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Biomedical subjects

A R Gould

Publications and source records attributed to A R Gould.

At least 19 recordsLinked to original sources
PubMed1992

Linear and cyclic peptide analogues of the polypeptide cardiac stimulant, anthopleurin-A. 1H-NMR and biological activity studies.

A loop corresponding to residues 8-17 in the polypeptide cardiac stimulant anthopleurin-A is known to be important for the cardiostimulant activity of this molecule. To investigate the activity and possible conformations of this loop in isolation, two synthetic peptides have been studied. The first corresponds to residues 6-20 of anthopleurin-A with Cys6 replaced by Thr, and the second to residues 6-21 of anthopleurin-A, with Thr21 replaced by Cys. The introduction of an additional cysteine in the latter peptide enabled an intramolecular disulfide to be formed between the N- and C-terminal residues. Both linear peptides and the disulfide-containing analogue lack the cardiostimulant and Na(+-)-channel binding activity in the parent molecule, anthopleurin-A, indicating that although the loop is important for the function of anthopleurin-A, other regions of the molecule must also be involved in activity. Assignments of the 1H-NMR spectra of both peptides are presented, and their pH and temperature dependences investigated. The results show that the amide protons of Gly5 and Asn11 (corresponding to Gly10 and Asn16 in anthopleurin-A) sample hydrogen-bonded conformations in solution. Based on these NMR data, two regions of non-random structure, encompassing residues 2-5 and 8-11, respectively, are proposed, and the possible involvement of such structures in the activity of anthopleurin-A is discussed.

Amino Acid Sequence↗
PubMed1992

Cinnamon-induced stomatitis venenata, Clinical and characteristic histopathologic features.

Fourteen new cases of cinnamon-induced stomatitis are reported. Ten of these fourteen cases were first detected on the basis of histopathologic changes, which included hyperkeratosis, chronic lichenoid mucositis with plasmacytic infiltration, and marked chronic perivasculitis. Six cases of false-positive histopathologic findings are presented for comparison. It is recommended that when the histopathologic features described are recognized, cinnamon stomatitis should be considered.

Adult↗
PubMed1992

Application of a diagnostic DNA probe for the differentiation of the two types of Mycoplasma mycoides subspecies mycoides.

Contagious bovine pleuropneumonia (CBPP), which is caused by Mycoplasma mycoides subspecies mycoides, is still a serious disease in some parts of the world. There is also a commonly occurring mycoplasma which is sufficiently related to the CBPP organism to bear the same name, even though this organism does not cause CBPP. Thus it is very important to be able to distinguish between these organisms and identify either with certainty. Fragments derived from M mycoides subspecies capri by restriction enzyme digestion of genomic DNA were cloned into the vector M13mp8. One resulting clone CAP-21, with a 1.5 kb insert was used as a probe in Southern hybridisation assays where genomic DNA was digested with the restriction endonuclease TaqI. This probe could differentiate a strain of M mycoides subspecies mycoides which does not cause CBPP. Subsequent tests on 14 other strains from cattle and goats showed that although they were isolated from diverse geographical areas, CAP-21 could clearly differentiate between these two types of M mycoides subspecies mycoides.

Animals↗
PubMed1992

Relationships between members of the Mycoplasma mycoides cluster as shown by DNA probes and sequence analysis.

A gene probe, CAP-21, which demonstrated interrelationships between the members of the Mycoplasma mycoides cluster was developed. The probe easily differentiated mycoplasmas in this cluster by clear and predictable hybridization patterns in Southern blots and separated the cluster into four groups. Strains of M. mycoides subsp. mycoides which were capable of causing contagious bovine pleuropneumonia composed one group. Strains of M. mycoides subsp. mycoides which did not cause contagious bovine pleuropneumonia together with strains of M. mycoides subsp. capri composed the second group. Mycoplasma capricolum and the F38 mycoplasmas formed a third group, while the bovine group 7 mycoplasmas composed a separate, fourth group. Further support for the above grouping of the cluster was obtained when amplified DNA analogous to the probe from one representative strain of each of the cluster members was sequenced and these data were used to construct a phylogenic tree. Contagious caprine pleuropneumonia is recognized as an important disease, and the etiological agent of this disease is now known to be the F38 mycoplasma. The CAP-21 probe did not differentiate between M. capricolum and the closely related F38 mycoplasma. A second probe, F38-12, which was capable of distinguishing these two mycoplasmas was made.

Animals↗
PubMed1991

A bluetongue serogroup-reactive epitope in the amino terminal half of the major core protein VP7 is accessible on the surface of bluetongue virus particles.

Immunoelectron microscopy has been used to confirm that the core protein VP7 is accessible on the surface of bluetongue virus (BTV) particles. Monospecific antibodies generated to vaccinia virus-expressed VP7 and an anti-VP7 monoclonal antibody (MAb 20E9) bound to native virus particles and were localized by protein A-gold. In contrast, MAb 20E9 labeled directly with gold failed to gain access and bind, suggesting that VP7 is neither adventitiously adsorbed to the virion surface nor exposed in a manner such as protrusion through the outer capsid. Thus the surface layer of BTV may be considered as a net which only partially obscures the underlying core particle. Sequencing of VP7 revealed it to be an extremely hydrophobic protein, 350 amino acids in length with cysteine residues at positions 15, 65, and 154. Examination of VP7 in the cytosol of cells infected with either BTV or a vaccinia virus recombinant expressing VP7 indicated that the protein may exist as an oligomer, whose constituent monomers are not linked by intermolecular disulfide bonds. The cysteine residues in sodium dodecyl sulfate (SDS)-denatured, dithiothreitol (DTT)-treated VP7 were labeled with the fluorescent iodoacetamide AEDANS and the protein was cleaved by V8 protease. The size of the labeled peptides and knowledge of the location of potential V8 cleavage sites suggested that the enzyme cleaved VP7 at three locations (glutamic acid residues at positions 61, 104 (or 108), and 132 (or 134 or 135). Analysis of the fluorescent peptides generated by V8 protease cleavage of VP7 labeled with AEDANS in the absence of DTT (i.e., with any putative intramolecular disulfide bonds intact) suggested that the cysteine at position 154 was the only one accessible to AEDANS. The cysteines at positions 15 and 65 may therefore be linked via a disulfide bond. Denaturation of VP7 with SDS did not eliminate the capacity of the protein to bind MAb 20E9. However, the sensitivity of the epitope to reduction and acetylation and its resistance to either of these processes alone suggest that it may be located near a disulfide bond linking cysteines at positions 15 and 65. Confirmation that the epitope lay in the amino-terminal half of the VP7 came from immunoelectron microscopy experiments in which thin sections of bacteria expressing the complete VP7 and the amino-terminal half were probed with MAb 20E9 and protein A-gold.

Amino Acid Sequence↗
PubMed1991

High level expression of the major core protein VP7 and the non-structural protein NS3 of bluetongue virus in yeast: use of expressed VP7 as a diagnostic, group-reactive antigen in a blocking ELISA.

The major core protein VP7 and a non-structural protein NS3 of bluetongue virus serotype 1 have been synthesized from recombinant plasmids using both an in vitro transcription/translation system and a yeast expression system. Bluetongue virus genes were transcribed under the control of the bacteriophage SP6 promoter and the regulatable yeast metallothionein promoter. An indirect ELISA showed that expression of NS3 in yeast was inducible with 1 mM CuSO4 and VP7 synthesis was constitutive but could be further induced. The preferred procedure for antigen extraction from yeast was sonication for VP7 and SDS/NaOH treatment for NS3. Yeast-expressed VP7 antigen and a monoclonal antibody were used in a blocking ELISA to distinguish sera raised against bluetongue virus serotypes from those generated to viruses of the epizootic haemorrhagic disease serogroup.

Antigens, Viral↗
PubMed1991

Phylogenetic analyses of the complete nucleotide sequence of the capsid protein (VP3) of Australian epizootic haemorrhagic disease of deer virus (serotype 2) and cognate genes from other orbiviruses.

The complete nucleotide sequence of the minor capsid protein (VP3) of epizootic haemorrhagic disease of deer virus (EHDV; Australian serotype 2) was determined using a combination of cloning and sequencing methods. Gene segment 3 that coded for the EHDV VP3 capsid protein was 2768 nucleotides in length with a coding region of 2697 nucleotides flanked by 5' and 3' non-coding regions of 17 and 53 nucleotides, respectively. A protein of 899 amino acids (Mr 103,160) having no overall charge at neutral pH was deduced from the nucleotide sequence. Comparisons with equivalent regions from the other Australian EHDV serotypes showed the VP3 genes and the segments that coded for them were similar, varying by a maximum of 5%. Comparisons with known cognate genes from bluetongue viruses showed that their VP3 genes and the proteins translated from them were remarkably similar to those of EHDV, having approximately 70% to 80% homology at either level, respectively. In an attempt to delineate the evolution of orbiviruses, we have obtained sequence data from the VP3 genes from representative members of all Australian orbiviruses now known. Computer analyses of this data enabled a phylogenetic tree for the orbiviruses to be proposed that incorporated the concept of topotypes.

Amino Acid Sequence↗
PubMed1991

Detection and characterisation of bluetongue virus using the polymerase chain reaction.

Pairs of oligodeoxynucleotide primers whose sequences were based on those of RNA segment 3 that encodes the bluetongue virus serogroup-reactive protein VP3, were synthesized for three BTVs from different geographic regions of the world and for seven Australian orbiviruses. Each pair of primers was then tested for the synthesis of cDNA and in subsequent polymerase chain reactions (PCR) with all ten virus groups. All primers were serogroup-specific at low or high stringency. One pair of primers was specifically designed for its ability to serogroup a BTV isolate irrespective of its geographic origin. At either high or low stringency, this primer-pair resulted in a common and specific PCR product for each of the BTVs tested but not for the other orbiviruses. Eight pairs of primers based on RNA2 sequences (the gene segment encoding the serotype-specific protein VP2) were also synthesized for the eight Australian serotypes of BTV. Each primer-pair was serotype-specific at low or high stringency except for the BTV16A pair, which cross-reacted with BTV3A and also gave a non-specific product that differed in Mr from the authentic PCR product. Using the PCR and BTV1A RNA3-based primers, BTV1A was detected in blood samples from two sheep at 9 days post inoculation. Virus was found in the platelet, buffy-coat and packed red blood cell fractions, but not in whole blood.

Animals↗
PubMed1991

Localization of the non-structural protein NS3 in bluetongue virus-infected cells.

The localization of the blue tongue virus (BTV) non-structural proteins NS3 and NS3a has been identified using immunoelectron microscopical techniques. NS3 and NS3a have been observed in the plasma membrane of BTV- and recombinant vaccinia virus (expressing NS3)-infected cells. The NS3 protein was associated with areas of membrane perturbation. There was a good correlation between the presence of NS3 and NS3a and BTV release. The NS3 protein was associated with membrane fragments and the inability to detect it on the extracellular aspect of intact cells suggested that the protein was not exposed extracellularly. Electron microscopical and biochemical evidence suggested that fragments of plasma membrane containing NS3 and NS3a were released from infected cells. Collectively, the data indicate that NS3 and NS3a may be involved in the final stages of BTV morphogenesis, i.e. the release of BTV from infected cells.

Animals↗
PubMed1990

Structure-function relationships in the polypeptide cardiac stimulant, anthopleurin-A. Effects of limited proteolysis by trypsin.

Selective proteolysis of the polypeptide cardiostimulant anthopleurin-A by trypsin introduces a single break in the polypeptide backbone on the C-terminal side of Arg14. The resulting derivative is devoid of any cardiostimulant activity. The structural changes which accompany this loss of activity have been examined by one- and two-dimensional 1H-NMR spectroscopy. It is shown that the overall backbone folding of anthopleurin-A is conserved on digestion, with some structural changes occurring for residues which are adjacent to the site of cleavage by trypsin. Thus, although previous NMR studies on anthopleurin-A indicate that the region surrounding Arg14 is devoid of any ordered structure, it appears that some degree of structural integrity is required to allow the essential side chains to adopt the conformation necessary to produce a cardiostimulant effect.

Amino Acid Sequence↗
PubMed1990

The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses.

Monoclonal antibodies which reacted with four different epitopes were used to select neutralization-resistant variants of Australian bluetongue virus serotype 1 (BTV1AUS; isolate CS156). Nucleotide sequencing of the VP2 outer coat protein gene of these variants showed that two of them contained alterations within the previously defined neutralization site at amino acids 328 to 335 (Gould et al., 1988). Comparison of VP2 sequences of several BTV serotypes, in addition to nucleotide sequence changes in a number of variants, suggested that this neutralization site was larger and contained 19 amino acids, the conformation of which could be affected by other regions of the VP2 protein. Nucleotide sequencing of neutralization-resistant variants revealed a total of four other regions of VP2 affecting the ability of monoclonal antibodies to neutralize the virus and these results support the notion that the neutralization site in VP2 was conformation dependent. The complete nucleotide sequence of the VP2 gene of virulent BTV1AUS (C5156) was determined directly from viral nucleic acid isolated from the blood of a sheep suffering clinical bluetongue disease. Comparison of the VP2 sequence of this virulent virus with that previously published for an avirulent, laboratory strain (Gould, 1988), indicated that the passage of virulent virus approximately 20 times in tissue culture over the last decade, not only led to attenuation but resulted in the appearance of ten nucleotide changes in the VP2 gene. Six of these nucleotide changes were silent, two resulted in conservative amino acid substitutions and two generated radical amino acid changes. However, in a separate experiment, a single passage of the virulent virus in tissue culture while leading to attenuation did not result in a nucleotide change in the VP2 outer coat protein gene.

Amino Acid Sequence↗
PubMed1990

Relationships amongst bluetongue viruses revealed by comparisons of capsid and outer coat protein nucleotide sequences.

Sequence data from the gene segments coding for the capsid protein. VP3, of all eight Australian bluetongue virus serotypes were compared. The high degree of nucleotide sequence homology for VP3 genes amongst BTV isolates from the same geographic region supported previous studies (Gould, 1987; 1988b, c; Gould et al., 1988b) and was proposed as a basis for "topotyping" a bluetongue virus isolate (Gould et al., 1989). The complete nucleotide sequences which coded for the VP2 outer coat proteins of South African BTV serotypes 1 and 3 (vaccine strains) were determined and compared to cognate gene sequences from North American and Australian BTVs. These VP2 comparisons demonstrated that BTVs of the same serotype, but from different geographical regions, were closely related at the nucleotide and amino acid levels. However, close inter-relationships were also demonstrated amongst other BTVs irrespective of serotype or geographic origin. These data enabled phylogenic relationships of the BTV serotypes to be analysed using VP2 nucleotide sequences as a determinant.

Amino Acid Sequence↗
PubMed1988

The use of recombinant DNA probes to group and type orbiviruses. A comparison of Australian and South African isolates.

Under the appropriate conditions, recombinant DNA (rDNA) probes to either RNA segment 2 or segment 6 may be used to mimic the serotyping of bluetongue virus (BTV), but this may be attempted only on isolates originating in the same geographical area. Using an rDNA probe from RNA segment 3, group reactivities between the South African and Australian BTV isolates have been observed within those defined by serological methods, as have cross reactivities within the orbivirus group. Comparative hybridisation data is presented to show that variation within the RNA genome segments of BTV renders rDNA probes of RNA3 inadequate for serogrouping the orbiviruses. However, rDNA probes to RNA3 may be used to delineate the geographical origin of a BTV isolate.

Africa, Southern↗
PubMed1988

Morphogenesis of a bluetongue virus variant with an amino acid alteration at a neutralization site in the outer coat protein, VP2.

Neutralization-resistant variants of bluetongue virus, selected with a monoclonal antibody to the outer coat protein VP2, have been used to delineate a neutralization epitope on the VP2 protein. Comparison of the RNA 2 sequence of four variants with that of the wild-type virus indicated that each variant contained a single nucleotide substitution which in turn resulted in a single amino acid alteration in VP2. The changes were clustered within a span of eight amino acids at positions 328 to 335 in the VP2 protein. In addition, analyses of cells infected with wild-type and a variant virus V35B2 have provided information on the site of VP2 addition to virus particles during morphogenesis. Electron microscopic examination revealed few virus-like particles around virus inclusion bodies (VIB) in wild-type virus-infected cells and cytoskeletons. In contrast, VIB in cells infected with the neutralization-resistant variant V35B2 were surrounded by particles identified as virus cores on the basis of their size and morphology. Probing of cytoskeletons with gold-labeled anti-VP2 monoclonal antibody revealed that in wild-type virus-infected cells the antibodies reacted weakly with VIB and only at locations where virus particles appeared to be leaving. The core-like particles surrounding VIB in V35B2-infected cells labeled very weakly with the anti-VP2 antibody. In contrast, wild-type and V35B2 virus particles which bound to the cytoskeleton at locations distal to VIB and those outside the infected cell bound significant amounts of antibody. These results suggest that although some VP2 may be added to developing virus particles at the periphery of VIB, the remainder of the VP2 protein is added outside the VIB either in the cytosol or following attachment of the particles to the cytoskeleton.

Amino Acid Sequence↗