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PubMed1990

The primary structure of six leucine isoacceptor tRNAs of yellow lupin seeds. The structural requirements for amber tRNA suppressor activity.

Six tRNA(Leu) isoacceptors from yellow lupin seeds were purified, sequenced, and their readthrough properties over the UAG stop codon were tested using TMV RNA as a messenger. The tested tRNAs(Leu) did not show amber suppressor activity. The partial structure of tRNA(Gln), a minor species in yellow lupin, was also determined. Comparison of the nucleotide sequence of all known isoacceptors of tRNA(Tyr), tRNA(Gln) and tRNA(Leu) from plants, mammals and ciliates enabled us to find general structural requirements for tRNA to be a UAG suppressor. From the partial sequence of lupin tRNA(Gln) we suggest that it will have readthrough properties.

Autoradiography↗
PubMed1983

Quantitative evaluation of high-resolution features in images of negatively stained Tobacco Mosaic Virus.

This study investigates the causes of the apparent differences between the optical diffraction pattern of a micrograph of a Tobacco Mosaic Virus (TMV) particle, the optical diffraction pattern of a ten-fold photographically averaged image, and the computed diffraction pattern of the original micrograph. Peak intensities along the layer lines in the transform of the averaged image appear to be quite unlike those in the diffraction pattern of the original micrograph, and the diffraction intensities for the averaged image extend to unexpectedly high resolution. A carefully controlled, quantitative comparison reveals, however, that the optical diffraction pattern of the original micrograph and that of the ten-fold averaged image are essentially equivalent. Using computer-based image processing, we discovered that the peak intensities on the 6th layer line have values very similar in magnitude to the neighboring noise, in contrast to what was expected from the optical diffraction pattern of the original micrograph. This discrepancy was resolved by recording a series of optical diffraction patterns when the original micrograph was immersed in oil. These patterns revealed the presence of a substantial phase grating effect, which exaggerated the peak intensities on the 6th layer line, causing an erroneous impression that the high resolution features possessed a good signal-to-noise ratio. This study thus reveals some pitfalls and misleading results that can be encountered when using optical diffraction patterns to evaluate image quality.

Computers↗
PubMed1986

Gene transfer into intact plant cells by electroinjection through cell walls and membranes.

Tobacco mosaic virus (TMV) RNA was introduced directly into mesophyll cells of Nicotiana tabacum var. Samsun using electric-field pulses (electroinjection). The injected gene was successfully expressed in the recipient cells as judged by the assay for the virus coat protein using immunofluorescence and by the virus infectivity assay of the homogenate of the electroinjected cells for local lesions on tobacco leaves. As much as 50% of the cells that survived 24 days after electroinjection showed immunofluorescent specks.

Capsid↗
PubMed1987

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

Acetyltransferases↗
PubMed1996

Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector.

A cDNA fragment encompassing the Aequorea victoria green fluorescent protein-encoding gene (gfp) was introduced into a genomic cDNA clone of tobacco mosaic virus (TMV). Infectious RNA transcripts produced in vitro were used to inoculate tobacco (Nicotiana benthamiana) leaves. After 1-2 days, bright green fluorescent areas could be visualized upon illumination with a long-wave ultraviolet (UV) light source. The virus was capable of infecting and expressing gfp in plant tissues both locally (in the inoculated leaf) and systemically (throughout the entire plant). Continued observation of inoculated plants indicated that systemic infection resulted from a combination of two forms of virus movement: a slow, cell-to-cell spread with concomitant virus replication and gfp expression, and a rapid, vascular-mediated form of transport without gfp expression.

Animals↗
PubMed1993

The effects of a cysteinyl leukotriene antagonist (ONO-1078) on antigen-induced responses in allergic sheep.

The cysteinyl leukotrienes (LTC4/D4/E4) are putative mediators of asthma. In this study we used sheep allergic to Ascaris suum antigen to examine the effects of a novel orally active cysteinyl LT antagonist, ONO-1078, on antigen-induced early and late responses, airway inflammation, post challenge (24 h) airway hyperresponsiveness (AHR) and mucociliary clearance. Airway responses to antigen were determined by measuring specific lung resistance (SRL) before and for 8 h after challenge, bronchoalveolar lavage (BAL) was used to estimate airway inflammation, and airway responsiveness was measured by determining the carbachol dose that increased SRL by 400% (PC400). We also used a radiographic technique to measure the antigen-induced change in tracheal mucus velocity (TMV), a marker of mucociliary clearance. In two trials separated by at least 21 days, sheep were treated once with ONO-1078 (30 mg/kg, p.o.) and once with placebo (0.5% methylcellulose), 2 h before and 4 h after antigen challenge. Treatment with ONO-1078 (n = 7) provided 40% protection (p < 0.10) against the peak early increase in SRL, resulted in a more rapid reversal of the early response, and provided 96% protection against the peak late (6-8 h) increase in SRL. ONO-1078 also inhibited the AHR 24 h after challenge. In the drug trial, PC400 was unchanged as compared to pre-challenge, whereas in the placebo trial, PC400 was decreased 1.4-fold (p < 0.05). Treatment however, did not affect BAL cell numbers or differential.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗
PubMed2006

Bath-water scalds in children and thermostatic mixer valves.

BACKGROUND: Scalds in children caused by hot bath-water have been virtually eliminated in countries where thermostatic mixer valve (TMV) devices have been introduced. We aimed to determine the frequency and severity of these injuries in our region and estimate reduction in workload and cost if TMVs were introduced in the future. METHODS: A retrospective review was conducted of case-notes for all bath-water scalds patients aged under 16 years, admitted to the Northern Regional Burn Centre from the 1 January 1997 until 1 January 2005. RESULTS: One hundred and twenty-five patients were admitted over this period. Their average+/-S.D. age was 35.7+/-27.23 months. Scalds ranged from 0.5 to 45% total burn surface area (mean 9.3+/-9.15%). These patients stayed in hospital for an average of 6.3+/-5.58 days and 18 (14.4%) required tangential excision and split skin grafting. DISCUSSION: These injuries are: painful, psychologically disturbing and potentially preventable. The care of these children has significant cost implications, which could be reduced with the introduction of proposed legislation to enforce the installation of TMVs into all newly built houses. We are in favour of such legislation to prevent any further unnecessary suffering and cost arising from these accidents.

Adolescent↗
PubMed2005

A novel pathogenesis-related protein (SsPR10) from Solanum surattense with ribonucleolytic and antimicrobial activity is stress- and pathogen-inducible.

A cDNA clone (designated as SsPR10, GenBank Accession Number AY660753 ) encoding a PR10 protein from yellow-fruit nightshade (Solanum surattense) was isolated and characterized. SsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17.58 kDa and pI of 5.29. Sequence alignments showed that SsPR10 had high identity (68.1%) with CaPR10, but had only about 31.7% identity with JIOsPR10 at the amino acid level. Genomic DNA gel blot analysis indicated that SsPR10 belonged to a multigene family. The constitutively expressed SsPR10 was detected to be the highest in roots of the sterile seedlings cultured in jars, while SsPR10 expression was the highest in old yellow leaves from the seedlings incubated with sap containing TMV. SsPR10 always expressed at slightly higher level in senescent leaves than in tender ones under both conditions. Further expression analysis revealed that the signaling components of defense/stress pathways (MeJA, SA, ABA, GA3, H2O2 and Cu2+) up-regulated significantly the SsPR10 mRNA levels over the control. However, darkness failed to induce SsPR10 expression and its expression was also inhibited by cold treatment. The SsPR10 was successfully expressed in Eschericha coli and the expressed protein was purified to near homogeneity. The dialytically renatured SsPR10 protein without phosphorylation exhibited ribonucleolytic activity against S. surattense leaf total RNA preparations and could inhibit hyphal growth of Pyricularia oryzae. Our findings suggest that the novel stress- and pathogen-inducible SsPR10 with ribonucleolytic and antimicrobial activity participates not only in the defense/stress response pathways but also in plants' growth, development and senescence.

Amino Acid Sequence↗
PubMedDate not supplied

Windex: a toolset for indexing helices.

We describe here a set of procedures and algorithms that may be used as an aid in determining the indexing rule of a helical specimen. Crystallizing macromolecules into helical arrays has the potential to speed up and simplify the process of three-dimensional reconstruction of the macromolecular structure. The process of helical reconstruction has been largely automated except for the critical first step of indexing the helical diffraction pattern. This is quite often the rate-limiting step in the overall process, particularly in the case of large helical tubes, which have complicated helical diffraction patterns that may vary from tube to tube. We have developed a set of procedures, supported by a graphical user interface, that provide a straightforward and semi-automated approach to indexing a helical structure. The new procedures have been tested using a number of helical specimens, including TMV, acto-myosin, decorated microtubules, and a variety of helical tubes of a bacterial membrane protein.

ATP-Binding Cassette Transporters↗
PubMed2004

Detection of plant viruses using a surface plasmon resonance via complexing with specific antibodies.

The use of instrumental systems based on the surface plasmon resonance (SPR) for rapid diagnosis of intact plant viruses (in particular, tobacco mosaic virus (TMV)) is considered. A new approach using detection of viral antigen and antibody (IgG) complexes formed during the preincubation step (instead of their consecutive application in classical approach) is discussed. A comparison between signal level registered from the mixture of virus and specific serum and that from the sample without virus (samples deposited onto the sensor surface treated with thiocyanate and protein A Staphylococcus aureus) allows unambiguous detection of viral particles in the material studied. The performance capabilities of the method are discussed and illustrated by quantitative detection of virus in the actual samples (cells homogenate) at high concentration.

Antibodies, Viral↗
PubMed2005

An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water.

Dielectrophoresis (DEP), the motion of a particle caused by an applied electric field gradient, can concentrate microorganisms non-destructively. In insulator-based dielectrophoresis (iDEP) insulating microstructures produce non-uniform electric fields to drive DEP in microsystems. This article describes the performance of an iDEP device in removing and concentrating bacterial cells, spores and viruses while operated with a DC applied electric field and pressure gradient. Such a device can selectively trap particles when dielectrophoresis overcomes electrokinesis or advection. The dielectrophoretic trapping behavior of labeled microorganisms in a glass-etched iDEP device was observed over a wide range of DC applied electric fields. When fields higher than a particle-specific threshold are applied, particles are reversibly trapped in the device. Experiments with Bacillus subtilis spores and the Tobacco Mosaic Virus (TMV) exhibited higher trapping thresholds than those of bacterial cells. The iDEP device was characterized in terms of concentration factor and removal efficiency. Under the experimental conditions used in this study with an initial dilution of 1 x 105 cells/ml, concentration factors of the order of 3000x and removal efficiencies approaching 100% were observed with Escherichia coli cells. These results are the first characterization of an iDEP device for the concentration and removal of microbes in water.

Bacillus subtilis↗
PubMed2004

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique.

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.

Chlorogenic Acid↗
PubMed2006

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli.

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).

Amino Acid Sequence↗
PubMed2006

Isolation of a novel Ser/Thr protein kinase gene from oligochitosan-induced tobacco and its role in resistance against tobacco mosaic virus.

Oligochitosan induces defense responses to pathogenic microbes in a wide variety of plants by acting as an elicitor. In the present study, mRNA differential display was used to investigate oligochitosan-induced transcriptional activation of defense-related genes. Accordingly, a novel Ser/Thr protein kinase gene was isolated and designated as oligochitosan-induced protein kinase (oipk). Molecular cloning showed that oipk contains six introns interrupted by seven exons. The open reading frame (ORF) of the gene is 1848 bp, which encodes a putative protein of 615 amino acids with the predicted molecular mass of 70.96 kDa and a pI of 6.32. A plant oipk antisense expression vector was constructed and transformed into tobacco by Agrobacterium tumefaciens. Decreased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity and decreased resistance to tobacco mosaic virus (TMV) were observed in transgenic tobacco. RT-PCR analysis revealed that oipk was expressed at high levels after oligochitosan induction in wild-type tobacco, but not in transgenic tobacco. These results indicated that oipk is involved in the signal pathway of oligochitosan-induced resistance in tobacco.

Amino Acid Sequence↗
PubMed2004

Visualizing the RNA molecule in the bacterially expressed vesicular stomatitis virus nucleoprotein-RNA complex.

Packaging of the RNA molecule in viruses is important for the preservation and expression of viral genomic information. The vesicular stomatitis virus (VSV) nucleoproteins are kept associated with its negative-strand RNA during the mRNA synthesis and replication, in contrast to the tobacco mosaic virus whose nucleoproteins are released from RNA. It has been a puzzle how the VSV RNA is packaged to meet the contradicting requirements of protection and the accessibility to the polymerase. We report an 18 A resolution structure of the recombinant nucleoprotein-RNA complex determined by single-particle electron microscopy. In the 3D density map, a ring of density is resolved on the inner surface and the density is proposed to be the RNA. The RNA is located on the inner surface of the decameric complex near the top end. This is dramatically different from the RNA packaging in TMV, but consistent with previously published biochemical findings.

Amino Acid Sequence↗
PubMed2005

Plant based HIV-1 vaccine candidate: Tat protein produced in spinach.

The HIV-1 Tat protein has been recently explored as a prospective vaccine candidate with broad, subtype non-specific action. We approached the problem of delivery of Tat through the mucosal route by expressing Tat in an edible plant. The tat gene was assembled from synthetic overlapping oligonucleotides, and was subsequently cloned into a plant virus-based vector tobacco mosaic virus (TMV). Spinach plants inoculated with the Tat-producing constructs were collected and fed to mice 7-14 days post inoculation. DNA vaccinations were performed using a gene gun. Codon optimization of the Tat gene expressed in spinach plants resulted in several-fold yield increase as detected in immunoblots, and did not cause severe symptoms in inoculated plants. Mice were fed with the Tat-producing or control vector-inoculated spinach. After three feedings, 1 week apart, 1g per mice, no differences were detected in the growth rate or behavior of the animals fed with these three types of spinach. None of the animals developed measurable Tat antibodies. Following DNA vaccination, however, mice having previously received oral Tat developed higher antibody titers to Tat than did the controls, with the titers peaking at 4 weeks post-vaccination. Codon optimization allows production of up to 300-500 microg of Tat antigen per 1 g of leaf tissue in spinach using a plant virus-based expression system. The plant produced Tat does not seem to have any apparent adverse effect on mice growth or behavior, when fed with spinach for 4 weeks. ELISA data suggested that oral Tat primed for the development of Tat antibodies when mice were subsequently vaccinated with plasmid DNA designed for Tat expression.

AIDS Vaccines↗
PubMed2006

Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector.

A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 microg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector.

Antibodies, Monoclonal↗
PubMed1962

Action of urea on tobacco mosaic virus. II. The bonds between protein subunits.

The rate of urea degradation is not uniform over the tobacco mosaic virus (TMV) rod, as might be expected since the protein subunits appear to be chemically identical. Only two stable intermediates are formed; the four components with sedimentation constants, S(40) (20), about 80, 120, 140, and 165 correspond to the two intermediates with and without RNA tails. Analysis of the reaction kinetics for the disappearance of whole virus and of the first intermediate was made in terms of a new hypothesis concerning protein bonding.

Protein Subunits↗