行PCR和反转录PCR检测,获得9个阳性株系,Southern印记分析进一步确定了6株转基因百合中携带蓝色基因F3′5′H,为后续进一步获得蓝色百合奠定了基础。