Effect of cyclopentene ��,�¡�-triketone disodium salt on the activity of hydrolases and the state of tobacco mosaic virus particles in tobacco leaves

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.     

Influence of a few coat protein subunits on the base-paired structure of 3′-terminal fragments of RNA 4 of alfalfa mosaic virus

In contrast to expectation (Srinivasan, S. and Jaspars, E.M.J. (1978) Biochim. Biophys. Acta 520, 237–241) differentiated thermal melting profiles and fluorescence measurements show that the coat protein of alfalfa mosaic virus has a negligible effect on the base-paired structure of isolated 3′-terminal fragments (length about 90 nucleotides) of the coat protein messenger RNA (RNA 4) of this virus.     

Discovery of the first virus, the tobacco mosaic virus: 1892 or 1898?

Two scientists contributed to the discovery of the first virus, Tobacco mosaic virus. Ivanoski reported in 1892 that extracts from infected leaves were still infectious after filtration through a Chamberland filter-candle. Bacteria are retained by such filters, a new world was discovered: filterable pathogens. However, Ivanovski probably did not grasp the full meaning of his discovery. Beijerinck, in 1898, was the first to call 'virus', the incitant of the tobacco mosaic. He showed that the incitant was able to migrate in an agar gel, therefore being an infectious soluble agent, or a 'contagium vivum fluidum' and definitively not a 'contagium fixum' as would be a bacteria. Ivanovski and Beijerinck brought unequal but decisive and complementary contributions to the discovery of viruses. Since then, discoveries made on Tobacco mosaic virus have stood out as milestones of virology history.     

Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum ‘Xanthi’) plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.     

Effects of the cucumber mosaic virus 2a protein on aphid–plant interactions in Arabidopsis thaliana

The cucumber mosaic virus (CMV) 2a RNA-dependent RNA polymerase protein has an additional function in Arabidopsis thaliana, which is to stimulate feeding deterrence (antixenosis) against aphids. Antixenosis is thought to increase the probability that aphids, after acquiring CMV particles from brief probes of an infected plant's epidermal cells, will be discouraged from settling and instead will spread inoculum to neighbouring plants. The amino acid sequences of 2a proteins encoded by a CMV strain that induces antixenosis in A. thaliana (Fny-CMV) and one that does not (LS-CMV) were compared to identify residues that might determine the triggering of antixenosis. These data were used to design reassortant viruses comprising Fny-CMV RNAs 1 and 3, and recombinant CMV RNA 2 molecules encoding chimeric 2a proteins containing sequences derived from LS-CMV and Fny-CMV. Antixenosis induction was detected by measuring the mean relative growth rate and fecundity of aphids (Myzus persicae) confined on infected and on mock-inoculated plants. An amino acid sequence determining antixenosis induction by CMV was found to reside between 2a protein residues 200 and 300. Subsequent mutant analysis delineated this to residue 237. We conjecture that the Fny-CMV 2a protein valine-237 plays some role in 2a protein-induced antixenosis.     

Influence of some herbicides on the reproduction of tobacco mosaic virus inNicotiana tabacum cv. ‘Samsun’

The application of 10^?3 g 1^?1 of 2,4 dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 3-amino-1,2,4-triazole and 6-chloro-2-ethylamino-4-isopropyl-amino-1,3,5-triazine toNicotiana tabacum cv. ‘Samsun’ plants inoculated with tobacco mosaic virus results in an increase in the content of this virus in the tissues. When whole plants are used, TMV content increases by 20% after herbicide application; when leaf dises are used, the amount of the virus can be doubled by the herbicide in comparison with control untreated inoculated discs. The results clearly show that the used non-selective herbicides act as stimulators of virus biosynthesis, probably via enhanced pentose phosphate cycle activity which enables an enhanced formation of viral RNA.     

17β-estradiol inhibits the production of infectious particles of hepatitis C virus

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh‐7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β‐estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E₂, but not P₄, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E₂–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα‐selective agonist 4, 4′, 4″‐ (4‐propyl‐[1H]‐pyrazole‐1, 3, 5‐triyl)trisphenol (PPT), but not with the ERβ‐selective agonist 2, 3‐bis (4‐hydroxyphenyl)‐propionitrile (DPN) or the G protein‐coupled receptor 30 (GPR30)‐selective agonist 1‐(4‐[6‐bromobenzo 1, 3 dioxol‐5‐yl]‐3a, 4, 5, 9b‐tetrahydro‐3H‐cyclopenta [c] quinolin‐8‐yl)‐ethanone (G‐1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E₂, inhibits the production of HCV infectious particles in an ERα–dependent manner.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Influence of κ/β-carrageenan from red alga <Emphasis Type="Italic">Tichocarpus crinitus</Emphasis> on development of local infection induced by tobacco mosaic virus in Xanthi-nc tobacco leaves

The influence of κ/β-carrageenan from red marine alga Tichocarpus crinitus on the development of tobacco mosaic virus (TMV) infection in Xanthi-nc tobacco leaves was studied. It was shown that the number of necrotic lesions on the leaves inoculated with the mixture of TMV (2 μg/ml) and carrageenan (1 mg/ml) was reduced by 87%, compared to the leaves inoculated with the virus only. The suppression of virus infection was also observed when leaves were treated with carrageenan 24 h before or 24 h after leaf inoculation with TMV; however, in these cases, suppression was less evident than after inoculation with the virus-polysaccharide mixture. It is supposed that the antiviral activity of carrageenan applied together with TMV may be explained by its action not only on the plant but also on the virus itself. The inhibitory effect of carrageenan pretreatment can be explained by its favorable effect on tissue resistance to infection. The suppression of this resistance by actinomycin D indicates that carrageenan functions via its action on the cell genome.     

Immunohistochemical localization of the α and β subunits of S-100 protein in pleomorphic adenoma of the salivary glands

The immunohistochemical expression of the alpha and beta subunits of S-100 protein in reactive, modified and transformed of myoepithelial cells, salivary pleomorphic was investigated using monoclonal antibodies. With S-100 alpha, normal salivary glands showed strong staining in serous acinar cells and moderate to slight staining in ductal segments, and with S-100 beta staining was slight or negative in acinar cells, but strong in nerve fibres. In pleomorphic salivary adenomas, the immunohistochemical distribution of S-100 alpha and beta proteins indicated great variation in the tumour cells. Some neoplastic cells gave similar staining for both S-100 alpha and beta, others were strongly positive for S-100 alpha and stained only slightly for S-100 beta, or vice versa. Yet other cells were positive for S-100 alpha and negative for S-100 beta, or vice versa. Pleomorphic salivary adenomas were classified both by histopathological criteria and by their staining pattern for S-100 alpha and beta proteins. Great heterogeneity in S-100 alpha and beta protein expression was found in individual tumour cells of both ductal and myoepithelial origin, and no regular pattern was identified. The cellular origin of salivary pleomorphic adenomas is discussed in terms of S-100 alpha and beta protein immunohistochemistry. Pleomorphic adenoma cells may be transformed from reserve cells into tumour cells displaying biologic properties of myoepithelial cells, ductal cells, or a mixture of both.     

Bioenergetic consequences in the interaction of mammalian cell and virus for the multiplication of recombinant vaccinia and expression of heterologous protein (β-hCG)

Summary Energy generation in vaccinia virus infected Vero cell is investigated. The specific ATP pool in infected cells increases in the range of 40–80%, in contrast to uninfected cells. Virus replication and -hCG expression are associated with the drop in ATP/ADP ratio from 7.0 to 2.5. High initial infection level leads to more energy generation and also more product formation.     

β-1,3-glucanase and chitinase as pathogenesis-related proteins in the defense reaction of twoCapsicum annuum cultivars infected with cucumber mosaic virus

Pathogenesis-related (PR) proteins from pepper (Capsicum annuum L.) cv. Americano (tolerant) and cv. Smith-5 (sensitive), both elicited by infection with cucumber mosaic virus (CMV), were assayed for chitinase and glucanase activities. Two basic PR-proteins, M_r 49.0 and 28.0 kD, were elicicited from the intracellular fraction (INTRA-F) of both cvs. by CMV infection, while four acidic M_r 15, 19, 36 and 40 kD and two basic M_r 21.2 and 24 kD PR-proteins were elicited from the intercellular fluid (IF) of cv. Americano leaves. Five acidic M_r 21.5, 23.2, 24.4, 25.2 and 36 kD and five basic M_r 23.3, 26, 28.8, 30 and 32.3 kD PR proteins were elicited from the IF of cv. Smith-5. Isoelectric focusing (IEF) of the IF and the INTRA-F proteins revealed the occurrence, in both pepper cultivars, of one acidic M_r 36 kD and one basic M_r 25 kD PR-protein with glucanase activity. After native-PAGE for acidic proteins, the acidic PR-protein of Rf 0.7 and M_r 36 kD present in the IF of both pepper cvs. showed glucanase activity. Native-PAGE for basic proteins of the INTRA-F showed the presence of one band (Rf 0.61, M_r 25 kD) common to both cvs. and two additional bands (Rf 0.49, M_r 26 kD and Rf 0.79, M_r 33 kD) in the cv. Americano with glucanase activity. The specificity shown by the basic PR-proteins suggests glucanase activity is involved in the mechanisms of resistance to CMV in the cv. Americano. There was no difference in chitinase isoform patterns between the two pepper cultivars analyzed. After IEF of the IF proteins, one acidic chitinase isoform was detected. Native-PAGE separation of the IF showed one band (M_r 30 kD) with chitinase activity. Chitinase activity was not detected in the INTRA-F of either cultivar.     

How thick is the layer of thermal volume surrounding the protein?

Investigation on the volume properties of protein hydration layers is reported. Presented results are based on combination of Monte Carlo modeling and available experimental data. Six globular proteins with known data are chosen for analysis. Analyzing the model and the experimental results we found that water molecules bound to proteins by hydrogen bond are preferentially located at the places with local depressions on the protein surface. Consequently, the hydration level is not strictly proportional to the area of charged and polar surfaces, but also depends on the shape of the molecular surface. The thickness of the thermal volume layer as calculated in the framework of the scaled particle theory is 0.6-0.65 A for chosen proteins. The obtained value is significantly lower than that presented for proteins in earlier papers (where proportionality between the hydration level and the area of charged and polar surfaces was assumed), but is close to the value published for small solute molecules. Discussion including the influence of protein size and the thermal motion of the surface is presented.     

A simple fluorimetric method for the estimation of DNA–DNA relatedness between closely related microorganisms by thermal denaturation temperatures

Determination of whole-genome DNA–DNA similarity is today a standard technique for species delineation in microbial taxonomy. However, these studies demand hard-to-perform and time-consuming experiments. Herein, we present an easy and rapid fluorimetric method to estimate DNA–DNA relatedness between microbial strains from differences of the thermal denaturation temperatures of hybrid and homologous genomic DNA. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence. A quantitative, real-time PCR thermocycler was used to perform the experiment and obtain fluorescence determinations at increasing temperatures. The proposed method was validated by comparing species of the hyperthermophilic genera Pyrococcus and Thermococcus. The method proves to be an easy, rapid, and inexpensive alternative to estimate DNA–DNA relatedness between closely related species.