CRISPR/Cas技术及其在发酵菌株上的应用

发酵菌株的改良在发酵工业生产上通常是一项非常重要的内容,而传统的驯化模式周期长、效率低、稳定性差且成本高,很难满足工业化快速生产的要求。规律成簇间隔短回文重复Cas(Clustered Regularly Interspaced Short Palindromic Repeats/Cas,CRISPR/Cas)是细菌及古生菌中的一种适应性免疫系统,在此系统上改进的基因编辑技术能实行RNA导向的DNA精准编辑。因而利用CRISPR/Cas基因编辑技术对菌株的快速构建和优化,是一条快速获得高产菌株的新途径。本文综述了CRISPR/Cas系统组成、工作原理、分类以及该技术在发酵菌株上的应用研究进展,并探索了该技术存在的问题以及目前的解决办法,最后对CRISPR/Cas技术在发酵工程上的潜力进行了展望。     

Protective Antioxidant Effects of Amentoflavone and Total Flavonoids from Hedyotis diffusa on H2O2‐Induced HL‐O2 Cells through ASK1/p38 MAPK Pathway

In this study, total flavonoids and total triterpenoid acid were extracted with ethyl acetate from Hedyotis diffusa Willd, and hepatoprotective activities of them and five compounds from total flavonoids against H₂O₂ induced hepatocyte damage on HL‐02 cells were determined. In particular, amentoflavone and total flavonoids had influence on the leakage of ALT, AST, LDH, the activities of SOD and the content of MDA. They effectively reduced the loss of MMP, the release of Cyt C, and then inhibited activation of caspase‐3/caspase‐9 cascade in hepatotoxic cells. The contents of ROS were significantly reduced to inhibit p38 in amentoflavone and flavonoids groups which decreased ASK1 and p‐p38 levels through increasing thioredoxin Trx1 and reductase TrxR1. These results suggesting that the antioxidant protection of amentoflavone and flavonoids might be reducing ROS to inhibit the H₂O₂‐induced upstream of pathway via increasing levels of Trx1 and TrxR1, which were pivotal in blocking the down streaming effectors of ASK1/p38 MAPK pathway and alleviating hepatotoxicity.     

Enzymes involved in metabolism of extracellular nucleotides and nucleosides: Functional implications and measurement of activities

Abstract

Extracellular nucleotides and nucleosides mediate diverse signaling effects in virtually all organs and tissues. Most models of purinergic signaling depend on functional interactions between distinct processes, including (i) the release of endogenous ATP and other nucleotides, (ii) triggering of signaling events via a series of nucleotide-selective ligand-gated P2X and metabotropic P2Y receptors as well as adenosine receptors and (iii) ectoenzymatic interconversion of purinergic agonists. The duration and magnitude of purinergic signaling is governed by a network of ectoenzymes, including the enzymes of the nucleoside triphosphate diphosphohydrolase (NTPDase) family, the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, ecto-5′-nucleotidase/CD73, tissue-nonspecific alkaline phosphatase (TNAP), prostatic acid phosphatase (PAP) and other alkaline and acid phosphatases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). Along with “classical” inactivating ectoenzymes, recent data provide evidence for the co-existence of a counteracting ATP-regenerating pathway comprising the enzymes of the adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK/NME/NM23) families and ATP synthase. This review describes recent advances in this field, with special emphasis on purine-converting ectoenzymes as a complex and integrated network regulating purinergic signaling in such (patho)physiological states as immunomodulation, inflammation, tumorigenesis, arterial calcification and other diseases. The second part of this review provides a comprehensive overview and basic principles of major approaches employed for studying purinergic activities, including spectrophotometric P_i-liberating assays, high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analyses of purine substrates and metabolites, capillary electrophoresis, bioluminescent, fluorometric and electrochemical enzyme-coupled assays, histochemical staining, and further emphasizes their advantages, drawbacks and suitability for assaying a particular catalytic reaction.     

Divergent patterns of photosynthetic phosphorus-use efficiency versus nitrogen-use efficiency of tree leaves along nutrient-availability gradients

1.  Nitrogen (N) and phosphorus (P) are essential nutrients for photosynthetic carbon assimilation and most frequently limit primary productivity in terrestrial ecosystems. Efficient use of those nutrients is important for plants growing in nutrient-poor environments.
2.  We investigated the pattern of photosynthetic phosphorus-use efficiency (PPUE) in comparison with photosynthetic nitrogen-use efficiency (PNUE) along gradients of P and N availability across biomes with 340 tree and shrub species. We used both total soil N and P concentration and foliar N/P ratios for indicating nutrient-availability gradients.
3.  Photosynthetic phosphorus-use efficiency increased with greater leaf mass per area (LMA) toward decreasing P availability. By contrast, PNUE decreased with greater LMA towards decreasing N and P availability.
4.  The increase in PPUE with decreasing P availability was caused by the net effects of a relatively greater reduction in foliar P concentration and a relatively constant photosynthetic carbon assimilation rate. The decrease in PNUE with decreasing N availability was caused by the effects of a reduction in photosynthetic carbon assimilation rate with greater LMA.
5. Synthesis . Our results suggest that higher PPUE may be an effective leaf-level adaptation to P-poor soils, especially in tropical tree species. Future research should focus on the difference between PPUE and PNUE in relation to leaf economics, physiology and strategy.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Targeted delivery of NRASQ61R and Cre-recombinase to post-natal melanocytes induces melanoma in Ink4a/Arflox/lox mice

We have developed a somatic cell gene delivery mouse model of melanoma that allows for the rapid validation of genetic alterations identified in this disease. A major advantage of this system is the ability to model the multi-step process of carcinogenesis in immune-competent mice without the generation and cross breeding of multiple strains. We have used this model to evaluate the role of RAS isoforms in melanoma initiation in the context of conditional Ink4a/Arf loss. Mice expressing the tumor virus A (TVA) receptor specifically in melanocytes under control of the dopachrome tautomerase (DCT) promoter were crossed to Ink4a/Arf^lox/lox mice and newborn DCT-TVA/Ink4a/Arf^lox/lox mice were injected with retroviruses containing activated KRAS, NRAS and/or Cre-recombinase. No mice injected with viruses containing KRAS and Cre or NRAS alone developed tumors; however, more than one-third of DCT-TVA/Ink4a/Arf^lox/lox mice injected with NRAS and Cre viruses developed melanoma and two-thirds developed melanoma when NRAS and Cre expression was linked.     

猪瘟病毒E2蛋白A/D抗原区基因在酵母中的分泌表达与鉴定

基于猪瘟病毒主要保护性抗原E2囊膜糖蛋白有两个相对独立的抗原结构单位-B/C抗原区和A/D抗原区,设计一对特异性的引物扩增猪瘟病毒E2蛋白的A/D抗原区基因,并将PCR产物克隆入含有强启动子PAox1和α-MF信号肽序列的巴斯德毕赤酵母表达载体pPICZαC中,构建成重组质粒pPICZα-AD,酶切线性化后电穿孔导入巴斯德毕赤酵母X33菌中,经ZeocinTM筛选得到5株高拷贝转化子,甲醇诱导表达.SDS-PAGE和Westernblot试验表明酵母培养上清液中含有具有良好反应原性的E2蛋白,蛋白表达量达175.8μg/mL.N-糖基化分析显示该表达蛋白在分泌过程中发生糖基化.该研究为研制防治猪瘟的亚单位疫苗与诊断试剂盒奠定基础.