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PubMed1989

Edema formation and degranulation of mast cells by a basic phospholipase A2 purified from Trimeresurus mucrosquamatus snake venom.

The basic phospholipase A2 (PLA2) purified from Trimeresurus mucrosquamatus snake venom was injected into the subplantar in order to induce edema formation in the rat hind paw. The maximum edema induced by PLA2 was induced at 1-2 hr after injection, and the per cent swelling curve showed a dose-dependent increase by PLA2 injection (2.5-10.0 micrograms). The rate of edema formation is different from the acute swelling induced by T. mucrosquamatus venom (TMV). Pretreatment with dexamethasone (4 mg/kg, s.c.), indomethacin (10 mg/kg, per 05) and diphenhydramine (100 mg/kg s.c.) inhibited the edema induced by the purified phospholipase A2. The injection of purified PLA2 or venom into rabbit skin resulted in an increase in vascular permeability which could be decreased by pretreatment with three antiinflammatory drugs. However, the pharmacological effect of dexamethasone (4 mg/kg) demonstrated a more effective inhibition than the other drugs in the PLA2-induced edema and vascular permeability change. Injection (i.p.) of PLA2 caused marked degranulation of mast cells in the rat mesentery which was facilitated by addition of calcium ion (10 mM) but antagonized by pretreating with three antiinflammatory agents. After incubating peritoneal mast cells with PLA2 (1.0 micrograms/ml), the release of histamine from the mast cell was approximately 36%, this effect was inhibited by preincubating the mast cell with three antiinflammatory agents.

Animals↗
PubMed1986

Encapsidation of 18 S rRNA by tobacco mosaic virus coat protein.

It has been reported that tobacco mosaic virus capsid protein encapsidates discretesized truncated portions of host 18 S rRNA in vitro. This paper presents further information concerning the nature and specificity of this reaction. We have found that it is only the 5' portions of 18 S rRNA that are encapsidated. The structure recognized by capsid protein is highly conserved; bovine as well as plant 18 S rRNA becomes encapsidated. It is further demonstrated that assembly of 18 S rRNA is slow in comparison to assembly of TMV RNA and that this is due to a slow rate of initiation. Synthetic 18 S rRNA, prepared by in vitro transcription of an 18 S rRNA coding sequence, differs from native 18 S rRNA in that full length, rather than a truncated portion, is encapsidated. The possible reasons for this are discussed.

Base Sequence↗
PubMed1989

Mutational analysis of the coat protein gene of tobacco mosaic virus in relation to hypersensitive response in tobacco plants with the N' gene.

Tomato strain L of tobacco mosaic virus (TMV-L) induces a hypersensitive response (necrotic local lesions) on tobacco plants with the N' gene. A factor responsible for induction of the hypersensitive response has been mapped to the coat protein gene. We have constructed several mutants which have insertions or deletions in the coat protein gene. Frame-shift mutants which cause premature termination of translation of the coat protein caused no necrotic local lesions on N' plants. Mutants which result in the expression of coat protein derivatives with one amino acid inserted after residue 56, 101, or 152 caused necrotic local lesions on N' plants. Deletion mutants lacking the coding region for fewer than the C-terminal 13 amino acid residues caused necrotic local lesions, whereas mutants lacking the coding region for the C-terminal 38 residues caused no necrotic local lesions. These results show that modifications of the coat protein gene affect its ability to induce the hypersensitive response in N' plants.

Amino Acid Sequence↗
PubMed1989

Virus-ribosome complexes from cell-free translation systems supplemented with cowpea chlorotic mottle virus particles.

When particles of cowpea chlorotic mottle virus (CCMV) were added to cell-free extracts from wheat germ, the encapsidated viral genome was translated into polypeptides similar to the translation products specified by unencapsidated viral RNA (as shown before by M.J. Brisco, R. Hull, and T.M.A. Wilson, 1986, Virology 148, 210-217). The rate of protein synthesis observed upon addition of virus particles was much slower than that of extracted RNA and the quantity of protein formed was only 10% of that of extracted RNA. Using sucrose and cesium-chloride gradient analysis, virus-ribosome complexes, containing up to four ribosomes per virus particle, were isolated from translation mixtures supplemented with CCMV particles. These complexes, with densities intermediate of those of virus (1.36 g cm-3) and ribosomes (1.58 g cm-3), were analyzed and quantified in the electron microscope. Less than 5% of the particles was found in association with ribosomes. To verify whether these complexes were involved in the process of cotranslational disassembly, tobacco mosaic virus was analyzed with the same techniques and methods. The results found for TMV were similar to those found for CCMV except that virus-ribosome complexes with up to 20 ribosomes per virus particle were observed. The implications of the process of virion-directed translation for the structure of the particle as well as the role of this process in vivo are discussed.

Centrifugation, Density Gradient↗
PubMed1989

Nucleotide sequence of barley stripe mosaic virus RNA alpha: RNA alpha encodes a single polypeptide with homology to corresponding proteins from other viruses.

The complete nucleotide sequence of RNA alpha from the Type strain of barley stripe mosaic virus has been determined. The RNA is 3768 nucleotides long and contains a single open reading frame which codes for a polypeptide of 1139 amino acids (mw 129,634). The open reading frame is flanked by a 5'-terminal sequence of 91 nucleotides and a 3'-nontranslated region composed of a short poly(A) tract followed by a 238-nucleotide tRNA-like structure. The amino acid sequence of the polypeptide (alpha a) encoded by the open reading frame has homology with the TMV 126K protein and with related polypeptides from other viruses. The carboxy-terminal portion of the alpha a polypeptide also has limited homology with the 58K (beta b) protein encoded by BSMV RNA beta and includes a consensus sequence found in mononucleotide-binding polypeptides.

Amino Acid Sequence↗
PubMed1990

Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement.

The regulation of the internal open reading frame (ORF) of tobacco mosaic virus (TMV) that encodes the 30K movement protein was examined by constructing mutants in vitro with the putative coat protein subgenomic RNA promoter and leader sequences inserted upstream of the 30K ORF. A mutant with a 49-nucleotide fragment of the promoter region inserted replicated only transiently before being overtaken by a progeny wild-type virus with the insert deleted. A mutant with a 253-nucleotide promoter region fragment inserted replicated stably, and the inserted promoter was active in its new location. The production of 30K protein was not enhanced by this promoter/leader insertion to a level similar to that of coat protein. However, the accumulation of 30K protein was delayed, suggesting that different promoters/leader sequences determine the time of expression of the genes. This mutant was deficient in movement. A similar mutant, but with increased production of 30K protein, overcame the movement deficiency, suggesting that 30K protein is needed during the early stages of infection for efficient cell-to-cell movement of the virus.

Base Sequence↗
PubMed1990

Polarity of binding of monoclonal antibodies to tobacco mosaic virus rods and stacked disks.

Monoclonal antibodies to tobacco mosaic virus that bind only to one end of the viral rods have been shown to recognize the surface of the protein subunit designated as the bottom, which contains the right radial and left radial alpha-helices. The specificity of the antibody binding was established by immunoelectron microscopy of complexes in which the 5' end of the RNA had been exposed at the bottom of the helical virus particle. These antibodies have been shown to bind to both ends of the stacked disk aggregate of TMV protein, which is therefore bipolar. The observations on the bipolarity of this structure are inconsistent with the presumption that stacked disks are formed by aggregation of polar two-layer disks.

Antibodies, Monoclonal↗
PubMed1990

Deletion of repeated sequences from tobacco mosaic virus mutants with two coat protein genes.

Tobacco mosaic virus (TMV) hybrids were constructed in vitro with an extra coat protein gene (gene order 5'- 126K/183K gene-30K gene-first coat protein gene-subgenomic mRNA promoter:second coat protein gene-3'). One coat protein gene was wild type and the other was derived from one of three coat protein mutants that elicit distinctive phenotypic responses. The hybrids contained large sequence repeats (up to 747 nucleotides). RNA transcripts of each of seven hybrid constructs initiated infections in host plants, but the progeny virus contained only one coat protein gene with no additional or missing nucleotides. Progeny of mutants with the two functional coat protein genes in different order within the genome retained coat protein gene sequence predicted by the number of repeated nucleotides 3' and 5' of the differential nucleotides. In contrast, progeny that resulted from virus hybrids with a wild-type and a nonfunctional coat protein gene possessed only the wild-type coat protein gene, even when the predicted progeny was a free-RNA virus. This demonstrated that selection strongly affected which progeny became predominant in the resulting populations.

Base Sequence↗
PubMed1992

Tobamovirus-plant interactions.

It is clear that the genetic information responsible for the phenomenon we think of as TMV not only consists of the genes carried in the viral genome, but that numerous plant genes are equally important in viral gene functions. These gene products not only allow the virus to replicate, but may effect functions of evolution that determine what the virus is. Even the processes of pathogenesis and resistance appear to involve similarly precise plant interactions. The challenge of the future is to identify the plant genes involved in these precise interactions and to understand both components of genetic information that comprise plant viruses.

Biological Evolution↗
PubMed1987

Polyamine changes during senescence and tumorogenesis in plants.

Putrescine, spermidine, spermine and two unknowns designated as A and B were detected in first seedling leaves of barley (Hordeum vulgare L. var. Wolfe). The levels of these polyamines in first seedling leaves from 4-day-old barley plants grown in darkness or in light were comparable and did not change significantly after exposure of dark grown plants to light for 24 h. No significant consistent changes in the amounts of above polyamines, except perhaps decline in spermidine, were noted during senescence of intact or excised first seedling leaves of barley and this spermidine decline was suppressed during retardation of senescence of excised leaves by 10 mg/l kinetin in the dark. In addition, putrescine, spermidine, spermine, cadaverine and diaminopropane (0.2 mM, 1 mM, 10 mM) had no effect on senescence of excised barley leaves in the dark and both spermine and spermidine induced bleaching of the leaves in the light. Both spermine and spermidine (approx. 10 mM) inhibited RNase and DNase activities but stimulated phosphodiesterase activity (assayed with bis-p-nitrophenyl phosphate as substrate) in crude soluble extracts from barley leaves. Purified snake venom phosphodiesterase activity assayed with RNA as substrate was, however, stimulated by 300-400% by 7-14 mM spermine or spermidine indicating similar possibilities for barley phosphodiesterase. These results together with the presence of multiple species of these enzymes and a decline in net soluble RNase and DNase activities during senescence in barley leaves reported previously, make it unlikely that inhibition of RNase activity in vitro by polyamines could be correlated with their effect on senescence. Putrescine, spermidine and spermine were detected in normal and crown gall tumor tissue cultures of tobacco (Nicotiana tabacum var Wisconsin 38) and in tobacco mosaic virus (TMV)-infected freshly excised pith tissue from tobacco which represented non-proliferating tissue. The level of all three polyamines was several-fold higher in cultured tissues compared to the non-dividing freshly excised pith tissue and the tumor cultures had several-fold higher spermidine and putrescine respectively compared to normal tissue cultures. These results indicate high levels of polyamines in growing tissues but no consistent pivotal changes in polyamines during senescence. The results also do not support polyamines being natural anti-senescent compounds in plants or that their anti-senescent compounds effect could result from inhibition of RNase activity.

Adenine↗
PubMed1984

The antibody response to a single antigenic determinant of the tobacco mosaic virus protein: analysis using monoclonal antibodies, mutant proteins and synthetic peptides.

Three hybridomas were selected which secreted monoclonal antibodies specific to a decapeptide determinant representing residues 103-112 of the tobacco mosaic virus protein ( TMVP ). A series of proteins from several strains of TMV which differ in the amino acid sequence in this region of the protein were used as probes for specificity analysis. The fine-specificity analysis was extended by assessing the binding of the antibodies with a panel of synthetic peptide analogues of the native decapeptide with amino acid substitutions at different locations. The binding of each synthetic peptide with each of the monoclonal antibodies was determined by the ability of the radiolabeled peptide to bind with the antibody. The binding of the decapeptide with antibodies was determined by equilibrium dialysis; the relative binding affinity of each peptide of the panel was determined by the capacity of the peptide to inhibit the binding between the antibody and the radiolabeled native decapeptide. The results demonstrated that a panel of synthetic peptide analogues constitutes a powerful tool for discerning the fine specificity of antibodies directed to a given determinant of a protein antigen. The data indicated that, although all of the antibodies recognized the same nominal decapeptide determinant, their binding with the different mutant proteins or with the synthetic peptides of the panel differed greatly, indicating dramatic differences in their fine specificity. The existence of such differences should be taken into consideration when assessing residues of a protein antigen that are involved in antibody binding. The differences which were found in monoclonal antibodies produced following immunization with the whole TMVP reflect differences which occur in heterogeneous serum antibody populations and point out the complexity of antigenic recognition even of as small an epitope as a decapeptide.

Amino Acid Sequence↗
PubMed1992

Measurement of affinity of viral monoclonal antibodies using Fab'-peroxidase conjugate. Influence of antibody concentration on apparent affinity.

The binding affinity of a monoclonal antibody to tobacco mosaic virus (TMV) was studied using a Fab'-peroxidase conjugate. Measurement of the enzymatic activity allowed the determination of the amount of free antibody present after ultracentrifugation of virus-antibody complexes at equilibrium. The method was very sensitive and allowed measurements over a 1000-fold range of antibody concns. The calculated affinity constant decreased about 25 fold when the antibody concn used in the binding assay was increased from 30 ng/ml to 35 micrograms/ml.

Antibodies, Monoclonal↗
PubMed1980

Hen egg yolk as a source of antiviral antibodies in the enzyme-linked immunosorbent assay (ELISA): a comparison of two plant viruses.

A method of raising antibodies against plant viruses in hen egg yolk is described. Laying hens were immunized with citrus tristeza virus (CTV) or tobacco mosaic virus-avocado isolate (TMV-A). Anti-viral antibodies in the yolks of sequentially laid eggs as well as in the serum were titrated by the (heterologous) antiglobulin double antibody sandwich form of the enzyme-linked immunosorbent assay (HADAS-ELISA). Antibodies first appeared in yolk 7 days after injection and peak levels were attained on day 9-11; these levels persisted for about 6-12 days. Non-specific yolk antibodies were removed by absorption with an extract of uninfected plant tissue. Using the HADAS-ELISA technique we found that yolk titres were equal to, or higher than those in serum. The benefits of using laying hens over conventional laboratory animals as a source of antiviral antibody are discussed.

Animals↗
PubMed1982

Serological differentiation of tobamoviruses by means of monoclonal antibodies.

Several serologically related plant viruses have been differentiated for the first time by means of monoclonal antibodies. Two orchid strains of tobacco mosaic virus that were previously considered to be serologically identical with the type strain were shown to be distinguishable with certain monoclonal antibodies. In indirect ELISA, none of the antibodies showed any binding to tobamoviruses that differed from TMV (common strain) by a serological differentiation index larger than 2. Monoclonal antibodies are likely to find widespread use as diagnostic tools in plant virology.

Animals↗
PubMed1983

Autoradiography of 32P and 14C incorporation into protoplasts as a means of determining the percentage of virus infected protoplasts.

Stripping Film Autoradiography (SFA) was used to measure percentage protoplast infection. Control and infected protoplasts were incubated in the presence of [32P]orthophosphate or [14C]leucine fixed onto glass slides and placed in contact with a sensitive photographic emulsion. Infected protoplasts were distinguished by blackened areas of exposed film which coincided with protoplasts containing labelled virus products. The use of SFA is demonstrated for two different virus/protoplast systems, namely tobacco mosaic virus (TMV) in tobacco and turnip yellow mosaic virus (TYMV) in rape. The method detects infection as early as 12-16 h post-inoculation and UV irradiation is not necessary for reducing background interference from labelled host proteins.

Autoradiography↗
PubMed1989

Anomalous electrophoretic behavior of the major potato virus X RNA translation product.

The major potato virus X (PVS) RNA translation product migrates in Laemmli's electrophoresis system as a 210 kDa polypeptide ('p210'). If a Tris-phosphate-SDS buffer system is used instead of a Tris-glycine-SDS one, the mobility of p210 is higher than that of the largest TMV RNA translation product, the 183 kDa protein. It is suggested that anomalous electrophoretic behavior of the largest PVX polypeptide during SDS-electrophoresis is due to its primary structure, namely to the presence of hydrophilic domains.

Centrifugation, Density Gradient↗
PubMed1987

Antiviral effects of dihydroxypropyladenine [RS)-DHPA) and bromovinyldeoxyuridine (BVDU) on plant viruses.

Two established antiviral agents, dihydroxypropyladenine (RS)-DHPA and bromovinyldeoxyuridine (BVDU) were evaluated for their inhibitory effects on 4 plant viruses: Tobacco mosaic virus, potato virus X (PVX), eggplant mosaic virus (EMV) and a potyvirus isolated from Solanum palinacanthum (Poty-Sp). Using the leaf disc incubation test, BVDU proved virtually inactive while (RS)-DHPA efficiently inhibited EMV and Poty-Sp when applied at concentrations as low as 5 mg/l. TMV was less susceptible to the chemical while PVX could be inhibited at drug concentrations of 100 mg/l. To achieve a similar inhibitory effect in the leaf spray test, concentrations up to 250 and 500 mg/l were required. Using these tests no phytotoxicity was observed with (RS)-DHPA at any of the concentrations used.

Adenine↗
PubMed1985

Preparation and properties of an improved cell-free protein synthesis system from mammalian liver.

A cell-free protein synthesis system derived from mouse liver has been developed which faithfully translates endogenous and exogenous mRNA. The system is based on the unfractionated post-mitochondrial supernatant. The main measures taken to improve the activity of the system were: the use of high levels (30 mM) of creatine phosphate as an energy-generating system to counteract a hyperactive nucleoside triphosphatase activity in the extracts, the choice of homgenisation buffer, and the use of potassium acetate rather than KCl in the assay. The system exhibits a high initial rate of amino acid incorporation, and reinitiates translation on endogenous mRNA. Added tobacco mosaic virus (TMV) RNA is faithfully translated into full-length products at a rate of 60-80 amino acid residues per min at 30 degrees. The rate of overall amino acid incorporation slows after about 20 min and eventually ceases due to a failure in the re-initiation of translation, and not because of degradation of mRNA. Over a limited period of time, this improved cell-free translation system is comparable in activity to other eukaryotic systems generated to date, and should be useful in studies of the control of translation rates in mammalian liver.

Adenosine Triphosphate↗