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。     

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.     

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     

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.     

Synthesis of tobacco mosaic virus coat protein following migration of viral RNA into isolated mouse liver mitochondria.

RNA of Tobacco Mosaic Virus is shown to be able to migrate into isolated mouse liver mitochondria, whence it can be reisolated intact. The migration of RNA is accompanied by enhanced rate of protein synthesis, which is sensitive to chloramphenicol, but not to cycloheximide. Evidence is presented showing that, among the products formed is the coat protein of Tobacco Mosaic Virus.     

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.     

Ion channels in icosahedral virus: a comparative analysis of the structures and binding sites at their fivefold axes.

An analysis of the crystallographically determined structures of the icosahedral protein coats of Tomato Bushy Stunt Virus, Southern Bean Mosaic Virus, Satellite Tobacco Necrosis Virus, Human Rhinovirus 14 and Mengovirus around their fivefold axes is presented. Accessibilities surfaces, electrostatic energy profile calculations, ion-protein interaction energy calculations, free energy perturbation methods and comparisons with structures of chelating agents are used in this study. It is concluded that the structures built around the viral fivefold axes would be adequate for ion binding and transport. Relative ion preferences are derived for the binding sites, using free energy perturbation methods, which are consistent with the experimental data when available. In the cases where crystallographic studies determined the existence of ions on the fivefold axes, our results indicate that they would correspond to ions in crystallization or purification buffers. The environment of the fivefold axes are rich in polar residues in all icosahedral viral structures whose atomic coordinates are available, including some that are not being analyzed in detail in this work. The fivefold channel-like structures have most of the basic properties expected for real ion channels including a funnel at the entrance, a polar internal environment with frequent alternation of acidic and basic residues, ion binding sites, the capability to induce ion dehydration and ion transit from the external viral surface to the binding sites.     

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.     

Plant disease and the regulation of enzymes involved in lignification

Tobacco varieties carrying the N gene from Nicotiana glutinosa respond to infection by Tobacco Mosaic Virus (TMV) by forming necrotic local lesions (hypersensitive reaction), thereby localizing the infection. In this study, infected mesophyll leaf tissue of N. tabacum Samsun NN was treated with the non-permeating, non-metabolizable carbohydrate mannitol. The local lesions developed under iso-osmotic conditions (0.28 M mannitol), though with a slight delay and with a reduced rate of growth, as compared to those on attached leaves. At increasing plasmolysing concentrations of mannitol, necrotization was progressively inhibited, but not completely suppressed. The leaf tissue produced tiny translucent zones, with a delay that increased between the virus inoculation and application of the plasmolytica. Activities of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and O-methyltransferase (OMT, EC 2.1.1.6) are strongly stimulated in hypersensitively reacting tobacco and were used as biochemical markers in the present study. This study was done to determine whether the inhibitory effect of plasmolysis on the elicitation of the hypersensitive response is due to a decrease in virus spread, resulting from the rupture of plasmodesmata or, at least in part, to metabolic alterations of the host cell exposed to osmotic stress. Since necrotization is normally preceded by intense virus multiplication, the inhibitory effects found for early applications (i.e., before local lesion appearance) of plasmolytica could easily be related to an inhibition of virus spread which also occurred in similarly treated leaf tissue of the systemically reacting variety Samsun. The most meaningful data were obtained from mannitol treatments performed on leaf tissue already carrying local lesions, i.e., in which the elicitor(s) and/or the factor(s) of necrotization were already operating. Under iso-osmotic conditions, we found the stimulated PAL and OMT activities characteristic of the hypersensitive response. At plasmolysing concentrations of mannitol, we observed the counteracting effects of two different mechanisms controlling the phenylpropanoid enzymes. Floating the leaf material on the liquid medium induced an ageing-like effect with a continuous increase in enzyme activities that was independent on osmotic pressure and sensitive to cycloheximide. At the same time, the stimulated enzyme activities related to hypersensitivity decreased at a rate related to osmotic pressure. Since PAL and OMT of tobacco leaves are long-lived enzymes, it is likely that the increased de novo synthesis of the enzymes was suppressed by plasmolysis while their degradation and/or inactivation was maintained or even increased. From these results it is concluded that the apparent inhibition of the hypersensitive response by plasmolysis is due to both a decrease in virus spead (artificially caused by the rupture of connections between cells) and to drastic metabolic alterations of the host cell exposed to high osmotic pressure.     

Beet necrotic yellow vein virus coat protein-mediated protection in sugarbeet (Beta vulgaris L.) protoplasts

Transformed Beta vulgaris L. suspension cultures were obtained after cocultivation of sugarbeet cells with Agrobacterium tumefaciens harbouring a binary vector containing the coat protein gene of beet necrotic yellow vein virus inserted between the kanamycin resistance gene and a ß-glucuronidase reporter gene. Protoplasts were isolated both from untransformed cells, and from transformed cells expressing the viral coat protein, and both were then infected with beet necrotic yellow vein virus. Comparison of the levels of infectivity shows that the expression of the coat protein gene in sugarbeet protoplasts mediates high levels of protection against infection by beet necrotic yellow vein virus.Abbreviations TMV Tobacco Mosaic Virus - CP Coat Protein - BNYVV Beet Necrotic Yellow Vein Virus - ß-Glu ß-glucuronidase - MS Murashige and Skoog (1962) - PEG Polyethylene glycol - npt neomycin phosphotransferase - nos nopaline synthase - FITC fluoresceine isothiocyanate - IAA indole acetic acid - BAP benzyl amino purine - MES 2-[N-Morpholino]ethane sulfonic acid - IgG Immunoglobulin G - nt nucleotide     

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.     

TOBACCO MOSAIC VIRUS IN CHLOROPLAST AND CYTOPLASM OF INFECTED TOBACCO LEAF

Chloroplasts containing Tobacco Mosaic Virus (TMV) were isolatedfrom TMV inoculated tobacco leaves, and TMV was extracted fromthem. The preparations from isolated chloroplasts of infectedleaves showed 30% increase in the optical density at 260 mµover those from healthy leaves. Most of infectivity was observedin chloroplast fraction 40 hr after inoculation. Thereafter,infectivity in the chloroplast fraction decreased and that ofcytoplasm increased with time. (Received April 6, 1964; )     

Suppression of Tobacco Mosaic Virus by Bacillus amyloliquefaciens Strain Ba33

Suppression of Tobacco Mosaic Virus (TMV) by B. amyloliquefaciens Ba33 was evaluated on Nicotiana tabacum by spraying before (①), after (②) and simultaneously with (③) TMV inocula. The results suggested that Ba33 treatments reduced local necrotic lesion number and disease index, showing ③ treatment was the best and ① treatment was better than ② treatment in TMV suppression. It also showed Ba33 virus‐contaminated scissors could be disinfected by dipping. Field trials showed that Ba33 had an inhibitory effect of 48.59% in 2009 and 50.54% in 2010, close to the effect of Ningnanmycin, a registered antiviral agent in tobacco. In conclusion, Ba33 might be used as a soil disinfector and an antiviral agent against TMV.     

A Survey of German Hops for the Presence of American Hop Latent Virus (AHLV)

The presence of rod-shaped viruses in German hops has been known for a long time and Hop Mosaic Virus (HMV) and Hop Latent Virus (HLV) were proved to be present by serological techniques. When another CARLA-Virus, the American Hop Latent Virus was detected in England an extensive survey was carried out in the German hop growing regions and among wild hops. No natural occurrence of the virus could be detected. Only recently introduced plants of the American clone USDA 21055 in an isolated breeding garden were infected. Spread of the virus to German seedlings could not be detected.     

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.     

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.