植物乳杆菌ZS2058生物转化共轭亚油酸的反应动力学

摘要：【目的】对本实验室从泡菜中筛选到的植物乳杆菌ZS2058完整细胞生物转化共轭亚油酸的反应动力学进行研究。【方法】探讨底物浓度、细胞浓度、反应体系pH值等因素对生物转化共轭亚油酸反应速度的影响,并通过双倒数和Hanes-Woolf作图法拟合反应初始阶段的速度方程。【结果】生物转化共轭亚油酸时存在明显的底物抑制现象,当亚油酸浓度为0.4 mg/mL时产c9, t11-共轭亚油酸的反应速度达最大值15.99 μg/(mL?h);反应速度随细胞浓度增加而上升,当细胞浓度为5×1010 cfu/mL时反应速度达到最高;最适pH值和最适反应温度分别为6.5和40 ℃。利用双倒数和Hanes-Woolf作图法求得米氏常数和最大反应速度,在低底物浓度下,反应初始阶段的反应规律与经典的米氏方程相符,而在高底物浓度下,存在明显的底物抑制现象。【结论】通过对植物乳杆菌ZS2058完整细胞催化合成共轭亚油酸各因素的考察,在得到最佳反应条件的同时建立了不同底物浓度范围内的反应速度方程,这对于实现共轭亚油酸的生产和研究其生理功能具有十分重要的理论价值。     

纯化标签的不同位置对Δ6脂肪酸脱饱和酶异源表达的影响

【背景】目前利用酵母表达系统已鉴定了多种物种中的Δ6脂肪酸脱饱和酶(FADS6)。由于FADS6是一种具有多个跨膜螺旋的膜蛋白,使得其大量表达和纯化具有挑战性。【目的】探索FADS6的高效表达策略,研究纯化标签添加的位置对高山被孢霉FADS6I (Ma FADS6I)重组表达效率的影响。【方法】在毕赤酵母表达载体中插入串联亲和标签HRV 3C-Protein A-His,利用改造后的载体构建带有N端或C端标签的Ma FADS6I表达载体;通过电转化获得毕赤酵母重组表达菌株;利用斑点印迹杂交(DotBlot)、聚丙烯酰胺凝胶电泳(SDS-PolyacrylamideGelElectrophoresis,SDS-PAGE)和免疫印迹(Western Blot)分析重组蛋白的表达水平,并利用气相色谱-质谱(Gas Chromatography-Mass Spectrometry,GC-MS)分析检测Ma FADS6I催化生成的脂肪酸。【结果】通过大量的毕赤酵母转化子筛选,最终获得高效表达Ma FADS6I的毕赤酵母重组菌,证实各转化子的表达具有差异性,Ma FADS6I的C端带有纯化标签较N端更有利于表达。【结论】在Ma FADS6I的C端添加纯化标签比在N端添加更有利于该蛋白在酵母系统中的表达以及底物的转化,为进一步探究FADS6高效表达和结构功能奠定了基础。     

溶氧影响土壤杆菌ATCC 31749发酵生产热凝胶的蛋白质组学

摘要:【目的】土壤杆菌(Agrobacterium sp.)ATCC 31749在氮源限制条件下生物合成热凝胶是一个专性好氧过程,在微氧和缺氧条件下,热凝胶的合成受到严重限制。为探寻溶氧影响微生物多糖合成的代谢途径和调控机制,本研究比较了不同溶氧条件下(75%,50%,25%,5%)土壤杆菌发酵生产热凝胶的蛋白质组差异。【方法】利用蛋白质二维电泳技术,分离出不同溶氧水平下土壤杆菌显著表达差异的胞内蛋白,利用质谱MALDI-TOF/TOF鉴定二维电泳表达差异蛋白点,并分析热凝胶合成过程中溶氧对相关蛋白表达的影响。【结果】在4个溶氧水平下成功鉴定出15个显著差异蛋白,主要参与多糖合成、脂肪酸合成、氨基酸合成等途径。其中葡萄糖磷酸变位酶和乳清苷5-磷酸脱羧酶直接参与调控热凝胶合成。【结论】溶氧可显著影响与热凝胶合成途径相关蛋白的表达,高溶氧水平可增加热凝胶前体物质UDP-葡萄糖的积累,使更多的UDP-葡萄糖用来合成热凝胶。     

Cloning and heterologous expression of a bacteriocin sakacin P from <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.     

多能干细胞体外分化为类神经管模型的研究进展

近年来多能干细胞(PSCs)的体外培养与分化技术发展迅速,并广泛应用于再生医学和发育生物学等领域。PSCs能够在体外神经诱导的条件下分化为类神经管模型,这为探索体内早期神经发育与中枢神经系统发育疾病的形成机制提供了全新的实验平台。本文总结了近年来应用小鼠和人PSCs建立体外类神经管模型的研究进展,其中体外模型主要包括在不同培养体系下诱导获得的二维(2D)与三维(3D)类神经管模型,并针对早期类神经管模型在神经系统发育性疾病机制研究中的前景和挑战作进一步探讨,同时为疾病预防和治疗提供新的思路。     

Role of Ca2+/Calmodulin-dependent Protein Kinase II in Drosophila Photoreceptors

Ca²⁺ modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca²⁺ mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca²⁺-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.Visual transduction is the process that converts the signal of light (photons) into a change of membrane potential in photoreceptors (see Ref. 1 for review). Visual signaling is initiated upon the activation of rhodopsins by light: light switches on rhodopsin to generate metarhodopsin, which activates the heterotrimeric G_q in Drosophila (2). Subsequently, the GTP-bound Gα_q subunit activates phospholipase Cβ4 encoded by the norpA (no receptor potential A) gene (3). Phospholipase Cβ4 catalyzes the breakdown of phosphoinositol 4,5-bisphosphate to generate diacylglycerol, which or its metabolite has been implicated in gating the transient receptor potential (TRP)² and TRP-like channels (4, 5). TRP is the major Ca²⁺ channel that mediates the light-dependent depolarization response leading to an increase of cytosolic Ca²⁺ in photoreceptors. The rise of intracellular Ca²⁺ modulates several aspects of the visual response including activation, deactivation, and light adaptation (6). For example, Ca²⁺ together with diacylglycerol activates a classical protein kinase C, eye-PKC, which is critical for the negative regulation of visual signaling by modulating deactivation and light adaptation (7–11).Light adaptation is the process by which photoreceptors adjust the visual sensitivity in response to ambient background light by down-regulating rhodopsin-mediated signaling. Light adaptation can be arbitrarily subdivided into long term and short term adaptation and may involve multiple regulations to reduce the efficiency of rhodopsin, G protein, or cation channels. For example, translocation of both G_q (12, 13) and TRP-like channels (14, 15) out of the visual organelle may contribute to long term adaptation in Drosophila. In contrast, short term adaptation may be orchestrated by modulating the activity of signaling proteins by protein kinases. Hardie and co-workers (16) demonstrated that an increase of cytoplasmic [Ca²⁺] mimicked light adaptation, leading to inhibition of the light-induced current. These authors also showed that light adaptation is independent of eye-PKC. Thus the effect of cytoplasmic Ca²⁺ to control light adaptation is likely mediated via calmodulin and CaMKII. The contribution of CaMKII to light adaptation has not been explored.CaMKII is a multimeric Ca²⁺/calmodulin-dependent protein kinase that modulates diverse signaling processes (17). Drosophila contains one CaMKII gene (18) that gives rise to at least four protein isoforms (19). These CaMKII isoforms share over 85% sequence identities with the α isoform of vertebrate CaMKII. For insights into the in vivo physiological role of CaMKII, Griffith et al. (20) generated transgenic flies (ala) expressing an inhibitory domain of the rat CaMKII under the control of a heat shock promoter, hsp70. They demonstrated that, upon heat shock treatment, the overexpression of the inhibitory peptide resulted in a suppression of the endogenous CaMKII activity in the transgenic flies (20). It has been shown that inhibition of CaMKII affects learning and memory (20) and neuronal functions (21–24). In photoreceptors, CaMKII has been implicated in the phosphorylation of the major visual arrestin, Arr2 (25, 26). However, how phosphorylation of Arr2 by CaMKII modifies the visual signaling remains to be elucidated.Here we report the biochemical and electrophysiological analyses of CaMKII in Drosophila retina. We demonstrate that suppression of CaMKII in ala¹ transgenic flies leads to a phenotype indicative of defective light adaptation. The ala¹ flies also display greater visual response, suggesting a defect in Arr2. These results support the notion that CaMKII plays a role in the negative regulation of the visual response. Our biochemical analyses demonstrate that dephosphorylation of CaMKII is mediated by protein phosphatase 2A (PP2A). Importantly, we show that PP2A interacts with CaMKII, indicating that CaMKII forms a stable protein complex with PP2A to ensure a tight regulation of the kinase activity. Thus a partial loss of function in PP2A would elevate the CaMKII activity. Indeed, we show that mts heterozygotes display reduced prolonged depolarizing potential (PDA) amplitude. This PDA phenotype strongly suggests that Arr2 becomes more effective to terminate the visual signaling in mts flies. Together, our findings indicate that the ability of Arr2 to terminate metarhodopsin is increased upon phosphorylation by CaMKII, and the retinal CaMKII activity is regulated by PP2A.     

Molecular phylogeny and maternal progenitor implication in the genus Kengyilia (Triticeae: Poaceae): Evidence from COXII intron sequences

Kengyilia is a perennial genus distributing in central and western Asia. Here, the levels of nucleotide diversity for COXII intron were obtained. The estimates of nucleotide diversity for different genome constitution ranged from θ = 0.00082 and π = 0.00082 for St genome species to π = 0.01227 and θ = 0.01229 for P genome species. Employing COXII intron sequences, the phylogenetic relationships within Kengyilia and between Kengyilia genus and its closely related genera were examined. The Maximum Parsimony analysis demonstrated that Kengyilia species were positioned into two clades corresponding to different maternal genomic donor. Kengyilia stenachyra, Kengyilia grandiglumis, Kengyilia hirsuta, Kengyilia melanthera, Kengyilia thoroldiana, Kengyilia alatavica and Kengyilia zhaosuensis were related to species of Agropyron, while Kengyilia kokonorica, Kengyilia rigidula, Kengyilia nana, Kengyilia mutica, Kengyilia longiglumis, Kengyilia laxiflora and Kengyilia gobicola were close to species of Roegneria and Pseudoroegneria. In addition, other three species of Kengyilia, such as Kengyilia batalinii, Kengyilia tahelacana and Kengyilia kaschgarica, were related to Douglasdeweya deweyi, Pseudoroegneria strigosa and Roegneria tibetica. This result indicated that there had been two phylogenetically divergent maternal donors within Kengyilia. Our new finding will help to understand the evolutionary history of the genus Kengyilia.     

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.