棕曲霉毒素A的制备

本文建立了一般实验室条件下棕曲霉毒素A大的制备方法.以从粮食上分离的棕曲霉菌Ly8604作为生产菌株,接种于灭菌的湿全麦粉上,于25℃培养三周.培养物以氯仿:甲醇(5:1,V/V)提取,用碳酸氢钠水溶液进行液液分配,水相酸化后再用氯仿萃取,经硅胶柱层析净化.用苯:乙酸(9:1)洗脱,紫外光灯下收集黄绿色荧光溜分,浓缩后进行磋胶薄层层析,用甲苯:乙酸乙脂:90甲 酸(5:4:1)展开,紫外光灯下刮取黄绿色荧光谱带,再在色谱柱上用氯仿洗脱后于苯中结晶.产品为无色针状晶体.熔点为89℃～91℃,荧光最大激发波长为340nm ,最大发射波长为470nm.UV、IR、NMR和MS谱图证明产品是棕曲霉毒索A.

探讨老年肺炎患者临床诊断中检验血清C反应蛋白(CRP)、血沉(ESR)、以及D-二聚体(D-D)水平的临床价值

目的探讨老年肺炎患者临床诊断中检验血清C反应蛋白(CRP)、血沉(ESR)、以及D-二聚体(D-D)水平的临床价值。方法随机选择该院2015年1月—2016年12月期间收治的50例老年肺炎病例为观察组研究对象,并以同期自愿参与研究的50名健康老年体检者为对照组研究对象,比较两组血清C反应蛋白(CRP)、血沉(ESR)、以及D-二聚体(D-D)水平。结果观察组老年患者血清CRP(103.2±23.4)mg/L、ESR(35.8±6.5)μg/L、D-D(1 152.4±43.1)mm/h水平均显著高于对照组(8.9±1.2)mg/L、(10.2±1.4)μg/L、(182.6±14.8)mm/h,差异有统计学意义(P<0.05)。将观察组以80岁为临界值分组,80岁以上患者CRP(102.4±21.8)mg/L、ESR(34.8±5.8)μg/L、D-D(1 148.7±45.2)mm/h与80岁以下患者(103.8±22.1)mg/L、(35.4±6.2)μg/L、(1 142.3±43.9)mm/h,差异无统计学意义(P>0.05)。结论临床上可行血清C反应蛋白、血沉及D-二聚体检测来诊断老年肺炎的情况,为治疗和预后提供有效依据。

Molecular Cloning and Sequence Analyses of cDNAs Encoding Seven C-type Lectin-like Protein Subunits from Daboia russellii siamensis

Total mRNA was extracted from a venom gland of snake Daboia russellii siamensis following the manufacturer＇s protocol of the PolyATtract System 1000 kit purchased from Promega Biotech. cDNAs encoding C-type lectins were amplified by RT-PCR and subcloned into a pMD18-T vector. Fourteen positive clones were selected for nucleotide sequencing and seven cDNAs encoding various snake venom C-type lectin-like protein precursors, designated as DRS-L1, DRS-L2, DRS-L3, DRS-L4 DRS-L5, DRS-L6 and DRS-L7, were obtained. Amino acid sequences of these proteins were deduced and each contains a carbohydrate recognition domain. Of all the deduced protein sequences, only DRS-L1 seemed to represent a closer sequence similarity to α subunits of other known snake venom C-type lectin-like proteins using the BLAST program. Homology comparison combined with analysis of cysteine position indicate that DRS-L1 and DRS-L2 are probably the light chain LC2 and LC1 of factor X activator from Daboia russellii siamensis venom, respectively. DRS-L3 and DRS-L4 might be the β subunits of higher molecular weight C-type lectin-like proteins while DRS-L5 and DRS-L6 might be β subunits of lower molecular weight C-type lectin-like proteins. DRS-L7 might be the β subunit of a platelet membrane glycoprotein Ib-binding protein similar to echicetin.

Alpha-picolinic acid,a fungal toxin and mammal apoptosis-inducing agent,elicits hypersensitive-like response and enhances disease resistance in rice

Alpha-picolinic acid (PA),a metabolite of tryptophan and an inducer of apoptosis in the animal cell,has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here,we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS),rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride,indicating that the generation of reactive oxygen species was NADPHoxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence,it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice,which also provides a simple eliciting tool for studying apoptosis in the plant cell.

The Nucleolus and Viral Infection

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.

The Trichoderma-plant interaction is mediated by avirulence proteins produced by this fungus

The molecular basis of Trichoderma -plant interaction is very complex and still not completely understood. The colonization of the root system by rhizosphere competent strains of Trichoderma results in increased development of root/aerial systems, in improved yields and in plant disease control. Other beneficial effects, such as the induction of plant systemic resistance, have also been described. To understand the mechanisms involved we are using different approaches, including the making of transformants expressing genes that encode for compounds able to affect plant response to pathogens. Trichoderma transformants carrying the avirulence gene Avr4 from Cladosporium fulvum under the control of constitutive and inducible promoters were obtained and tested on tomato plants having the Cf4 resistance gene. Necrosis and suberification zones, similar to the symptoms appearing during Cladosporium-tomato interaction, were found when the roots of the Cf4 plants were treated with Avr4-Trichoderma. This demonstrates that selected Trichoderma strains are able to transfer to the plant molecules that may deeply affect metabolism, disease resistance etc. Therefore, these beneficial fungi can be regarded as biotechnological tools to provide a variety of crops with useful compounds. Moreover, in in vitro competition assays the transformants were found to be more effective as antagonists against Alternaria alternata than the wild type. Trichoderma sends a variety of biochemical signals to the plants including avirulence molecules; therefore the presence of avr-like proteins in the fungus proteome was investigated. Proteome analysis has permitted us to isolate and sequence many proteins potentially having this function. From the extracellular protein extracts, we have purified and sequenced a protein with structural characteristics similar to Avr4 of C. fulvum. The protein, Hytra1, was found to be a hydrophobin with chitin binding activity, the typical 8 cysteine residues, and 4 disulfide bridges. Infiltrations of the extracellular protein fractions containing Hytra1 resulted in a strong HR reaction on tobacco and tomato leaves, and induction of a novel phytoalexin.

Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

Objective： To construct a lentiviral expression vector for RNA interference （RNAi） of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods： Three pairs of human VIM gene short hairpin RNA（shRNA） sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides （dsOligo） were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction （PCR） and DNA sequencing analysis. Real-time PCR and Westemblotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 calls were validated by real-time PCR and Western-blotting. Results： An effective Lv-VIM-shRNA was successfully constructed. The titer of lentivirus was determined on 2× 10^9TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion： An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.

Toxigenic Fungi and Mycotoxins in Organic Spelt and Its Products

This study shows that the main cause of Fusarium head blight of spelt was F. poae. In 2007 deoxynivalenol was found up to 0.27 mg/kg in 2 of 18 samples of winter spelt kernels from organic farms. Also in 3 samples T-2 toxin was found in amount below 0.075 mg/kg. Aflatoxins and ochratoxin A were not found in kernels. Among nine of the examined samples of winter spelt in 2008, DON was identified in all samples （up to 0.31 mg/kg）, while T-2 toxin, aflatoxins and OTA were not found. Among twenty of the examined cultivars of winter spelt, deoxynivalenol was identified in 6 samples （up to 0.3 mg/kg）, T-2 toxin was identified in one sample in very low amount （below 75 μg/kg） while aflatoxins and ochratoxin A were not found. Deoxynivalenol was found in following winter spelt cultivars： T. spelta L. album, T. spelta BG, T. spelta BG 1166, T. spelta, Schwabenspelz and Franckenkorn. T-2 toxin was identified in T. spelta L. album BG 31. Among 13 products from spelt, DON was detected in 1 sample, OTA in 1 sample and zearalenone in 1 sample, T-2 toxins and aflatoxins were not found.

Reduction of Patulin Content in Apple Juice Using Activated Carbone

The mycotoxin, patulin （4-hydroxy-4H-furo [3, 2c] pyran-2 [6H]-one）, is produced by a number of fungi common to fruit and vegetable-based products, most notably apples. Patulin contamination within apple products poses a serious health risk to consumers. Studies done on laboratory animals have demonstrated that patulin has a broad spectrum of toxicity, including mutagen city and carcinogenicity. The aim of the experiment was studying influence of selectively acting activated carbon powder--Ercarbon SH （Erbsloh, Germany） which is special produced for lowering HMF （hydroxy methyl furfural）, on reduction of patulin content in clear apple juice. Industrial apple row material with some damaged parts was pressed, juice was pasteurized at 95 ℃ during 2 min. After cooling on 55 ℃, enzymatic treated and clarified juice were filtered by 0.45 [am pore sizes membrane filter, Apple clear juice sample was divided for five parts. The samples of apple juice were diluted to 11.5° Brix and contacted with concentrations of 2, 2.5, 3 and 3.5 g/L activated carbon powder for 30 min. After filtration in the experimental samples, putulin was quantitatively determined by HPLC （high performance liquid chromatography with UV） detector at 276 nm. The research revealed that the best results were achieved by treatment with activated carbon in its powder form at concentration of 2.5 g/L with 30 min contact time.

Wheat Genetic Transformation as Efficient Tools to Fight against Fungal Diseases

Wheat ranks first among cereal crops cultivated in the world. In its production, diseases like powdery mildew, fusarium head blight and rusts caused by fungal pathogens represent a major problem. They produce different symptoms that cause severe crop damage by infecting the spikes, leaves, roots, stems and grains. They are causing losses both by reducing the quantity of the harvested crop and the quality of the product. Quality problems of the harvested product can be due to shrivelled seed, which are frequently found as a consequence of the infection by leaf pathogens, such as mildews, rusts and Septoria. Fusarium head blight is the major culprit for mycotoxin contamination from the harvested grain, causing economic losses and in the worst casing human and animal health problems. In severe epidemics, all these fungal diseases can significantly reduce yield. Resistance to fungi is beneficial not only from a commercial point of view （yield）, but also because of the reduced levels of mycotoxins. The integration of transgenic approaches offers a potential chemical-free and environment-friendly solution for controlling fungal pathogens. This is an essential asset for wheat world food security.

Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression

In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse genetic research of SARS-CoV.

Genes and Transcriptional Factors in Chili Plant with Aspect to Metabolism and Resistance against Virus, Bacteria and Fungi： A Review

These days, there is a lot of discussion about genetically modified plants. There are different schools of thoughts in public, and some people adjusted while others are reluctant to accept genetically modified organism foods. Many vegetables are transformed and are used in daily life. Chili is one of those which is genetically modified and used in our food. Race specific genes can be used more efficiently for disease resistance and improving metabolic pathways. Different genes and transcriptional factors are available in Capsicum for this purpose. We can optimize and use the better expressed genes while engineering the chili plants, Genetic modifications causing significant changes are related with metabolism, which cause disease resistance.

Modeling active worm propagation on the P2P network

This paper analyzes the characteristics of the Peer-to-Peer （P2P） active worm and its attacking mechanism, and then proposes a mathematical model of propagation of the P2P active worm applying Epidemiology. Based on the analysis on the protocols of realistic P2P systems, a software which can be used to simulate the P2P network environment and the propagation of P2P active worm is imple- mented in this paper. A large number of simulation experiments are performed using the developed simulation software. The results from these simulation experiments validate the proposed model, which means that the model can be used to analyze the spreading behaviors of the P2P active worm and predict its trend.

Carbendazim resistance and calculation effective concentration of carbendazim for Trichoderma harzianum

There is a method for investigating the transformation of resistance gene of carbendazim into Trichoderma harzianum. In order to introduce the resistance to benzimidazole fungicide into bio-control microorganism Trichoderma harzianum was transformed with the resistance gene. In this study, we investigate resistance level and calculate EC 50 (effective concentration of carbendazim that can survive 50% of Trichoderma harzianum in that concentration) and stability of the resistance for the transformant isolate of Trichoderma harzianum. Results show the transformants can growth on the medium containing more than 1 000 μg/ml carbendazim and the resistance is stabled after 10 times transfer on non-selective medium and have EC 50 average about, 1 200 μg/ml.

Fungal Population Dynamics in Ready-to-eat Salads During a Shelf-life in Italy

The aim of this work was to investigate the fungal population dynamics in ready-to-eat bagged samples of rocket （Diplotaxis spp.）, lettuce baby leaf （Lactuca sativa L.） and ＂songino＂ （Valerianella olitoria L.） during a shelf-life, in order to evaluate the effects of the storage length and season of production on the spoilage processes. The incidence of toxigenic moulds was particularity studied in order to evaluate a potential production of mycotoxins and allergenic conidia. A total of 900 samples collected from 10 Italian trademarks were analyzed at the 2nd, 5th and 8th day after the packaging in the spring and summer. A very high number of fungi was found and a great variability of moulds and yeasts at the 1 st day of sampling was observed. Regarding to season of production, any seasonal effect on the moulds and yeasts has been observed, but the moulds detected belonged to different species in relation to season. Regarding to storage length, the yeasts and moulds did not showed significant variations during a shelf-life. In relation to vegetable species, the lettuce resulted always less contaminated with respect to other salads, and the rocket presented 1-2 Log cfu/g of increasing in the level of moulds. Regarding to fungi species, the yeasts were significantly predominant respect to moulds. Finally, the toxigenic moulds Aspergillusflavus and Penicillium italicum were found in all the types of salad in the summer, and their growth during the storage at low temperature represented a potential hazard for the mycotoxins and allergenic conidia production in these commodities.

AcMNPV As A Model for Baculovirus DNA Replication

Baculoviruses were first identified as insect-specific pathogens, and it was this specificity that lead to their use as safe, target specific biological pesticides. For the past 30 years, AcMNPV has served as the subject of intense basic molecular research into the baculovirus infectious cycle including the interaction of the virus with a continuous insect cell line derived from Spodoptera frugiperda. The studies on baculoviruese have led to an in-depth understanding of the physical organization of the viral genomes including many complete genomic sequences, the time course of gene expression, and the application of this basic research to the use of baculoviruses not only as insecticides, but also as a universal eukaryotic protein expression system, and a potential vector in gene therapy. A great deal has also been discovered about the viral genes required for the replication of the baculovirus genome, while much remains to be learned about the mechanism of viral DNA replication. This report outlines the current knowledge of the factors involved in baculovirus DNA replication, using data on AcMNPV as a model for most members of the Baculoviridae.

Expression and semi-quantification of hepatitis B virus reverse transcriptase protein in a prokaryotic system

The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.

沸腾的狐狸

一 太阳真毒.柏油路被烤化了,路上的小石子化了,微微乱窜的风和我也化了.唯一高兴的是麦子,一片一片,钉在路边.刚收获的小麦,一边喝柏油一边被太阳炙烤.路两侧群山环绕,山谷间一丛丛绿树,遮挡着或有或无的村庄.前方一百米终于看到了一个人.女人,确切讲应该是农村妇女.她头戴斗笠,长袖长裤,正拎着一只靶子,在麦子上跳舞,为成千上万麦粒翻身.走到离她二十米时看清了她的容貌,皱纹乍起,脸上一团云.至少三十岁吧,也可能更大.

Potential natural exposure of endangered red-crowned crane(Grus japonensis) to mycotoxins aflatoxin B_1, deoxynivalenol, zearalenone, T-2 toxin, and ochratoxin A

A survey was conducted to determine whether mycotoxins were present in the foods consumed by red-crowned cranes（Grus japonensis） in the Yancheng Biosphere Reserve, China. Collected in the reserve＇s core, buffer, and experimental zones during overwintering periods of 2013 to 2015, a total of 113 food samples were analyzed for aflatoxin B1, deoxynivalenol, zearalenone, T-2 toxin, and ochratoxin A using high performance liquid chromatography（HPLC）. The contamination incidences vary among different zones and the mycotoxins levels of different food samples also presented disparity. Average mycotoxin concentration from rice grain was greater than that from other food types. Among mycotoxin-positive samples, 59.3% were simultaneously contaminated with more than one toxin. This study demonstrated for the first time that red-crowned cranes were exposed to mycotoxins in the Yancheng Biosphere Reserve and suggested that artificial wetlands could not be considered good habitats for the birds in this reserve, especially rice fields.

Identification of Pns12 as the second silencing suppressor of Rice gall dwarf virus

RNA silencing is a conserved mechanism found ubiquitously in eukaryotic organisms.It has been used to regulate gene expression and development.In addition,RNA silencing serves as an important mechanism in plants' defense against invasive nucleic acids,such as viruses,transposons,and transgenes.As a counter-defense,most plants,and some animal viruses,encode RNA silencing suppressors to interfere at one or several points of the silencing pathway.In this study,we showed that Pns12 of RGDV (Rice gall dwarf virus) exhibits silencing suppressor activity on the reporter green fluorescent protein in transgenic Nicotiana benthamiana line 16c.Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA.Expression of Pns12 also enhanced Potato virus X pathogenicity in N.benthamiana.Collectively,these results suggested that RGDV Pns12 functions as a virus suppressor of RNA silencing,which might target an upstream step of dsRNA formation in the RNA silencing pathway.Furthermore,we showed that Pns12 is localized mainly in the nucleus of N.benthamiana leaf cells.