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Thermal modulation voltammetry with laser heating at an aqueous|nitrobenzene solution microinterface: determination of the standard entropy changes of transfer for tetraalkylammonium ions.

Thermal modulation voltammetry (TMV) with laser heating was successfully performed at an aqueous|nitrobenzene (NB) solution microinterface, by taking advantage of the fact that laser light with a wavelength of 325.0 nm is optically transparent to the aqueous solution but opaque to the NB solution. When the laser beam impinges upon the interface from the aqueous solution side, a temperature is raised around the interface through the thermal diffusion subsequent to the light-to-heat conversion following the optical absorption by the NB solution near the interface. Based on such a principle, we achieved a fluctuating temperature perturbation around the interface for TMV by periodically irradiating the interface with the laser beam. On the other hand, the fluctuating temperature perturbation has influence on currents for transfer of an ion across the interface to produce fluctuating currents synchronized with the perturbation through temperature coefficients of several variables concerning the transfer, such as the standard transfer potential and the diffusion coefficient of the ion. Consequently, TMV has the possibility of providing information about the standard entropy change of transfer corresponding to a temperature coefficient of the standard transfer potential and a temperature coefficient of the diffusion coefficient. In this work, the aqueous|NB solution interface of 30 microm in diameter was irradiated with the laser beam at 10 Hz, and the currents synchronized with the periodical irradiation were recorded as a function of the potential difference across the interface in order to construct a TM voltammogram. TM voltammograms were measured for transfer of tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetra-n-butylammonium ions from the aqueous solution to the NB solution, and the standard entropy change of transfer was determined for each ion, according to an analytical procedure based on a mathematical expression of the TM voltammogram. Comparison of the values obtained in this work with the literature values has proved that TMV with laser heating is available for the determination of the standard entropy change of transfer for an ion.

Journal Article↗

Salicylate activity. 3. Structure relationship to systemic acquired resistance.

Salicylic acid (2-hydroxybenzoic acid; SA) is a primary signal inducing plant defenses against pathogens. This plant disease resistance, known as systemic acquired resistance (SAR), is an attractive target for the development of new plant protection agents. SAR induction is a multistep process that includes accumulation of pathogenesis-related (PR) proteins. The structure-activity profile of salicylates and related compounds has been evaluated using an inducible PR protein (PR-1a) and plant resistance to tobacco mosaic virus (TMV) as markers. Among the 47 selected monosubstituted and multiple-substituted salicylate derivatives tested, all 8 derivatives that induced more PR-1a protein than SA were fluorinated or chlorinated in the 3- and/or 5-position (3,5-difluorosalicylate > 3-chlorosalicylate > 5-chlorosalicylate > 3,5-dichlorosalicylate > 3-chloro-5-fluorosalicylate > 3-fluorosalicylate > 3-fluoro-5-chlorosalicylate > 3,5-dichloro-6-hydroxysalicylate > SA). In general, substitutions for or on the 2-hydroxyl group or at the 4-position of the ring reduced or eliminated PR-1a protein induction. In contrast, substitutions in positions ortho (3-position) or para (5-position) to the hydroxyl group with electron-withdrawing groups other than chlorine or fluorine decreased induction, and electron-donating groups in these positions also had a deleterious effect on PR-1a induction. PR-1a protein accumulation and reduction in TMV lesion diameter exhibited a log-linear relationship. The seven salicylate derivatives that were the most active TMV resistance inducers were all halogenated in the 3- and/or 5-position (3-chlorosalicylate > 3,5-difluorosalicylate > 3,5-dichloro-6-hydroxysalicylate > 3,5,6-trichlorosalicylate > 5-chlorosalicylate > 5-fluorosalicylate > 3,5-dichlorosalicylate > 4-fluorosalicylate > 3-fluorosalicylate > 3-chloro-5-fluorosalicylate > 4-chlorosalicylate > SA). The correlation between PR-1a protein induction and resistance to TMV confirms the value of using PR-1a induction as a screening tool for developing new plant disease control agents.

Plant Diseases↗

Induction of pepper cDNA encoding a lipid transfer protein during the resistance response to tobacco mosaic virus.

Pepper (Capsicum annuum) plants exhibit hypersensitive response (HR) against infection by many tobamoviruses. A clone encoding a putative nonspecific lipid transfer protein (CaLTP1) was isolated by differential screening of a cDNA library from resistant pepper leaves when inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaLTP1 is highly similar to that of the other plant LTPs. Southern blot analysis showed that a small gene family of LTP-related sequences was present in the pepper genome. Transcripts homologous to CaLTP1 accumulated abundantly in old leaves and flowers. CaLTP1 expression was induced in the incompatible interaction with TMV-P0 but was not induced in the compatible interaction with TMV-P1.2. In correlation with the temporal progression of HR in the inoculated leaves, CaLTP1 transcripts started to accumulate at 24 h after TMV-P0 inoculation, reaching a maximal level at 48 h. A strain of Xanthomonas campestris pv. vesicatoria (Xcv) that carries the bacterial avirulence gene, avrBs2, was infiltrated into leaves of a pepper cultivar containing the Bs2 resistance gene. A marked induction of CaLTP1 expression was observed in Xcv-infiltrated leaves. Effects of exogenously applied abiotic elicitors on CaLTP1 expression were also examined. Salicylic acid caused a rapid accumulation of CaLTP1 transcripts in pepper leaves and ethephon treatment also induced the expression of the CaLTP1 gene. Transient expression in the detached pepper leaves by biolistic gene bombardment indicated that CaLTP1 is localized mostly at the plant cell surface, possibly in the cell wall. These results suggest possible role(s) for LTPs in plant defense against pathogens including viruses.

Amino Acid Sequence↗

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching.

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5 degrees C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation at 20 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.

Cold Temperature↗

Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus.

Using malaria as a model disease, we engineered the surface of tobacco mosaic tobamovirus (TMV) for presentation of selected epitopes to the mammalian immune system. The TMV coat protein is a well-characterized and abundant self-assembling polymer previously shown to be a highly immunogenic carrier. Selected B-cell epitopes were either inserted into the surface loop region of the TMV coat protein or fused to the C terminus using the leaky stop signal derived from the replicase protein reading frame. Tobacco plants systemically infected with each of these constructs contained high titers of genetically stable recombinant virus, enabling purification of the chimeric particles in high yield. Symptoms induced in tobacco ranged from a normal mosaic pattern similar to that induced by the parental U1 strain to a unique bright yellow mosaic. As measured by quantitative ELISA against synthetic peptide standards, wild type TMV coat protein and fusion protein synthesized by the leaky stop mechanism coassembled into virus particles at the predicted ratio of approximately 20:1. Recombinant plant viruses have the potential to meet the need for scalable and cost effective production of subunit vaccines that can be easily stored and administered.

Amino Acid Sequence↗

Systemic movement of a tobamovirus requires host cell pectin methylesterase.

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.

Carboxylic Ester Hydrolases↗

Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization.

The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.

Amino Acid Sequence↗

Tobacco mosaic virus RNA enters chloroplasts in vivo.

Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast in vivo. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces. Northern blot analysis of RNA extracted from these chloroplasts demonstrated that full-length TMV RNA was present within the chloroplasts prepared from both directly inoculated and systemically invaded leaves. Only genomic length, but not subgenomic length, RNA was found in the chloroplast extracts, indicating a selectivity of the transport of the viral RNA into the chloroplast. A temperature-sensitive TMV mutant (Ts 38), in which no virions are formed at 35 degrees C, was used to demonstrate that at that restrictive temperature viral RNA is detected in the chloroplast, indicating that free viral RNA can enter the chloroplast rather than intact virions. To our knowledge, the transport of a foreign RNA species into chloroplasts has not been reported previously.

Journal Article↗

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.

Capsid↗

Viral RNA trafficking is inhibited in replicase-mediated resistant transgenic tobacco plants.

Transgenic tobacco (Nicotiana tabacum cv. Turkish Samsun NN) plants expressing a truncated replicase gene sequence from RNA-2 of strain Fny of cucumber mosaic virus (CMV) are resistant to systemic CMV disease. This is due to suppression of virus replication and cell-to-cell movement in the inoculated leaves of these plants. In this study, microinjection protocols were used to directly examine cell-to-cell trafficking of CMV viral RNA in these resistant plants. CMV RNA fluorescently labeled with the nucleotide-specific TOTO-1 iodide dye, when coinjected with unlabeled CMV 3a movement protein (MP), moved rapidly into the surrounding mesophyll cells in mature tobacco leaves of vector control and untransformed plants. Such trafficking required the presence of functional CMV 3a MP. In contrast, coinjection of CMV 3a MP and CMV TOTO-RNA failed to move in transgenic resistant plants expressing the CMV truncated replicase gene. Furthermore, coinjection of 9.4-kDa fluorescein-conjugated dextran (F-dextran) along with unlabeled CMV 3a MP resulted in cell-to-cell movement of the F-dextran in control plants, but not in the transgenic plants. Similar results were obtained with viral RNA when the 30-kDa MP of tobacco mosaic virus (TMV) was coinjected with TMV TOTO-RNA into replicase-resistant transgenic tobacco expressing the 54-kDa gene sequence of TMV. However, in these transgenic plants, the TMV-MP was still capable of mediating cell-to-cell movement of itself and the 9.4-kDa F-dextran. These results indicate that an inhibition of cell-to-cell viral RNA trafficking is correlated with replicase-mediated resistance. This raises the possibility that the RNA-2 product is potentially involved in the regulation of cell-to-cell movement of viral infectious material during CMV replication.

Cell Communication↗

Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection.

Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene N because infection of susceptible tobacco not carrying the N gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and de novo synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an N gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.

Calcium-Calmodulin-Dependent Protein Kinases↗

Isolation and characterization of the 102-kilodalton RNA-binding protein that binds to the 5' and 3' translational enhancers of tobacco mosaic virus RNA.

Tobacco mosaic virus (TMV) is a positive-sense, single-stranded RNA virus the genome of which acts as a mRNA in the cytoplasm. On infection, TMV mRNA is efficiently and selectively translated by the host translation machinery despite the lack of a poly(A) tail, which is normally required for efficient translation. Both the 68-base 5' leader (Omega) and the 205-base 3' untranslated region of TMV promote efficient translation. A 25-base poly(CAA) region within Omega and the upstream pseudoknot domain, a 72-base region composed of three RNA pseudoknots, are responsible for the translational regulation. We have identified, purified, and characterized a 102-kDa RNA-binding protein (p102) from wheat that binds specifically to the poly(CAA) region within Omega and the upstream pseudoknot domain within the TMV 3' untranslated region. Polyclonal antibodies raised against wheat p102 were used to demonstrate that p102 is widely conserved in plant species. Moreover, specific RNA binding activity was detected in all plant species tested. Addition of anti-p102 antibodies to an in vitro translation lysate derived from wheat germ repressed translation, which was subsequently reversed by supplementing the lysate with p102. These findings suggest that this protein may play an important role in determining translational efficiency in plants.

Antibodies↗

The non-virus proteins associated with tobacco mosaic virus.

1. Exhaustive fractionation of leaves from tobacco plants systemically infected with TMV has led to the isolation of two non-virus proteins, B3 and B6, and the detection of a third, A4, which do not occur in comparable uninfected plants. 2. Components B3 and B6 have been found consistently in a series of ten extracts from plants grown over an 18 month period in all seasons of the year. It is concluded that these components are as characteristic of the infected plant as TMV itself. 3. As they occur in the initial extracts, the non-virus proteins are of low molecular weight (S(20)ca. 3). On treatment, each component tends to form a high molecular weight polymer with an electrophoretic mobility considerably greater than that of the starting material. The high molecular weight derivatives of A4, B3, and B6 have been designated A8, B8, and B7 respectively. There is no evidence that these high molecular weight components occur as such in the infected leaf. 4. The non-virus proteins are free of nucleic acid and are not infectious. They cross-react immunochemically with TMV. 5. Compared with TMV content, the amounts of the non-virus proteins found in infected leaf are relatively small, falling in the range of 10 to 150 micrograms per gm. of tissue.

Proteins↗

Variation in continuous reaction norms: quantifying directions of biological interest.

Thermal performance curves are an example of continuous reaction norm curves of common shape. Three modes of variation in these curves--vertical shift, horizontal shift, and generalist-specialist trade-offs--are of special interest to evolutionary biologists. Since two of these modes are nonlinear, traditional methods such as principal components analysis fail to decompose the variation into biological modes and to quantify the variation associated with each mode. Here we present the results of a new method, template mode of variation (TMV), that decomposes the variation into predetermined modes of variation for a particular set of thermal performance curves. We illustrate the method using data on thermal sensitivity of growth rate in Pieris rapae caterpillars. The TMV model explains 67% of the variation in thermal performance curves among families; generalist-specialist trade-offs account for 38% of the total between-family variation. The TMV method implemented here is applicable to both differences in mean and patterns of variation, and it can be used with either phenotypic or quantitative genetic data for thermal performance curves or other continuous reaction norms that have a template shape with a single maximum. The TMV approach may also apply to growth trajectories, age-specific life-history traits, and other function-valued traits.

Animals↗

Effect of 131 iodine therapy on the course of Graves' ophthalmopathy: a quantitative analysis of extraocular muscle volumes using orbital magnetic resonance imaging.

There remains uncertainty as to the effect of radioactive iodine (131I) therapy on the associated ophthalmopathy (GO). Twenty newly diagnosed patients with Graves' hyperthyroidism treated with 131I (median dose, 15.5 mCi) were followed with ophthalmologic evaluations (OE) and magnetic resonance imaging (MRI) at baseline, 2, and 6 months, and with OE alone at 3 years. For MRI, the superior, inferior, and medial rectus muscle volumes and total muscle volumes (TMV) were measured. Replacement levothyroxine was initiated as low thyroxine (T4) levels were noted. At baseline, 10 patients (50%) showed evidence of mild GO by OE and/or MRI. There was a significant difference in TMV between the 20 patients with Graves' hyperthyroidism and 10 controls (mean +/- standard error [SE]; 2,652 +/- 118 vs. 2,046 +/- 96 mm3; P = 0.002) and between the 10 patients with and 10 without GO (3,006 +/- 96 vs. 2,298 +/- 61 mm3; P = 0.001). TMV correlated with the Hertel score (r = 0.56, P = 0.01). TMV showed no significant change at 2 or 6 months posttreatment. The inferior rectus volume increased slightly at 2 months posttreatment (P = 0.03) but remained stable at 6 months. Furthermore, no significant changes occurred in Hertel scores or in clinical assessments up to 3 years posttreatment and none showed worsening or new development of GO. In conclusion, our results show no significant risk for radioiodine-induced initiation or progression of mild GO.

Adult↗

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 mug) and labeled amino acid, 68% of all the available (14)C leucine is incorporated in 70 min. at 30 degrees 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 (14)C leucine incorporated into protein in a 50 mul 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. Samples of crude polysomal RNA from hen oviducts (3% mRNA) and chorionating moth follicular cells (1% mRNA) are also translated in the wheat germ cell free system with high efficiency.

Animals↗

Multiple proteins and subgenomic mRNAs may be derived from a single open reading frame on tobacco mosaic virus RNA.

It has previously been shown that messenger activity for a protein of Mr = ca. 30k exists in RNA fractions extracted from particles of either native or alkali stripped U1 TMV, or from cowpea strain TMV, that are smaller than full genomic length. Analysis of sucrose gradient fractions containing this activity reveals a number of slightly smaller template activities directing synthesis of proteins between 18.5k and 29k in size. All of these messenger activities, including that for the 30k protein, respond to cap analogues in anomalous ways. Discrete RNA species that include active mRNAs for these proteins can be demonstrated in the same fractions by labelling with preparations of vaccinia capping enzyme and [alpha-32P] GTP without prior beta-elimination. Detailed analysis of three of these proteins (of Mr's ca. 30k, 29k and 23k) by peptide mapping and translation of purified vaccinia-labelled RNA demonstrates that all three are unrelated to the large early TMV proteins, but are related to each other in such a way as to form a nested set with staggered N termini and identical C termini. mRNAs of chain lengths ca. 1900 and 1500 bases direct synthesis of the 30k and 23k proteins respectively, an mRNA of about 1850 bases directs both 29k and (perhaps because of cross-contamination) 30k synthesis. Initiation codons for the 29k and 23k proteins have been mapped at positions 4960-4962 and 5191-5193 respectively on TMV RNA. Since all three encapsidated templates have similar properties we conclude that either there is a family of 30k-related proteins with unusual mRNAs, or that none of these in vitro translation products are directed by physiological templates.

Amino Acid Sequence↗

A comparison of eukaryotic viral 5'-leader sequences as enhancers of mRNA expression in vivo.

The 5'-untranslated leader sequences of several plant RNA viruses, and a portion of the 5'-leader of an animal retrovirus, were tested for their ability to enhance expression of contiguous open reading frames for chloramphenicol acetyltransferase (CAT) or beta-glucuronidase (GUS) in tobacco mesophyll protoplasts, Escherichia coli and oocytes of Xenopus laevis. Translation of capped or uncapped transcripts was substantially enhanced in almost all systems by the leader sequence of either the U1 or SPS strain of TMV. All leader sequences, except that of TYMV, stimulated expression of 5'-capped GUS mRNA with the native prokaryotic initiation codon context, in electroporated protoplasts. Only the TMV leaders enhanced translation of uncapped GUS mRNAs in protoplasts and increased expression of uncapped CAT mRNA in microinjected X. laevis oocytes. In oocytes, the TYMV leader sequence was inhibitory. In transformed E. coli, the TMV-U1 leader enhanced expression of both the native and eukaryotic context forms of GUS mRNA about 7.5-fold, despite the absence of a Shine-Dalgarno region in any of the transcripts. The absolute levels of GUS activity were all about 6-fold higher with mRNAs containing the native initiation codon context. In E. coli, the leaders of AlMV RNA4 and TYMV were moderately stimulatory whereas those of BMV RNA3, RSV and the SPS strain of TMV enhanced GUS expression by only 2- to 3-fold.

Animals↗