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Biomedical subjects

I M Roberts

Publications and source records attributed to I M Roberts.

At least 19 recordsLinked to original sources

Host-specific cell-to-cell and long-distance movements of cucumber mosaic virus are facilitated by the movement protein of groundnut rosette virus.

The cucumovirus, cucumber mosaic virus (CMV), requires both the 3a movement protein (MP) and the capsid protein (CP) for cell-to-cell movement. Replacement of the MP of CMV with the MP of the umbravirus, groundnut rosette virus (GRV), which does not encode a CP, resulted in a hybrid virus, CMV(ORF4), which could move cell to cell in Nicotiana tabacum and long distance in N. benthamiana. After replacement of the CMV CP in CMV(ORF4) with the gene encoding the green fluorescent protein (GFP), the hybrid virus, CMV(ORF4.GFP), expressing both the GRV MP and the GFP, could move cell to cell but not systemically in either Nicotiana species. Immunoelectron microscopic analysis of cells infected by the hybrid viruses showed different cellular barriers in the vasculature preventing long-distance movement of CMV(ORF4) in N. tabacum and CMV(ORF4.GFP) in N. benthamiana. Thus the GRV MP, which shows limited sequence similarity to the CMV MP, was able to support CP-independent cell-to-cell movement of the hybrid virus, but CP was still required for long-distance movement and entry of particular vascular cells required functions encoded by different proteins.

Capsid↗

Satellite RNA is essential for encapsidation of groundnut rosette umbravirus RNA by groundnut rosette assistor luteovirus coat protein.

Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed.

Animals↗

Small bowel diseases in the elderly.

Small bowel diseases in the elderly are discussed including small bowel gastrointestinal bleeding and malabsorption syndromes such as celiac disease. Crohn's disease and nonsteroidal enteropathy also cause considerable morbidity in the elderly population and are reviewed. Finally, a brief discussion of malignancies of the small bowel (adenocarcinoma, carcinoid, and so forth) occurring in the elderly is presented.

Aged↗

Production of a functional single chain antibody attached to the surface of a plant virus.

A potato virus X (PVX) vector was used to express a single chain antibody fragment (scFv) against the herbicide diuron, as a fusion to the viral coat protein. The modified virus accumulated in inoculated Nicotiana clevelandii plants and assembled to give virus particles carrying the antibody fragment. Electron microscopy was used to show that virus particles from infected leaf sap were specifically trapped on grids coated with a diuron-BSA conjugate. The results demonstrate that the PVX vector can be used as a presentation system for functional scFv.

Cloning, Molecular↗

Ultrastructural and temporal observations of the potyvirus cylindrical inclusions (Cls) show that the Cl protein acts transiently in aiding virus movement.

A systematic ultrastructural study across the edge of an advancing infection in pea seed-borne mosaic potyvirus-infected pea cotyledons showed the cylindrical inclusion (CI) protein to exist in transient functional states. Initially, the characteristic CI pinwheel inclusion bodies were positioned centrally over the plasmodesmal apertures (including those of plasmodesmata connected to the previously infected cell), in agreement with a proposed role in virus movement (Carrington et al., 1998, Plant J., 13, in press). The viral coat protein was associated with these structures and was seen within the modified plasmodesma, most notably in a continuous channel that passed along the axis of the pinwheel and through the plasmodesma. The CI protein was not detected within the plasmodesmal cavities. Later in the infection (i.e., behind the zone of active virus replication) the CI was no longer associated with cell walls, or with coat protein, and showed signs of structural degeneration. In contrast, the coat protein remained within plasmodesmal cavities. The role of the CI in assisting virus movement is not known but the presence of the CI was linked with an apparent transient reduction in callose in the vicinity of the plasmodesmata.

Inclusion Bodies, Viral↗

Assembly and movement of a plant virus carrying a green fluorescent protein overcoat.

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.

Amino Acid Sequence↗

Absorption and imagery locate immune responses in the body.

Imagery instructions specifying mucosal immunity should alter mucosal immunoglobulin A (m-IgA) levels in high absorbers, whose intent concentration evokes intense physiological responses. After screening for health status, 121 high or low absorbers were randomly assigned to either Relaxation Alone (R), Relaxation with Mucosal Immune Imagery (RI), or Vigilance Task control (VT). Before and after one 60-min intervention, subjects reported theory-relevant psychological variables and provided 5 ml whole saliva, which was immediately frozen and assayed later en masse with enzyme-linked immunoabsorbence (ELISA). MANOVA analysis of psychological variables replicated past research. ANOVA on residualized m-IgA found Time x Absorption interaction and Condition main effects. High more than low absorbers responded to relaxation with mucosal immune imagery by producing higher m-IgA. High absorbers appear able to locate where their immune systems will respond. Individual differences like absorption level need to be emphasized in diagnosis and treatment responsiveness.

Absorption↗

Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli.

The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.

Base Sequence↗

Antigenic structure of the coat protein of potato mop-top furovirus.

The antigenic structure of the coat protein (CP) of potato mop-top furovirus (PMTV) was studied by electron microscopy of virus particles labeled with gold-conjugated monoclonal antibodies (MAbs) and by the reactions of MAbs with overlapping octapeptides (Pepscan) representing the complete amino acid sequence of the CP. A total of seven epitopes were identified in the CP. MAb SCR 69 detected a continuous epitope, which was located at the extreme N-terminus of the CP, was exposed at the surface along the sides of PMTV particles, and was removed by treating them with trypsin. MAb SCR 68 detected a discontinuous epitope found at the concave end of PMTV particles. Five other epitopes, which were detected by Pepscan tests, were located internally in, and at intervals along, the CP amino acid sequence. A tentative model of the PMTV CP subunit was produced, based on computer-aided prediction of its secondary structure and apparent similarities with the CP of tobacco mosaic virus. In this model, four of the epitopes occur at high radius in each of the pairs of parallel and anti-parallel alpha-helices in the CP subunit. The fifth is at low radius in the putative left radial alpha-helix. The epitope detected by MAb SCR 77, although amenable to study by Pepscan, contains three reactive elements, separated by short runs of nonessential residues, in a sequence of 13 amino acids. In intact virus particles, the CPs of beet necrotic yellow vein furovirus and PMTV apparently differ in the accessibility of their N- and C-termini.

Amino Acid Sequence↗

Assembly of tobacco mosaic virus and TMV-like pseudovirus particles in Escherichia coli.

High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli. Native tobacco mosaic virus (TMV) CP expressed in E. coli remains soluble but has a non-acetylated N-terminal Ser residue and following extraction, is unable to package TMV RNA in vitro under standard assembly conditions. Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV. All TMV CPs made in E. coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP. TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4 kb ssRNA genome. Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence. Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles. When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2-18 h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4 micrograms/mg total bacterial protein). In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E. coli system also offers a rapid approach for studying the molecular requirements for plant virus "self-assembly" in vivo. Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.

Capsid↗

Factors affecting the efficiency of immunogold labelling of plant virus antigens in thin sections.

Sections of pellets of six purified plant viruses with three different morphologies were used to examine different technical aspects of the immunogold labelling (IGL) technique. The results showed that fixation by glutaraldehyde alone was better than with osmium tetroxide post-fixation, and that Decon 75 was the best of the pretreatments tried. The study showed that different virus homologous antisera gave different results in IGL tests, and that longer incubation times with both antiserum and gold probe gave higher label densities without any increase in background label. Also, cross-absorption of the virus antisera with healthy host protein before use gave cleaner backgrounds and thus higher specificity. The work also examined the relationship between label density and amounts of visible virus. There was no correlation between the numbers of virus particles seen in sections and the numbers of gold particles; moreover, there was no apparent relationship between label density and the orientation or distribution of the virus particles in the section. The role of the embedding resin and its polymerisation temperature are also discussed.

Antigens, Viral↗

Signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus X.

A British isolate of potato aucuba mosaic potexvirus (PAMV) was transmitted by aphids (Myzus persicae) which had fed previously on a source of potato Y potyvirus (PVY). Nucleotide sequence analysis of the PAMV coat protein gene indicated that amino acid residues 14 to 16 from the N terminus of the coat protein have the sequence DAG, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. A recombinant virus isolate (TXPA7) was produced in which a segment of the coat protein gene of PAMV encoding the 40 N-terminal amino acids was inserted in the genome of potato X potexvirus (PVX) in place of the segment encoding the 28 N-terminal amino acids of PVX coat protein. This isolate, and a second similar recombinant (TXPA5) in which the DAG motif was changed to YTS, were mechanically transmissible to intact plants, in which they caused slightly milder symptoms than PVX. Particles of TXPA7 reacted in immunosorbent electron microscopy with PVX- and PAMV-specific antibodies and so were antigenically distinguishable from PAMV and PVX particles, which reacted only with their homologous antibody, and from TXPA5 particles, which reacted only with the PVX antibody. Recombinant TXPA7 was transmitted by aphids that had already fed on a source of PVY whereas TXPA5 and PVX were not. TXPA7 was not transmitted by aphids that had not fed on a PVY source. It is concluded that (i) the potyvirus-dependent aphid transmissibility of PAMV results from possession of a domain which includes the DAG motif and is located near the N terminus of the virus coat protein, and (ii) potyvirus-dependent aphid transmissibility can be conferred on PVX, a non-aphid-borne potexvirus, by substituting this domain for the N-terminal part of its coat protein.

Amino Acid Sequence↗

Diffraction studies of the particles of two closteroviruses: heracleum latent virus and heracleum virus 6.

X-ray diffraction from oriented specimens of purified preparations of particles of heracleum latent closterovirus (HLV) showed that they have a helical arrangement of protein subunits, and that the structure repeats in five helical turns in which there are 5q +/- 1 protein subunits, where q is an integer. The pitch of the helix was estimated to be 3.26 (+/- 0.10) nm. Optical diffraction patterns from electron micrographs of HLV particles give an estimated pitch of 3.3 (+/- 0.2) nm and show that the number of subunits in the repeat period is 5q-1, where q has a value of 8 or 9. Optical diffraction from electron micrographs of particles of a second closterovirus, heracleum virus 6, shows that they too have a helical structure which repeats in five turns, in which there are 5q +/- 1 protein subunits. The estimated pitch of the primary helix is 3.6 (+/- 0.2) nm, and the estimate of q is 9.

Microscopy, Electron↗

PC GI board review. A ToolBook application for postgraduate review of Gastroenterology.

"Hypertext" applications have become an important educational resource for medical teaching. ToolBook version 1.5 allows the import of SVGA 256 color, 640 x 480 pixel images. We developed a program for the review of Gastroenterology by Subspecialty Board applicants which included digitized pathology, endoscopy, dermatology, and radiology images interfaced with textual description. 5 cases were presented with 6 questions per case to test the user's comprehension of the material. A scoring function was included to give feedback to the users. An evaluation questionnaire was also completed to survey user satisfaction with the program. A similar "shell" could be applied to other teaching programs.

Computer-Assisted Instruction↗

A comparison of the sensitivity and specificity of enzyme immunoassays and time-resolved fluoroimmunoassay.

Time-resolved fluoroimmunoassay (TR-FIA) and various enzyme immunoassays (EIA) were compared in order to determine the detection system which showed the greatest degree of sensitivity without sacrificing specificity. The system chosen for the evaluation of these assays was the detection of antibodies to human immunodeficiency virus (HIV). For EIA, horseradish peroxidase (HRP) and alkaline phosphatase (AP) were investigated, each with a number of different substrates. HRP with its fluorogenic substrate, 3-(p-hydroxyphenyl)propionic acid (HPPA) was 1.6 times (p less than 0.01) more sensitive than with 3,3',5,5'-tetramethylbenzidine (TMB) and four times (p less than 0.001) more sensitive than with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). AP with its fluorogenic substrate, 4-methylumbelliferyl phosphate (4MeUP), was 6-7 times (p less than 0.001) more sensitive than with phenolphthalein monophosphate (PMP) and 8-13 times (p less than 0.001) more sensitive than with p-nitrophenyl phosphate (pNPP). TR-FIA with Eu3(+)-labelled anti-human IgG was equivalent in sensitivity to HRP with TMB and AP with 4MeUP.

Acquired Immunodeficiency Syndrome↗