植物磷酸烯醇式丙酮酸羧化酶的功能及其在基因工程中的应用

植物磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC,EC 4.1.1.31)是广泛存在的一种细胞质酶,催化磷酸烯醇式丙酮酸(PEP)和HCO3-生成草酰乙酸(OAA),后者可转化生成三羧酸循环的多种中间产物.PEPC在植物细胞中参与植物的光合碳同化等重要代谢途径,并且在不同组织中具有多种生理功能.PEPC同时也参与调控植物种子的营养物质合成与代谢过程,控制糖类物质流向脂肪酸合成或蛋白质合成途径.以下介绍了植物PEPC的种类、蛋白质结构特点及其在植物组织中的调控方式,并重点论述了PEPC在生物基因工程中的应用方面的进展,随着对其功能机制和应用研究的深入,将有助于植物PEPC在高产优质农作物育种、能源植物和工业微生物等的开发利用等方面得到更好的发展与应用.     

Genetic and metabolic engineering of microorganisms for the development of new flavor compounds from terpenic substrates

Throughout human history, natural products have been the basis for the discovery and development of therapeutics, cosmetic and food compounds used in industry. Many compounds found in natural organisms are rather difficult to chemically synthesize and to extract in large amounts, and in this respect, genetic and metabolic engineering are playing an increasingly important role in the production of these compounds, such as new terpenes and terpenoids, which may potentially be used to create aromas in industry. Terpenes belong to the largest class of natural compounds, are produced by all living organisms and play a fundamental role in human nutrition, cosmetics and medicine. Recent advances in systems biology and synthetic biology are allowing us to perform metabolic engineering at the whole-cell level, thus enabling the optimal design of microorganisms for the efficient production of drugs, cosmetic and food additives. This review describes the recent advances made in the genetic and metabolic engineering of the terpenes pathway with a particular focus on systems biotechnology.     

The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: organization and implications for biotechnology and phylogenetic relationships amongst angiosperms

The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25 943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA . Furthermore, whole-genome comparisons identified large indels (> 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)– trnT (GGU) spacer, ycf4 – cemA spacer, trnI (GAU) intron and rrn5 – trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop.     

A general method for the quantitative analysis of functional chimeras: applications from site-directed mutagenesis and macromolecular association

Two new parameters, I: and C:, are introduced for the quantitative evaluation of functional chimeras: I: (impact) and C: (context dependence) are the free energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by substitution of a part "a" from ensemble A into the analogous position of homologous ensemble B (S:(B --> A)). The C: value is a measure of the interaction of the interrogated position with its surroundings, whereas I: is an expression of the quantitative importance of the probed position. Both I: and C: vary with the evaluated property, for example, kinetics, binding, thermostability, and so forth. The retro chimera is the reverse substitution of the analogous part "b" from B into A, S:(A --> B). The I: and C: values derived from original data for forward and retro mutations in aspartate and tyrosine aminotransferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tat with bovine and human TAR are evaluated. The most salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity by these two enzymes (I: < -2.9 kcal/mol). The T109S mutation in AATase produces a nearly equal and opposite effect to S109T in TATase (C: < 0.4 kcal/mol). Second, an I: value of 5.5 kcal/mol describes the effects of mirror mutations D94S (site 1) and S55D (site 2) in the Ca(2+) binding sites of oncomodulin on Ca(2+) affinity. The second mirror set, G98D (site 1) and D59G (site 2), yields a smaller impact (I: = -3.4 kcal/mol) on Ca(2+) binding; however, the effect is significantly more nearly context independent (C: = -0.6 versus C: = -2.7 kcal/mol). Third, the stem and loop regions of HIV and BIV TAR are predominantly responsible for the species specific interaction with BIV Tat(65-81) (I: = -1.5 to -1.6 kcal/mol), whereas I: = 0.1 kcal/mol for bulge TAR chimeras. The C: values are from -0.3 to -1.2 kcal/mol. The analysis described should have important applications to protein design.     

Defying the activity–stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability

The biotechnological applications of enzymes are limited due to the activity–stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity–stability trade-off can be overcome. In this review, the origin of the activity–stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity–stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel–Danson equilibrium model, catalytic antibodies, and life on cold planets.     

Identification of the antigenic epitopes in staphylococcal enterotoxins A and E and design of a superantigen for human cancer therapy

Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.     

Evidence for the coevolution of axon guidance molecule Netrin and its receptor Frazzled

Coevolution of a ligand and its receptor is critical for maintaining their function in different species, but how ligand and its receptor coevolve is poorly understood. The axon guidance molecule Netrin and its receptor Frazzled (Fra) are useful to study the mechanisms of ligand–receptor coevolution. Here, we have applied codon substitution models to identify positive selection of the netrin and fra genes. The sites under positive selection in netrin and fra were detected in same lineage, such as nematode, dipteran, hymenopteran, hemichordate, and teleost. Several amino acid residues that are under positive selection were identified in the interaction domains. Here we provide evidence that positive selection is essential for the coevolution of Netrin and Fra during central nervous system evolution.     

腹腔镜与开腹胃癌根治术治疗胃癌患者的疗效及对免疫功能和炎性因子的影响

目的:探讨不同手术方式治疗胃癌患者的疗效及对免疫功能和炎性因子的影响。方法:选择2015年1月-2018年4月我院收治的择期行根治性切除术胃癌患者236例,根据不同的手术方式分为对照组(n=107)和实验组(n=129),对照组实施传统开腹胃癌根治术,实验组实施腹腔镜胃癌根治术。观察两组患者各项临床症状及并发症发生情况,比较两组患者术前、术后1 d、术后7 d免疫功能和炎性因子水平。结果:与对照组相比,实验组患者的手术时间明显升高,而其他临床症状指标以及并发症发生率显著下降(P0.05),两组淋巴结清除数目比较无统计学差异(P0.05)。两组患者术后1 d的IL-6、TNF-α、CRP水平均明显高于术前,但实验组低于对照组(P0.05)。术后7 d,对照组IL-6、TNF-α、CRP水平仍高于术前(P0.05),但实验组TNF-α、CRP水平与术前比较无统计学差异(P0.05),同时实验组IL-6、TNF-α、CRP水平均显著低于对照组(P0.05)。术后1 d、7 d,两组CD3~+、CD4~+、CD4~+/CD8~+均低于术前,CD8~+高于术前,且实验组CD3~+、CD4~+、CD4~+/CD8~+高于对照组,CD8~+低于对照组(P0.05)。结论:与传统开腹胃癌根治术比较,腹腔镜胃癌根治术治疗胃癌患者疗效更好,可减轻炎性反应,对患者的免疫功能影响较小,安全可靠。     

A review of white rust (Puccinia horiana Henn.) disease on chrysanthemum and the potential for its biological control with Verticillium lecanii (Zimm.) Viégas

The biology, aetiology and epidemiology of Puccinia horiana, the cause of white rust disease of chrysanthemum (Chrysanthemum spp.) is reviewed in relation to current environmental, cultural and chemical methods for its control. Importantly, basidiospore release, germination and infection can take as little as 5 h at optimum r.h. (96%) and temperature (between 17–24°C). Recent developments using the fungus Verticillium lecanii for the control of insects on glasshouse-grown all-year-round chrysanthemums rely upon the maintenance of r.h. during night periods in excess of 95%, thus predisposing plants to white rust attack. However, V. lecanii is unusual in that it can also parasitise spores and fruiting structures of a range of rust fungi including P. horiana. This mycoparasitic ability is also reviewed, and against this background, the potential for an integrated insect and white rust control programme on all-year-round chrysanthemums is assessed.     

Discovery of multiple interacting partners of gankyrin,a proteasomal chaperone and an oncoprotein—Evidence for a common hot spot site at the interface and its functional relevance

Gankyrin, a non‐ATPase component of the proteasome and a chaperone of proteasome assembly, is also an oncoprotein. Gankyrin regulates a variety of oncogenic signaling pathways in cancer cells and accelerates degradation of tumor suppressor proteins p53 and Rb. Therefore gankyrin may be a unique hub integrating signaling networks with the degradation pathway. To identify new interactions that may be crucial in consolidating its role as an oncogenic hub, crystal structure of gankyrin‐proteasome ATPase complex was used to predict novel interacting partners. EEVD, a four amino acid linear sequence seems a hot spot site at this interface. By searching for EEVD in exposed regions of human proteins in PDB database, we predicted 34 novel interactions. Eight proteins were tested and seven of them were found to interact with gankyrin. Affinity of four interactions is high enough for endogenous detection. Others require gankyrin overexpression in HEK 293 cells or occur endogenously in breast cancer cell line‐ MDA‐MB‐435, reflecting lower affinity or presence of a deregulated network. Mutagenesis and peptide inhibition confirm that EEVD is the common hot spot site at these interfaces and therefore a potential polypharmacological drug target. In MDA‐MB‐231 cells in which the endogenous CLIC1 is silenced, trans‐expression of Wt protein (CLIC1_EEVD) and not the hot spot site mutant (CLIC1_AAVA) resulted in significant rescue of the migratory potential. Our approach can be extended to identify novel functionally relevant protein‐protein interactions, in expansion of oncogenic networks and in identifying potential therapeutic targets. Proteins 2014; 82:1283–1300. © 2013 Wiley Periodicals, Inc.     

A probabilistic model of biological ageing of the lungs for analysing the effects of smoking,asthma and COPD

Background

Although a large body of literature is available that describes the effects of smoking, asthma and COPD on lung function, most studies are restricted to a small age range and to one factor. As a consequence, available results are incomplete and often difficult to compare, also due to the ways the effects are expressed. Furthermore, current approaches consider one type of measurement only or several types separately.

Methods

We propose a probabilistic model that expresses the effects as number of years added to chronological age or, in other words, that estimates the biological age of the lungs. Using biological age as a measure of the effects has the advantage of facilitating the understanding of their severity and comparison of results. In our model, chronological age and other factors affecting the health status of the lungs generate biological age, which in turn generates lung function measurements. This structure enables the use of multiple types of measurement to obtain a more precise estimate of the effects and parameter sharing for characterization over large age ranges and of co-occurrence of factors with little data. We treat the parameters that model smoking habits and lung diseases as random variables to obtain uncertainty in the estimated effects.

Results

We use the model to investigate the effects of smoking, asthma and COPD on the TwinsUK Registry. Our results suggest that the combination of smoking with lung disease(s) has higher effect than smoking or lung disease(s) alone, and that in smokers, co-occurrence of asthma and COPD is more detrimental than asthma or COPD alone.

Conclusions

The proposed model or other models based on a similar approach could be of help in improving the understanding of factors affecting lung function by enabling characterizations over large age ranges and of co-occurrence of factors with little data and the use of multiple types of measurement. The software implementing the model can be downloaded at the first author’s webpage.     

A large anatomically preserved calamitean stem from the Upper Permian of southwest China and its implications for calamitean development and functional anatomy

A large permineralized calamitean stem, Arthropitys yunnanensis Tian et Gu from the Upper Permian of southwest China is reinvestigated and interpreted. The stem has a broad pith and well developed and large carinal canals. Secondary xylem is thick and characterized by wide parenchymatous interfascicular zones that remain constant in width throughout the wood. Striking features of the stem include the abundant leaf traces arranged in two whorls in the cortex with this arrangement previously unrecognized within calamitean stems, and the presence of growth rings in secondary xylem that suggest frequent fluctuations in environmental stress presumably due to variations in water availability. Features of A. yunnanensis infer the stem to be in the epidogenetical phase of calamitean development, and suggest it to be the basal part of a large trunk. Comparisons with biomechanical models for calamitean stems suggest this species had a semi-self supporting habit.     

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long‐chain polyunsaturated fatty acids (LC‐PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC‐PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC‐PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC‐OX and pNOC‐stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase‐encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC‐PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.     

Case studies on the use of biotechnologies and on biosafety provisions in four African countries

This review is based on a study commissioned by the European Commission on the evaluation of scientific, technical and institutional challenges, priorities and bottlenecks for biotechnologies and regional harmonisation of biosafety in Africa. Biotechnology was considered within four domains: agricultural biotechnologies (‘Green’), industrial biotechnologies and biotechnologies for environmental remediation (‘White’), biotechnologies in aquaculture (‘Blue’) and biotechnologies for healthcare (‘Red’). An important consideration was the decline in partnerships between the EU and developing countries because of the original public antipathy to some green biotechnologies, particularly genetically modified organisms (GMOs) and food from GM crops in Europe. The study focus reported here was West Africa (Ghana, Senegal, Mali and Burkina Faso).The overall conclusion was that whereas high-quality research was proceeding in the countries visited, funding is not sustained and there is little evidence of practical application of biotechnology and benefit to farmers and the wider community. Research and development that was being carried out on genetically modified crop varieties was concentrating on improving food security and therefore unlikely to have significant impact on EU markets and consumers. However, there is much non-controversial green biotechnology such as molecular diagnostics for plant and animal disease and marker-assisted selection for breeding that has great potential application. Regarding white biotechnology, it is currently occupying only a very small industrial niche in West Africa, basically in the sole sector of the production of liquid biofuels (i.e., bio-ethanol) from indigenous and locally planted biomass (very often non-food crops). The presence of diffused small-scale fish production is the basis to develop and apply new (Blue) aquaculture technologies and, where the research conditions and the production sector can permit, to increase this type of production and the economy of this depressed areas. However, the problems bound to environmental protection must not be forgotten; priority should be given to monitor the risks of introduction of foreign species. Red biotechnologies potentially bring a vast domain of powerful tools and processes to achieve better human health, most notably improved diagnostics by molecular techniques, better targeting of pathogens and a better knowledge of their sensitivities to drugs to permit better treatment.Biosafety regulatory frameworks had been initiated in several countries, starting with primary biosafety law. However, disparate attitudes to the purpose of biosafety regulation (e.g., fostering informed decision-making versus ‘giving the green-light for a flood of GMOs’) currently prevent a needed consensus for sub-regional harmonisation. To date, most R&D funding has come from North America with some commercial interests from Asia, but African biotechnology workers expressed strong desire for (re-)engagement with interested parties from the European Union. Although in some of the visited countries there are very well qualified personnel in molecular biology and biosafety/regulation, the main message received is that human resources and capacity building in-house are still needed. This could be achieved through home-based courses and capacity-building including funds for post-degree research to motivate and retain trained staff.     

A novel big protein TPRBK possessing 25 units of TPR motif is essential for the progress of mitosis and cytokinesis

Through the comprehensive analysis of the genomic DNA sequence of human chromosome 22, we identified a novel gene of 702 kb encoding a big protein of 2481 amino acid residues, and named it as TPRBK (TPR containing big gene cloned at Keio). A novel protein TPRBK possesses 25 units of the TPR motif, which has been known to associate with a diverse range of biological functions. Orthologous genes of human TPRBK were found widely in animal species, from insecta to mammal, but not found in plants, fungi and nematoda. Northern blotting and RT-PCR analyses revealed that TPRBK gene is expressed ubiquitously in the human and mouse fetal tissues and various cell lines of human, monkey and mouse. Immunofluorescent staining of the synchronized monkey COS-7 cells with several relevant antibodies indicated that TPRBK changes its subcellular localization during the cell cycle: at interphase TPRBK locates on the centrosomes, during mitosis it translocates from spindle poles to mitotic spindles then to spindle midzone, and through a period of cytokinesis it stays on the midbody. Co-immunoprecipitation assay and immunofluorescent staining with adequate antibodies revealed that TPRBK binds to Aurora B, and those proteins together translocate throughout mitosis and cytokinesis. Treatments of cells with two drugs (Blebbistatin and Y-27632), that are known to inhibit the contractility of actin–myosin, disturbed the proper intracellular localization of TPRBK. Moreover, the knockdown of TPRBK expression by small interfering RNA (siRNA) suppressed the bundling of spindle midzone microtubules and disrupted the midbody formation, arresting the cells at G₂ + M phase. These observations indicated that a novel big protein TPRBK is essential for the formation and integrity of the midbody, hence we postulated that TPRBK plays a critical role in the progress of mitosis and cytokinesis during mammalian cell cycle.     

Construction of miRNA-mRNA network for the identification of key biological markers and their associated pathways in IgA nephropathy by employing the integrated bioinformatics analysis

BackgroundAbout half-century ago, Immunoglobulin A nephropathy (IgAN) was discovered as a complicated disease with frequent clinical symptoms. Until now, exact mechanism underlying the pathogenesis of IgAN is poorly known. Therefore, current study was aimed to understand the molecular mechanism of IgAN by identifying the key miRNAs and their targeted hub genes. The key miRNAs might contribute to the diagnosis and therapy of IgAN, and could turn out to be a new star in the field of IgAN.MethodsThe microarray datasets were downloaded from Gene Expresssion Omnibus (GEO) database and analyzed using R package (LIMMA) in order to obtain differential expressed genes (DEGs). Then, the hub genes were identified using cytoHubba plugin of cytoscpae tool and other bioinformatics approaches including protein-protein interaction (PPI) network analysis, module analysis, and miRNA-hub gene network construction was also performed.ResultsA total of 348 DEGs were identified, of which 107 were upregulated genes and 241 were downregulated genes. Subsequently, the 12 overlapped genes were predicted from cytoHubba, and considered as hub genes. Moreover, a network among miRNA-hub genes was created to explore the correlation between the hub genes and their targeted miRNAs. Network construction ultimately lead to the identification of nine gene named FN1, EGR1, FOS, JUN, SERPINE1, MMP2, ATF3, MYC, and IL1B and one novel key miRNA namely, has-miR-144-3p as biomarker for diagnosis and therapy of IgAN.ConclusionThis study updates the information and yield a new perspective in context of understanding the pathogenesis and development of IgAN. In future, key miRNAs might be capable of improving the personalized detection and therapies for IgAN. In vivo and in vitro investigation of miRNAs and pathway interaction is essential to delineate the specific roles of the novel miRNAs, which may help to further reveal the mechanisms underlying IgAN.