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At least 703 records · Page 39Linked to original sources
PubMed1997

Mucus quality on horse tracheal epithelium: microscopic grading based on transparency.

The aim of this ex-vivo study on excised tracheas of healthy horses was to characterise the microscopic heterogeneity of mucus quality by a visual grading system based on transparency and to determine whether differences in mucus quality, assessed by a visual grading system, influence tracheal mucus velocity (TMV). Small pieces of each trachea were mounted into a humidified chamber under a microscope. Mucus quality was visually subdivided into four grades (MG) and ciliary beat frequency and TMV were determined. Mucus on excised horse tracheal epithelium does not form a homogenous layer. We observed flakes and streams of a great heterogeneity, which by the characteristic of transparency can be qualified and quantified. Visual characterisation of mucus was able to explain a considerable part of TMV variation. Therefore, it can be considered as a suitable non-invasive method for the evaluation of mucus quality and transport effectiveness.

Animals↗
PubMed1998

Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses.

Immunocapture (IC) RT-PCR assays were developed for detection of tomato (ToMV) and tobacco mosaic (TMV) tobamoviruses in spruce and pine extracts. When purified viruses were diluted in root or needle extracts of virus-free conifer seedlings, both IC-RT-PCR assays detected their respective target viruses at concentrations of 10-100 fg ml(-1). This compared to ELISA detection sensitivities of 1 ng ml(-1). Primers were designed from regions of high sequence diversity. Specificity of all primer pairs was confirmed by sequencing of PCR products. PCR distinguished more reliably between the two viruses than ELISA. Moreover, a multiplex IC-RT-PCR assay for the simultaneous detection and differentiation of TMV and ToMV was developed. When root extracts were seeded with both viruses simultaneously, the multiplex assay detected each virus at concentrations of 1-10 pg ml(-1). Six TMV and 18 ToMV isolates from various hosts, water samples and a soil sample were amplified and differentiated by multiplex IC-RT-PCR. No amplifications were observed against pepper mild mottle and ribgrass mosaic tobamoviruses and against six viruses belonging to other taxonomic groups.

Enzyme-Linked Immunosorbent Assay↗
PubMed1975

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.

Genes↗
PubMed2003

A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein.

To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 degrees C, the TMV CP passed very rapidly (in less than 15s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70 mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein.

Capsid Proteins↗
PubMed1981

Structural studies of tobacco mosaic virus and its components by laser Raman spectroscopy.

Raman spectra have been recorded for oriented gels of tobacco mosaic virus, aqueous solutions of TMV protein in the form of capsid and disk, and protein-free TMV RNA. The protein-free RNA molecule contains about 80% ordered type-A structure, which is similar to other RNA molecules in solution. RNA in the virus particle does not exhibit the base stacking and pairing normally found in protein-free RNA but instead shows at least two distinct structures characterized by different phosphodiester group frequencies in the Raman spectrum. Approximately one out of three such linkages retains the type-A geometry characteristic of aqueous RNA, while two out of three appear to have a different geometry not encountered previously for RNA. The secondary structure of the TMV coat protein molecule is similar for the three aggregation states studied (virus, capsid, and disk) and consists of 40-50% alpha helix, 40-50% irregular structure, and 0-20% beta sheet. Two of the three tryptophan residues per protein molecule reside in hydrophobic regions, and the third is in contact with water. The distribution of the four tyrosine residues per protein molecule among hydrogen-bonding states is the same for capsid and virus but different for the disk. The latter could be a consequence of the presence of Cl- ions which may form hydrogen bonds with one or more p-hydroxyl groups of tyrosine residues.

Capsid↗
PubMed1979

Carbon-13 nuclear magnetic resonance studies on tobacco mosaic virus and its protein.

Fourier transform nuclear magnetic resonance studies on 12% 13C-enriched tobacco mosaic virus (TMV) and its rod-like protein oligomers in solution with molecular weights up to 42 X 10(6) are reported. In the virus approximately 17% of the carbons of the protein subunit have line widths of less than or equal to 300 Hz and T1 less than or equal to 1 s and are concluded to be mobile with more than one degree of freedom of internal rotation about a carbon--carbon bond. In the rodlike polymer of TMV protein at pH 5.3, 30% of the carbons are mobile, which implies rotational motions about carbon--carbon bonds and/or motions of the protein subunits within the polymer. The presence of internal mobility is supported by the observation that 20% of the carbons in the double disklike oligomer show decreasing line width upon increasing temperature; the remaining resonances have line widths which are temperature independent during the double disklike polymerization process. Since the molecular weight of TMV protein polymers increases with increasing temperature, this demonstrates that all nuclei within the double dislike oligomer are mobile. NMR and X-ray data on the double disklike polymer reveal differences with respect to internal mobility.

Fourier Analysis↗
PubMed2005

Influence of localized excited states on the transition moment directions of charge transfer complex absorptions.

The influence of localized excited (LE) states on the spectroscopy of charge transfer (CT) complexes has been examined for a series of complexes formed between methyl-substituted benzene donors and 1,2,4,5-tetracyanobenzene as acceptor in 1,2-dichloroethane and octanenitrile solvents. A molecular orbital model was used to describe the appearance of multiple CT absorption bands that occur in the spectra of these complexes. The influence of LE states in these CT absorptions was explored using time-resolved linear dichroism spectroscopy where the direction of the CT transition moment vector (TMV) was used to probe the magnitude of intensity borrowing. The TMV directions for each of the observed CT transitions within the absorption spectra were determined for several complexes. In some cases, the observed CT transitions were interpreted as being pure CT transitions; in others the observed transitions are influenced significantly by a LE transition. The correlation between the TMV directions and the transition energy suggests that the magnitude of intensity borrowing is influenced not only by the energy difference between the CT and LE transitions but also by the specific character of the transitions under consideration.

Benzene Derivatives↗
PubMed2004

Binding the tobacco mosaic virus to inorganic surfaces.

We studied the adsorption behavior and surface chemistry of the tobacco mosaic virus (TMV) on well-defined metal and insulator surfaces. TMV serves as a tubular supramolecular model system with precisely known surface termination. We show that if the surface chemistry of the substrate and the pH-dependent chemistry of the molecular surface match, for example, by hydrogen bonding, a strong adsorption occurs, and lateral movement is impeded. Due to the immobilization, the virion can be imaged by atomic force microscopy (AFM) in contact mode. We also used self-assembled monolayers with an acyl chloride group to induce covalent bonding via ester formation. Noncontact AFM proved that TMV keeps its cylindrical cross section only under weak adsorption conditions, that is, on hydrophobic surfaces, while on hydrophilic substrates a deformation occurs to maximize the number of interacting chemical groups.

Adsorption↗
PubMed1997

Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress.

Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens.

Apoptosis↗
PubMed1998

A glycine-rich RNA-binding protein gene is differentially expressed during acute hypersensitive response following Tobacco Mosaic Virus infection in tobacco.

During efforts for cloning disease resistance-responsive genes, a cDNA encoding a putative Nicotiana glutinosa glycine-rich RNA binding protein (ngRBP) was isolated from TMV induced cDNA library. Northern blot hybridization revealed that ngRBP gene is negatively regulated during early hours of TMV induced acute hypersensitive response (HR). Under greenhouse conditions induced expression of ngRBP gene was observed after 24 h following TMV infection. Salicylic acid and copper also induced ngRBP mRNA expression. Our findings are suggestive of some possible role for ngRBP in plant-pathogen interaction.

Amino Acid Sequence↗
PubMed1999

Signals controlling the expression of cytosolic ascorbate peroxidase during pathogen-induced programmed cell death in tobacco.

In plants ascorbate peroxidase (APX) is an important H2O2-detoxifying enzyme. The expression of APX is rapidly induced in response to stresses that result in the accumulation of reactive oxygen species (ROS). We have recently reported that the steady-state level of transcripts encoding cytosolic APX (cAPX) is dramatically induced during the hypersensitive response (HR) of tobacco plants infected with tobacco mosaic virus (TMV). Because cAPX expression is closely linked to the production of ROS in plant cells, studying the regulation cAPX mRNA can reveal some of the signal transduction events associated with ROS metabolism during the HR. Analysis of cAPX mRNA induction during the HR suggested that the expression of cAPX is under the control of the HR signal transduction pathway. The activation of cAPX expression followed signaling events such as changes in protein phosphorylation and induction of ion fluxes. Expression of cAPX was suppressed under conditions of low oxygen pressure, and could only be mimicked by enhancing the intracellular generation of ROS. Interestingly, salicylic acid (SA), which is thought to be involved in ROS metabolism during the HR, did not affect the induction of cAPX mRNA during TMV-induced HR. Using cAPX expression as a marker for the production of ROS, it is suggested that SA may not be involved in the formation of ROS during the HR of tobacco to TMV, and that ROS may not be involved in the induction of the pathogenesis-related protein, PR-1, during this process.

Anti-Infective Agents↗
PubMed1999

A cDNA from tobacco codes for an inhibitor of virus replication (IVR)-like protein.

We have shown previously that localization of tobacco mosaic virus (TMV) in tobacco is associated with a ca. 23 kDa protein that inhibits replication of several plant viruses. This protein, named 'inhibitor of virus replication' (IVR), was purified from the medium of TMV-inoculated protoplasts derived from Nicotiana tabacum cv. Samsun NN. IVR was shown to be present also in induced-resistant leaf tissue of N. tabacum cv. Samsun NN. We prepared an expression cDNA library from such induced-resistant tissue and screened it with a polyclonal antibody raised against the IVR protein. A 1016 bp clone (named NC330) containing a 597 bp open reading frame, coding for a 21.6 kDa polypeptide, was isolated. The NC330 clone hybridized with RNA from induced-resistant tissue from N. tabacum cv. Samsun NN but not with RNA from non-induced tissue. Likewise, it did not hybridize with RNA from infected or uninfected tissue of N. tabacum cv. Samsun nn. Similarly, the NC330 cloned probe hybridized with the RT-PCR products from RNA of the induced-resistant tissue only. In Southern blot hybridization the NC330 DNA probe detected several genomic DNA fragments in both N. tabacum cv. Samsun NN and Samsun nn. The size of the DNA fragments differed in Samsun NN and Samsun nn. We suggest that DNA encoding the IVR-like protein is present in resistant and susceptible N. tabacum genotypes, but is expressed only in NN. We have inserted the NC330 into the expression vector pET22b and a 21.6 kDa protein was produced in Escherichia coli that reacted in immunoblots with the IVR antibody. This protein greatly reduced replication of TMV in N. tabacum cv. Samsun nn leaf disk assays.

Amino Acid Sequence↗
PubMed2000

Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco.

A pathogen- and salicylic acid (SA)-induced DNA-binding activity has been recently identified in tobacco that is related to a previously identified class of WRKY DNA-binding proteins. To identify members of the WRKY gene family associated with this DNA-binding activity, we have attempted to isolate those WRKY genes that are induced by pathogen infection. Using a domain-specific differential display procedure, we have isolated two tobacco WRKY genes, tWRKY3 and tWRKY4, that are rapidly induced in resistant tobacco plants after infection by tobacco mosaic virus (TMV). Both tWRK3 and tWRKY4 encode proteins with a single WRKY domain that contain the conserved WRKYGQK sequence. Unlike other isolated WRKY proteins that contain the Cys2His2 zinc motif, tWRKY3 and tWRKY4 appear to contain the Cys2HisCys zinc motif. Nonetheless, both tWRKY3 and tWRKY4 are capable of binding DNA molecules with the W-box (TTGAC) element recognized by other WRKY proteins. Expression of the tWRKY3 and tWRKY4 genes could be rapidly induced not only by TMV infection but also by SA or its biologically active analogues that are capable of inducing pathogenesis-related genes and enhanced resistance. Interestingly, induction of both genes by TMV infection was still observed in resistant tobacco plants expressing the bacterial salicylate hydroxylase gene (nahG), although the levels of induction appeared to be reduced. Identification of pathogen- and SA-induced genes encoding WRKY DNA-binding proteins should facilitate future studies on the regulation and functions of this novel group of DNA-binding proteins.

Amino Acid Sequence↗
PubMed1998

Complete nucleotide sequence of the genomic RNA of tobacco mosaic virus strain Cg.

Tobacco mosaic virus (TMV)-Cg is a crucifer-infecting tobamovirus that was isolated from field-grown garlic. We determined the complete nucleotide sequence of the genomic RNA of TMV-Cg. The genomic RNA of TMV-Cg consists of 6303 nucleotides and encodes four large open reading frames, organized basically in the same way as that of other tobamoviruses. The nucleotide and deduced amino acid sequences are very similar to those of the other crucifer-infecting tobamoviruses that have been sequenced so far.

Amino Acid Sequence↗
PubMed1999

Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum.

We compared the expression of a functional recombinant TMV-specific full-size antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of full-size rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The full-size rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional full-size rAb29 expression were high in the apoplast (up to 8.5 micrograms per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody of scFv29, which was expressed in the periplasmic space of E. coli and showed the same binding specificity as full-size rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMV-coat protein monomers as rAb29.

Antibodies, Viral↗
PubMed2003

Structure of the tobacco caffeic acid O-methyltransferase (COMT) II gene: identification of promoter sequences involved in gene inducibility by various stimuli.

The tobacco gene encoding caffeic acid-O-methyltransferase of class II (COMT II) was isolated, including a 1.7 kb 5'-flanking region. Sequence motifs were identified in COMT II gene promoter which are present in many genes of the phenylpropanoid pathway or in stress-inducible pathogenesis-related (PR) genes. A 1215 bp COMT II promoter fragment was transcriptionally fused to the GUS coding region and its activity pattern studied by stable expression of the fusion gene in tobacco. Transgenic lines were analysed for GUS and OMT activities upon infection, UV irradiation, wounding and treatment by various signalling compounds. The promoter proved responsive to various biotic and abiotic elicitors and to infection by avirulent and virulent pathogens. During the course of the hypersensitive reaction of tobacco to TMV two peaks were detected, an early one induced by the inoculation process and a second one at the onset of lesion formation. Parallel changes were observed between GUS activity that reflected the activity of the COMT II promoter fragment and COMT II activity that mirrored expression of the endogenous COMT II gene, indicating that COMT II pattern of expression is established at the transcriptional level. Various promoter fragments were fused to the GUS gene and revealed that gene induction by MeJA or UV and by TMV or wounding requires different sequences included in a 74 bp fragment. When the 74 bp sequence was multimerized and inserted ahead of the CaMV 35S RNA minimal promoter, one construct was shown to be capable of driving expression of the reporter gene around the TMV-infected sites in transgenic tobacco plants.

Acetates↗
PubMed2004

From virus research to molecular biology: Tobacco mosaic virus in Germany, 1936-1956.

In 1937, a group of researchers in Nazi Germany began investigating tobacco mosaic virus (TMV) with the hope of using the virus as a model system for understanding gene behavior in higher organisms. They soon developed a creative and interdisciplinary work style and were able to continue their research in the postwar era, when they made significant contributions to the history of molecular biology. This group is significant for two major reasons. First, it provides an example of how researchers were able to produce excellent scientific research in the midst of dictatorship and war. Coupled with the group's ongoing success in postwar Germany, the German TMV investigators provide a dramatic example of how scientific communities deal with adversity as well as rapid political and social change. Second, since the researchers focused heavily (though no exclusively) on TMV, their story allows us to analyze how an experimental system other than phage contributed to the emergence of molecular biology.

Academies and Institutes↗
PubMed1982

Sequence specificity of trinucleoside diphosphate binding to polymerized tobacco mosaic virus protein.

The binding of trinucleoside diphosphates to long helical rods of tobacco mosaic virus (TMV) protein is shown to depend on base sequence, 5' AAG 3' binding being the strongest of the 25 trinucleoside diphosphate sequences measured. As TMV has a stoichiometry of three nucleotides per protein subunit, the sequence of TMV RNA suggested to be the nucleation site for self-assembly of the virus has three possible binding frames. From our binding constant data the most likely frame is predicted and shown to have two contiguous AAG sequences in a hairpin loop region.

Base Sequence↗