两株海洋真菌的鉴定及其次级代谢产物抑制烟草花叶病毒及抗肿瘤活性

摘要：【目的】通过对2株活性海洋真菌发酵产物提取物抑制烟草花叶病毒和抗肿瘤活性进行研究,为进一步得到活性纯品化合物作为抗病毒及抗肿瘤的先导化合物奠定基础。【方法】菌株发酵产物的粗提物是通过甲醇浸取并在真空条件下蒸干得到的。粗提物中溶于水的部分为水溶性部分,不溶于水的部分为脂溶性部分。通过间接酶联免疫法检测样品抑制烟草花叶病毒的活性,通过四甲基偶氮唑盐微量酶反应比色法（MTT法）检测样品抗肿瘤活性,通过形态及ITS rDNA序列法进行菌株鉴定。【结果】两株海洋真菌抑制烟草花叶病毒活性和抗肿瘤的活性均较高。分子鉴定结果显示,两株真菌分别与Penicillium oxalicum 和 Neosartorya fischeri 的同源性极高。菌株0312F1发酵液的水溶性部分具有抗病毒及抗肿瘤活性,菌株1008F1发酵液的脂溶性部分具有抑制烟草花叶病毒活性,而水溶性部分具有抗肿瘤活性。【结论】菌株0312F1和菌株1008F1发酵液的提取物抑制烟草花叶病毒的活性部位不同,而抗肿瘤活性部位相同。菌株0312F1发酵液提取物的水溶性活性部位对肝癌细胞BEL-7404的抑制效果比对胃癌细胞SGC-7901的抑制效果明显,而菌株1008F1发酵液提取物的水溶性活性部位对胃癌细胞SGC-7901的抑制效果比对肝癌细胞BEL-7404的抑制效果明显。     

Elastic properties of viruses

Viruses are compact biological nanoparticles whose elastic and dynamical properties are hardly known. Inelastic (Brillouin) light scattering was used to characterize these properties, from microcrystals of the Satellite Tobacco Mosaic Virus, a nearly spherical plant virus of 17-nm diameter. Longitudinal sound velocities in wet and dry Satellite Tobacco Mosaic Virus crystals were determined and compared to that of the well-known protein crystal, lysozyme. Localized vibrational modes of the viral particles (i.e., particle modes) were sought in the relevant frequency ranges, as derived assuming the viruses as full free nanospheres. Despite very favorable conditions, regarding virus concentration and expected low damping in dry microcrystals, no firm evidence of virus particle modes could be detected.     

Translation of tobacco mosaic virus RNA In Acetabularia cell cytoplasm.

Isolated Acetabularia nuclei were microinjected with Tobacco Mosaic Virus RNA and then implanted into an anucleate posterior fragment of an Acetabularia cell. Injected RNA was translated in the Acetabularia cytoplasm from the first to twelfth day after implantation of the nuclei. The production of specific virus proteins was detected and localized in the Acetabularia cytoplasm by an immunofluorescence precipitation technique.     

In vitro synthesis of turnip yellow mosaic virus coat protein in a wheat germ cell-free system

Turnip Yellow Mosaic Virus RNA directs the synthesis in vitro of its coat protein in a wheat germ cell-free extract. Optimum conditions for synthesis have been defined, and the effect of spermine on specifically enhancing coat protein formation has been examined. Identity between the in vitro synthesized coat protein and authentic coat protein of Turnip Yellow Mosaic Virus was established by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunoprecipitation.     

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.Abbreviations PPV Plum Pox Virus - CP coat protein - CaMV Cauliflower Mosaic Virus - CP+ coat protein expressing plant - CP– control plant = non coat protein expressing plant - TMV Tobacco Mosaic Virus - NPTII neomycin phosphotransferaseII - IBA indole-3-butyric acid - BAP 6-benzylaminopurine; - MS Murashige Skoog - ELISA enzyme linked immunosorbent assay     

Effects of two TMV strains on the synthesis and stability of chloroplast ribosomal RNA in tobacco leaves

Summary Multiplication of TMV-strains vulgare (light-green/dark-green mosaic symptoms) and flavum (severe yellow/green mosaic) had different effects on the ribosomal RNA of tobacco leaf chloroplasts. Vulgare inhibited chloroplast ribosomal RNA synthesis while having no effect on cytoplasmic ribosomal RNA synthesis (Fig. 2). Flavum inhibited chloroplast ribosomal RNA synthesis more severely than vulgare, and caused an earlier degradation of chloroplast ribosomal RNA than in control or vulgare-infected leaves (Fig. 1). Flavum also inhibited cytoplasmic ribosomal RNA synthesis. A connection between these differing effects on chloroplast ribosomal RNA metabolism and severity of visible symptoms is suggested, and discussed in relation to a possible influence on symptoms of denatured virus coat protein.Abbreviations TMV Tobacco Mosaic Virus - RNA Ribonucleic acid - DNA Deoxyribonucleic acid - m millions (in molecular weight values)     

Protein and virus crystal growth on international microgravity laboratory-2.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.     

Tobacco mosaic virus: the first century

Tobacco Mosaic Virus: Pioneering Research for a Century, organized by The Royal Society of Edinburgh, in conjunction with The Royal Society, was held at The Royal Society of Edinburgh, UK, 7–8 August 1998.     

Expression of the crucifer-infecting TMV-Cg movement protein in tobacco plants complements in trans a TMV-U1 trafficking-deficient mutant

Tobamovirus movement proteins play a determinant role in the establishment of infections in plants, allowing the local movement of viral RNA genome through plasmodesmatas. We expressed the movement protein (MP) of the crucifer- and garlic-infecting Tobacco Mosaic Virus strain Cg (TMV-Cg) in both resistant Xanthi NN and sensitive Xanthi nn Nicotiana tabacum plants. MP-Cg function was assayed by inoculating transgenic plants with a trafficking-deficient mutant of TMV strain U1. Following infection, local necrotic lesions were developed in resistant transgenic plants, and a systemic infection was produced in sensitive tobaccos. Thus, movement function of the mutant virus was complemented in trans by MP-Cg expressed in transgenic plants, causing the same symptoms as wild-type strain. We demonstrated that the function of MP-U1 could be replaced efficiently by MP-Cg, even though these proteins share only 36% of identity. Similar hydrophobic patterns of MP-Cg and MP-U1 suggests structure and function conservations of both proteins. This work is an example of how two tobamoviruses differing in their host range help to understand viral movement mechanism during the infection.     

Physiological effects of constitutive expression of Oilseed Rape Mosaic Tobamovirus (ORMV) movement protein in Arabidopsis thaliana

Movement proteins (MPs) are non-cell autonomous viral-encoded proteins that assist viruses in their cell-to-cell movement. The MP encoded by Tobamoviruses is the best characterized example among MPs of non-tubule-inducing plant RNA viruses. The MP of Oilseed Rape Mosaic Tobamovirus (ORMV) was transgenically expressed in Arabidopsis thaliana, ecotype RLD, under the expression of the 35S promoter from Cauliflower Mosaic Virus. Transgenic lines were obtained in sense and antisense orientations. One of the sense transgenic lines was further characterized turning out to carry one copy of the transgene inserted in the terminal region of the right arm of chromosome 1. The constitutive expression of ORMV-MP induced mild physiological effects in Arabidopsis. Plants of the transgenic line allowed a faster systemic movement of the phloem tracer carboxyfluorescein. The tracer was unloaded differentially in different flower parts, revealing differential effects of ORMV-MP on phloem unloading in sink organs. On the other hand, transgenic Arabidopsis did not show any effect on biomass partitioning or sugar availability, effects reported for equivalent transgenic solanaceous plants expressing the MP of Tobacco Mosaic Virus, another Tobamovirus. Finally, the transgenic Arabidopsis plants were susceptible to ORMV infection, although showing milder overall symptoms than non-transgenic controls. The results highlight the relevance of the specific host-virus system, in the physiological outcome of the molecular interactions established by MPs.C. Mansilla and I. Aguilar contributed equally.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

Dielectric spectroscopy of Tobacco Mosaic Virus

The dielectric properties of the Tobacco Mosaic Virus (TMV) have been measured using time domain dielectric spectroscopy (TDDS) in the temperature range from 1 to 40 degrees C. A single dielectric dispersion is observed in the MHz range. The activation energy of the process is found to be in the range 1-2 kcal/mol. The experimental data could not be completely accounted for by current theoretical models, but evidence indicates that the dielectric loss arises from polarisation of charge on and around the virus.     

毛头鬼伞(Coprinus comatus)中一种碱性蛋白的纯化及其活性

用离子交换层析(CM-sepharose FF)和凝胶层析(Superdex^TM 75)方法,从新鲜食用菌毛头鬼伞(Coprinus omatus)子实体中分离纯化出一碱性蛋白y3,经SDS-PAGE初步确定其分子量约为14．4kD。活性检测结果显示：当其浓度为12．5μg／mL时,对烟草花叶病毒(TMV)在心叶烟枯斑寄主上的侵染抑制率达83．0%;y3对兔血凝集活性滴度为2^5,对人血凝集活性滴度为26,其浓度分别为1．562μg／mL和0．78lμg／mL;利用胃癌细胞株MGC-803检测y3体外抗肿瘤活性,其IC50为12μg／mL。y3 N-端序列为NRDVAACARFIDDFCDTLTP,为一新的蛋白序列。在SWISS-PORT上登录号为P83477。     

Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ

A crude extract of commercial wheat germ is capable of translating mRNAs from widely different sources with high efficiencies. Of six wheat germs analyzed only one was found capable of a high level or incorporation with natural mRNAs. Under optimum conditions at a saturating level of Tobacco Mosaic Virus (TMV) RNA (4.5 μg) and labeled amino acid, 68% of all the available ¹⁴C leucine is incorporated in 70 min. at 30°C with a stimulation of 425 fold above background (with an efficiency of 252 moles leucine/mole TMV RNA). Thus this system which is 30 fold more efficient for TMV translation than previous reported wheat germ cell free systems is capable of yielding 568 pmoles of ¹⁴C leucine incorporated into protein in a 50 μl assay. 80% of the proteins produced have a molecular weight greater than TMV coat protein (17,400). This level of incorporation requires optimization of extract concentration, pH, Mg⁺², K⁺ and spermine concentration as well as the method of extract preparation.     

Birefringence of macromolecules. Wiener''s theory revisited, with applications to DNA and tobacco mosaic virus.

We summarize Wiener's theory of the dielectric constant of heterogeneous systems and extend its application to suspensions of particles with corrugated surfaces and interstitial solvent. We retain a simple geometrical shape for the particles and account specifically for the solvent associated with the particles. We calculate the birefringence of the rodshaped Tobacco Mosaic Virus (TMV) particle and of DNA and find excellent agreement between our numerical results and experimental values from the literature.     

Genomic fragments of Turnip Yellow Mosaic Virus: Appearance during infection

Turnip Yellow Mosaic Virus RNA was isolated at 5, 8, 11, 14, 20, and 24 days of infection and analyzed for yield, size distribution, 3′ adenosylation and valylation, and coat protein mRNA. Virus accumulation is slow from the third to tenth days and rapid to the third week of infection. Five-day RNA is composed of the 1.9×10⁶D genomic RNA (78%) and the 0.22×10⁶D coat protein in mRNA (6%). Later, intermediate size RNAs appear at 1.03, 0.46, 0.41, and 0.39×10⁶D. The valine tRNA of all RNAs has 80% CpC_OH and 6% CpCpA_OH 3′ termini.