灰霉菌及其胞外大分子对不同植物的伤害鉴定

[目的]探究灰霉菌及其胞外大分子毒素对不同植物的伤害情况。[方法]以灰霉菌分生孢子及其分泌的胞外大分子毒素为试验材料,选取21科29属30种植物为供试植物,观察了灰霉菌对其侵染性以及灰霉菌分泌至培养液中的大分子毒素对其伤害情况。[结果]30种供试植物中既被灰霉菌侵染又被毒素损害的植物有17种,占全部植物的56.7%;有2种植物均不能被灰霉菌侵染也不能被毒素伤害。[结论]为进一步了解植物病原真菌毒素致病机制提供了借鉴。     

Shellfish Toxins Targeting Voltage-Gated Sodium Channels

Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management.     

Exposure to the Neurotoxic Dinoflagellate,Alexandrium catenella,Induces Apoptosis of the Hemocytes of the Oyster,Crassostrea gigas

This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.     

转基因植物及其食品安全性评估方法

转基因产业的迅猛发展使转基因食品从实验室走向餐桌,转基因食品的安全性已为全世界所关注,它涉及到贸易壁垒和人体健康。文章从食品安全的内涵和外延出发,在实质等同性原则的基础上,从外源基因、遗传修饰、非预期效应等方面论述了转基因植物及其食品的安全性评估方法,以便为我国的转基因植物安全性评估和政府监管提供参考。     

镰刀菌毒素对植物形态和结构的影响

镰刀菌毒素是镰刀菌产生的非寄主选择性毒素,可导致植物萎蔫、根腐、穗腐等各种病症。本文综述了镰刀菌毒素的主要种类、产毒菌种及其侵染的主要植物,毒素作用机理及其对植株外观形态、细胞及细胞器结构、维管束等的影响,并提出了镰刀菌毒素研究在抗性材料筛选和抗性机理研究方面的应用前景。     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

Toxin levels and profiles in microalgae from the north-Western Adriatic Sea--15 years of studies on cultured species

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported.     

Augmenting Anti-Cancer Natural Products with a Small Molecule Adjuvant

Aquatic microbes produce diverse secondary metabolites with interesting biological activities. Cytotoxic metabolites have the potential to become lead compounds or drugs for cancer treatment. Many cytotoxic compounds, however, show undesirable toxicity at higher concentrations. Such undesirable activity may be reduced or eliminated by using lower doses of the cytotoxic compound in combination with another compound that modulates its activity. Here, we have examined the cytotoxicity of four microbial metabolites [ethyl N-(2-phenethyl) carbamate (NP-1), Euglenophycin, Anabaenopeptin, and Glycolipid 652] using three in vitro cell lines [human breast cancer cells (MCF-7), mouse neuroblastoma cells (N2a), and rat pituitary epithelial cells (GH4C1)]. The compounds showed variable cytotoxicity, with Euglenophycin displaying specificity for N2a cells. We have also examined the modulatory power of NP-1 on the cytotoxicity of the other three compounds and found that at a permissible concentration (125 µg/mL), NP-1 sensitized N2a and MCF-7 cells to Euglenophycin and Glycolipid 652 induced cytotoxicity.     

Bt Cry1类毒素共性结构域的分析、表达及鉴定

【目的】分析定位Bt Cry1类毒素Cry1Ab、Cry1Ac、Cry1B、Cry1C、Cry1F的共性结构域,克隆并表达共性结构域蛋白,为筛选Bt毒素广谱抗体及建立广谱检测方法打下基础。【方法】利用生物信息学和分子模拟技术,通过SWISS-MODEL同源建模分别对5种Cry1类毒素进行三维建模,并结合Ramachandran plot、ERRAT和Verify3D方法评价模型构象的合理性。通过分析比对5种Cry1类毒素的三维结构,确定DomainⅠ区域作为5种Cry1毒素的共性结构域。以含Cry1Ac基因的苏云金芽孢杆菌库斯塔克亚种为模板设计引物,PCR扩增获得共性结构域DomainⅠ基因,将其经NcoⅠ和NotⅠ双酶切连接至原核表达载体pET-26b(+),构建原核表达载体pET-26b-DomainⅠ。重组质粒经菌液PCR、双酶切以及测序鉴定验证正确后,转化至E.coli BL21(DE3),经终浓度为1 mmol·L~(-1)的IPTG在20℃下诱导表达16h后检测共性结构域蛋白的表达情况。离心收集诱导表达的大肠杆菌菌液,进行超声波破碎处理,收集上清及沉淀,采用SDS-PAGE分析融合蛋白的表达。利用His-Trap HP镍亲和柱纯化上清中的可溶性融合蛋白,经SDS-PAGE电泳、Western blot和ELISA试验验证纯化的共性结构域蛋白的生物活性。【结果】基于氨基酸序列及三维空间比对分析,发现5种Cry1类毒素的DomainⅠ的序列一致性最高,而且它们的DomainⅠ三维结构几乎完全重合,确定DomainⅠ区域作为5种Cry1毒素的共性结构域,通过PCR、双酶切及测序鉴定成功构建原核表达载体pET-26b-DomainⅠ,经IPTG诱导表达、His-Trap HP镍亲和柱纯化获得了可溶性的DomainⅠ共性结构域蛋白,SDS-PAGE和Western blot证实表达的共性结构域蛋白的分子量约为33.4 kD,且能与抗His标签鼠单克隆抗体发生特异性反应,ELISA试验证实共性结构域蛋白与5种Cry1类毒素特异性抗体均具有很强的结合能力,抗原表位分析结果显示共性结构域蛋白具有和完整的Cry蛋白存在多个潜在抗原表位位点的特征,抗原表位区域所占的比例分别为48.4%和63.6%,表明共性结构域蛋白具有良好的免疫原性和免疫反应性。【结论】基于分子模拟与分子克隆技术,成功定位及表达纯化获得共性结构域蛋白,为下一步利用共性结构域为靶标分子制备广谱特异性识别Cry1类毒素抗体打下基础。