枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定

通过PCR技术扩增得到dhbC基因,对其进行序列分析发现,dhbC基因片段长为1197bp,预期编码398个氨基酸,蛋白分子量大小为43.8kD。将目的片段连接到表达载体pET-30a(+),转化大肠杆菌Escherichia coli BL21(DE3)获得重组菌株BL21(DE3)/pET-30a-dhbC,以IPTG在30oC诱导4h实现高效表达,获得一个分子量为48.8kD的融合蛋白。重组蛋白可溶性分析结果表明:融合蛋白主要为可溶性蛋白。Western blotting分析结果表明:重组蛋白可与兔抗His-tag多克隆抗体发生特异性反应,在48.8kD处有特异条带,与预期结果一致,证明重组质粒中含有dhbC基因。通过同源重组的策略将dhbC基因敲除后重新导入,验证了dhbC基因与嗜铁素的生物合成密切相关。     

Neuritin Attenuates Neuronal Apoptosis Mediated by Endoplasmic Reticulum Stress In Vitro

Neuritin is an extracellular glycophosphatidylinositol-linked protein that promotes neuronal survival, differentiation, function, and repair, but the exact mechanism of this neuroprotective effect remains unclear. Meanwhile, endoplasmic reticulum stress (ERS) induced apoptosis is attracting increased attention. In this work, we hypothesized that neuritin inhibited ERS to protect cortical neurons. To check this hypothesis, we exposed primary cultured cortical neurons to oxygen and glucose deprivation (OGD) for 45 min followed by reperfusion (R) to activate ERS. We then performed resuscitation for 6, 12, 24, and 48 h. ERS-related factors such as glucose-regulated protein 78 (GRP78), caspase-12 and CHOP were detected by Western blotting and quantitative real-time polymerase chain reaction assay. Apoptosis was assessed by Annexin V binding and propidium iodide staining. Ultrastructural changes of endoplasmic reticulum were observed under a transmission electron microscope. Results showed that GRP78 expression significantly increased at 12, 24, and 48 h and peaked at 24 h. Caspase-12 and CHOP expression significantly increased in a time-dependent manner at 12, 24, and 48 h. GRP78, caspase-12 and CHOP expression as well as apoptosis rate of primary cultured neurons and the ultrastructural changes of endoplasmic reticulum in the OGD/R?+?neuritin group significantly improved compared with the OGD/R group. In conclusion, the neuroprotection function of neuritin may be involved in ERS pathways.     

真菌防御素plectasin研究进展

抗生素耐受现象日益严重,迫切需要研发新型抗菌药物。Plectasin是第一例报道的真菌防御素,其抗菌谱窄,仅对革兰氏阳性菌具有强大的杀菌活性,对其进行结构改造可进一步提高其抗菌作用特异性。Plectasin抗菌机制明晰,作用于细胞壁合成。其药物代谢动力学研究较为透彻,同时可在体外高产量表达且活性更高。这些研究为其应用提供了理论基础。综上,plectasin具有极大的临床应用潜力。     

药用植物栀子的组织培养

栀子(Gardenia jasminoides)为药用木本植物。以栀子果皮、种子团和种子为外植体,研究不同激素配比及不同培养方式对愈伤组织诱导和芽分化的影响。研究结果表明,培养基成分为MS+0.5 mg·L–12,4-D+0.25 mg·L–16-BA较适宜果皮和种子愈伤组织的诱导,诱导率分别为83.3%和88.5%;培养基成分为MS+1.0 mg·L–12,4-D+1.0 mg·L–16-BA较适宜种子团愈伤组织的诱导,诱导率为78.1%。3种外植体诱导的愈伤组织中,只有种子愈伤组织能通过液体培养分化出芽;TDZ对芽分化有明显的促进作用;最佳的芽分化培养基为MS+0.05 mg·L–1NAA+0.10 mg·L–1TDZ,其愈伤组织分化率为8.75%。该研究以栀子种子为外植体,并获得了再生植株,为药用植物栀子转基因体系的建立奠定了基础。     

Microbial 2,3-butanediol production: a state-of-the-art review

2,3-Butanediol is a promising bulk chemical due to its extensive industry applications. The state-of-the-art nature of microbial 2,3-butanediol production is reviewed in this paper. Various strategies for efficient and economical microbial 2,3-butanediol production, including strain improvement, substrate alternation, and process development, are reviewed and compared with regard to their pros and cons. This review also summarizes value added derivatives of biologically produced 2,3-butanediol and different strategies for downstream processing. The future prospects of microbial 2,3-butanediol production are discussed in light of the current progress, challenges, and trends in this field. Guidelines for future studies are also proposed.     

Layer-by-layer assembly to modify poly(l-lactic acid) surface toward improving its cytocompatibility to human endothelial cells

A novel technique to introduce free amino groups onto polyester scaffolds via aminolyzing the ester groups with diamine has been developed recently. Positively charged chitosan was then deposited onto the aminolyzed poly(l-lactic acid) (PLLA) membrane surface in a layer-by-layer assembly manner using poly(styrene sulfonate, sodium salt) (PSS) as a negatively charged polyelectrolyte. The layer-by-layer deposition process of PSS and chitosan was monitored by UV-vis absorbance spectroscopy, energy transfer by fluorescence spectroscopy, and advancing contact angle measurements. The existed chitosan obviously improved the cytocompatibility of PLLA to human endothelial cells. The cell attachment, activity, and proliferation on the PLLA membranes assembled with three or five bilayers of PSS/chitosan with chitosan as the outermost layer were better than those with one bilayer of PSS/chitosan or the control PLLA. The cells also showed morphology of an elongated shape with abundant cytoplasm, and a confluent cell layer was reached after being cultured for 4 days. Measurement of von Willebrand factor secreted by these endothelial cells (ECs) verified the endothelial function. Hence, better ECs compatible PLLA were produced.     

抗酶的研究进展

抗酶（antizyme）是当细胞内多胺水平升高时刺激机体合成的一种小分子量调节蛋白,能特异性地与鸟氨酸脱羧酶（omithine decarboxylase,ODC）结合,经泛素非依赖途径被26S蛋白酶体降解,从而使多胺合成减少;抗酶还可以调节多胺转运,以稳定细胞内多胺水平。近年来随着生物技术的不断发展,对抗酶的认识也逐步深入,本文综述了抗酶家族、合成、作用及定位等方面的研究进展。     

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.