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D R Sargan

Publications and source records attributed to D R Sargan.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cloning of canine rom-1 and its investigation as a candidate gene for generalized progressive retinal atrophies in dogs.

Generalized progressive retinal atrophy (gPRA) represents a genetically heterogenous group of retinal degenerations affecting pedigree dogs. Currently, we are using a candidate gene approach in an attempt to identify mutations causing gPRA in dogs. Here we report the cloning, sequencing and analysis of canine rom-1, a structural gene of the rod photoreceptor. Single-stranded conformation polymorphism (SSCP) analysis was used to look for polymorphisms segregating with gPRA in the English cocker spaniel, Labrador retriever, miniature poodle, miniature long-haired dachshund, Tibetan terrier, miniature schnauzer, Cardigan Welsh corgi and Irish wolfhound. Further investigation involved DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. Our studies revealed the presence of three polymorphisms, none of which segregated with disease phenotype. Haplotype analysis identified four rom-1 alleles. Our results indicate that rom-1 is unlikely to be a cause of gPRA in the breeds of dog examined.

Animals↗
PubMed1996

Recent advances in understanding the spectrum of canine generalised progressive retinal atrophy.

Canine generalised progressive retinal atrophy (gPRA) is a large and ever-increasing collection of naturally occurring, heterogeneous, progressive disorders. Most are inherited in an autosomal recessive manner and new, breed-specific forms continue to be described. The gPRAs cause photoreceptor cell death and subsequent retinal degeneration, culminating in blindness. In humans, similar inherited retinal dystrophies are recognised as retinitis pigmentosa and macular dystrophy. Molecular biological studies have revealed disease-causing mutations in several genes in humans and also in mice with retinal dystrophies. Recently, molecular genetic techniques have identified the cause of one form of gPRA in Irish setters while important candidate genes have been investigated in other breeds. Identification of mutations responsible for different forms of gPRA allows carrier and predegenerate animals to be detected using DNA-based tests. Such genetic tests will greatly facilitate the eradication of these diseases in different breeds.

Animals↗
PubMed1996

The molecular defect underlying canine fucosidosis.

Fucosidosis is a lysosomal storage disease which affects humans and English springer spaniel dogs. The disease is recessively inherited in both species and results from a deficiency of the enzyme alpha-L-fucosidase. We have recently cloned and sequenced the canine fucosidase gene (EMBL sequence admission number X92448 (cDNA) and X92671-X92678 (individual exonic data)). The gene spans 12 kb and consists of eight exons. SSCP based mutation analysis of affected animals was carried out on the coding region of this gene both with exonic primers, and intronic primer pairs for each exon. A 14 base pair deletion of the cDNA was identified at the 3' end of exon 1 in fucosidosis affected animals. Surprisingly, PCR based genomic cloning of DNA from these animals showed an identical deletion in this DNA, ending at the start of intron 1. This change causes a frameshift and, in consequence, 25 novel codons are transcribed in exon 2 before the first of two adjacent premature stop codons is encountered.

Animals↗
PubMed1995

Sequence and repeat structure variants in the long terminal repeat of maedi-visna virus EV1.

Diversity in the LTR of maedi-visna virus strain EV1 has been examined by PCR-based gene amplification using DNA from infected cells both in vitro and in experimentally infected animals. In vitro, several variant structures were found in the U3 regions of the LTR which contained repeats of sequences including presumed AP-1 and AP-4 binding sites. Although these repeat variants formed a minor fraction of the LTRs present in the proviral population, they were neither produced nor lost at a significant rate when PCR was performed on cloned viral DNA and so were unlikely to be artefacts of the isolation procedure. When LTRs were isolated from two experimentally EV1 infected sheep, repeat variant structures were found to be present in efferent lymph by 14 days postinfection (p.i.) (although not seen at 9 days p.i.). They were also present at later times and in blood. Overall sequence diversity at 9 days p.i. was reduced compared both with the infecting virus and with later times of infection. When a number of the variant LTR structures were used to drive CAT reporter gene constructs in chondrocytes, all were found to be active, although consistent differences of up to fourfold in activity were seen. However, there is no evidence from these data for strong selective pressure operating on the LTR in vivo.

Animals↗
PubMed1995

Cloning, sequencing and expression of the ovine interleukin 6 gene.

Gene amplification by reverse transcriptase PCR with heterologous primers has been used to obtain a cDNA clone encoding the structural sequences of ovine interleukin 6 from alveolar macrophages. This cDNA encodes a protein of M(r) = 23,429, which is 53% homologous in amino acid sequence to human IL = 6. The clone hybridizes to an RNA of size 1260 nt in alveolar macrophages, expression of which is potentiated by LPS. The ovine IL-6 structural gene has been cloned into the yeast expression vector pOGS40, and used to produce a recombinant protein. This protein is capable of causing increased immunoglobulin production in pokeweed mitogen stimulated ovine peripheral blood mononuclear cells at concentrations of 10-100 ng/ml, but it only causes very limited replication of B9 cells, a murine IL-6 dependent cell line. This is in contrast to recombinant human IL-6, which is capable of stimulating B9 cell proliferation, but not immunoglobulin production by ovine PBMC.

Animals↗
PubMed1995

Identification of a region of the alcelaphine herpesvirus-1 genome associated with virulence for rabbits.

The gammaherpesvirus Alcelaphine Herpesvirus 1 (AHV-1) causes the fatal lymphoproliferative disease known as malignant catarrhal fever (MCF), in susceptible hosts. The virulent C500 isolate of AHV-1 became attenuated for the laboratory model, the rabbit, as a result of serial passage in cells of bovine origin. This work describes the identification of a region of the central unique sequence of the C500 genome, located close to the terminal repeat units of the molecule, which is altered on attenuation. The virulent C500 genome contains two copies of a sequence of approximately 2 kbp, contained within a 7 kbp region of the unique DNA located adjacent to the terminal repeats at the left end of the molecule. In the genome of the attenuated virus, there are also two copies of the 2 kbp sequence but they are located at the ends of the attenuated genome unique region, adjacent to the terminally repeated sequences. One open reading frame (ORF), designated putative polypeptide 5, was altered on attenuation such that the 3' sequence was lost. The location of this ORF, coupled with the loss of its 3' sequence, suggests that this ORF may encode a gene involved in the virulent mechanisms of this virus, in a manner similar to that of the transforming proteins of Herpesvirus saimiri (HSV).

Amino Acid Sequence↗
PubMed1995

Investigation of the role of opsin gene polymorphism in generalized progressive retinal atrophies in dogs.

The generalized progressive retinal atrophies (gPRAs) form a group of retinal degenerations of pedigree dogs and cats, which have a variety of genetic origins (mostly unknown). We have examined the opsin gene for polymorphisms in several breeds of pedigree dog suffering from distinct forms of gPRA, by methods including single-strand conformation polymorphism analysis, microsatellite analysis and direct sequencing. The breeds examined included the Tibetan terrier, the miniature schnauzer, the Irish setter, the miniature poodle, the Labrador retriever and the English cocker spaniel, as well as individuals from breeds in which PRA has not been described and of mixed breed. Individuals from each of the named breeds suffering from PRA were compared with clinically normal dogs. Two polymorphisms were found. One, segregating within the Tibetan terrier population, but not seen in other breeds, was a synonymous transition at nucleotide position 780 in exon 3. Inheritance of this polymorphism suggests that opsin is unlikely to contain mutations causative of gPRA in this breed. The other polymorphism occurred between all miniature schnauzers examined and dogs of other breeds. It consisted of a single base insertion in intron 2. No polymorphisms in the opsin sequence were detected in any other breed. DNA sequencing allowed rigorous exclusion of mutations in opsin as a cause of gPRA in miniature poodles, English cocker spaniels or Labrador retrievers.

Animals↗
PubMed1995

Incidence of the gene mutation causal for rod-cone dysplasia type 1 in Irish setters in the UK.

A survey to establish the UK prevalence of the gene mutation causing the rod-cone dysplasia type one (rcd1) form of generalised progressive retinal atrophy (gPRA) in Irish setters was carried out. The dogs were selected by members of two Irish setter breed societies to provide examples from most of the main breeding lines in the UK. A total of 210 Irish setters were tested and one bitch was found to be a carrier of the rcd1 mutation. These results show that although a confirmed case of rcd1 has not been reported in Irish setters in the UK for over a decade the gene is still present in the gene pool.

Animals↗
PubMed1995

Initial lentivirus-host interactions within lymph nodes: a study of maedi-visna virus infection in sheep.

Reactive changes occurring within lymph nodes draining the subcutaneous site of acute infection with maedi-visna virus (MVV) were studied, and the appearance of infected cells correlated with the immune response. Cells infected with virus were detected in the node by cocultivation from day 4 postinfection (p.i.), with maximum numbers being seen between days 7 and 14, but even then infected cells were rare, with a maximum frequency of 23 50% tissue culture infective doses (TCID50) in 10(6) lymph node cells. At later times, infected cells were still detected, but their numbers fell to 1 to 2 TCID50 per 10(6) cells. Virus-specific CD8+ cytotoxic T-cell precursors (CTLp) were isolated from infected nodes from day 10 p.i. onwards, and T-cell proliferative responses to MVV were first detected on day 7 and consistently detected after day 18. Histological analysis showed a vigorous immune response in the node. There was a marked blast reaction in the T-cell-rich zones, which was greatest at the time when the number of virally infected cells was at its height. At this stage, large numbers of plasma cells were seen in the medullary cords, indicating that extensive T-cell-dependent B-cell activation was occurring in the T-cell-rich zones. Germinal centers were prominent shortly after the onset of the T-zone response and were still present at 40 days p.i. Phenotype studies of isolated lymph node cells failed to detect major changes in the proportion or phenotype of macrophages, CD1+ interdigitating cells, and CD4+ or CD8+ T cells despite the fact that CD8+ lymphoblasts form a major population leaving the node in efferent lymph. This suggests that there is a balanced increase in the number of all cell types in response to the virus within the node and selective migration of CD8+ lymphoblasts containing virus-specific CTLp from the node. Virus-specific immune responses are therefore present within the node when infectious virus isolation is maximal, but cellular immunity may act to control the level of infection from day 18 onwards.

Animals↗
PubMed1994

Sequence of the sheep interleukin-10-encoding cDNA.

The ovine interleukin-10 (oIL-10)-encoding cDNA has been cloned and sequenced using gene amplification by the polymerase chain reaction (PCR). We present the complete coding sequence of the ovine IL-10 gene, as well as the predicted amino acid (aa) sequence. The oIL10 DNA coding sequence is 531 nucleotides long and the mature protein product is predicted to be 18,367 Da, consisting of 158 aa, excluding a 19-aa N-terminal hydrophobic signal peptide. The oIL-10 protein is > 77% identical to pig and human IL-10, > 71% identical to rodent IL-10 and > 68% identical to viral IL-10.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence of the canine rod-opsin-encoding gene.

The major parts of two canine rod-specific opsin (Ops) transcripts have been cloned by polymerase chain reaction from retinal mRNA. Both transcripts are derived from the same gene. The 5' leader sequence of the transcripts was cloned from canine peripheral blood DNA. The transcripts code for a protein of 348 amino acids (aa), M(r) 38,962 (prior to any protein modification). The aa sequence suggests that in common with other sequenced Ops, canine rod Ops contains seven transmembrane domains, and residues believed essential for retinal pigment binding and for palmitate binding are conserved in the canine protein. Northern blotting using the central part of the ops gene as probe suggested that mature transcripts of three different sizes (about 1900, 2600 and 5500 bases) were found in retina. Of these, the 2600-base transcript was the most abundant. RACE cloning of the 3' end of ops showed that at least two of these size classes originate from differential transcript termination.

Amino Acid Sequence↗
PubMed1994

Interleukin-1 receptor expression by ovine afferent lymph dendritic cells: response to secondary antigen challenge.

Interleukin (IL)-1 is thought to enhance the function of antigen presenting cells of the dendritic cell lineage. To investigate the interaction of IL-1 and dendritic cells recombinant ovine IL-1 alpha and IL-1 beta have been used to determine IL-1 receptor (R) expression on fresh dendritic cells (ALDC) collected from cannulated sheep pseudoafferent lymph ducts, both prior to and in response to localized ovalbumin challenge. Resting ovine ALDC express approximately 510 IL-1R per cell for IL-1 alpha (Kd approximately 30 pM) and approximately 350 IL-1R/cell for IL-1 beta (Kd approximately 160 pM). Saturation binding and in situ analyses show an initial transient but dramatic increase in IL-1 alpha binding to ALDC by 4 h in response to ovalbumin challenge of primed sheep. Maximal IL-1R expression, reaching > or = 21700 IL-1R/cell for IL-1 alpha detected by around 48 h, was followed by a gradual return to resting level by 8 days post challenge. Fewer than 0.5% of resting ALDC expressed IL-1R but at least 5% of ALDC bound IL-1 alpha after ovalbumin challenge. There was no evidence of specific up-regulation of receptors for IL-1 beta on these cells. Fresh ovine alveolar macrophages, used as a positive control for specific IL-1R expression, were found to express approximately 2600 sites/cell for IL-1 alpha (Kd approximately 56 pM) and 16,500 sites/cell for IL-1 beta (Kd approximately 4.6 pM). In view of the differing IL-1 binding characteristics displayed by the receptors on the two cell types, it is postulated that afferent lymph dendritic cells and macrophages are not expressing the same form of IL-1R.

Animals↗
PubMed1994

A temporal study of RNAs produced in maedi-visna virus infection of choroid plexus cells.

We have examined the types and cellular distribution of transcripts of maedi-visna virus during infection of choroid plexus fibroblasts. Early in infection (19 hours post infection) only small spliced transcripts are found in the cytoplasm. Little virus specific RNA is detected in the nucleus at this stage. Later in infection structural gene transcripts are detectable in the cytoplasm as well as the nucleus. We have measured the half life of each type of transcript in total cellular RNA late in infection by two different methods (northern blotting or S1 protection assays after alpha-amanitin treatment, or specific hybridisation after pulse chase). Both of these methods suggest half lives for unspliced viral RNAs of 8-12 hours. Although the gel based methods were unsuitable for the accurate determination of half life for the shorter transcripts, they did confirm a long half life for these transcripts: pulse chase measurements suggested that this was similar to that seen for the unspliced transcripts. These observations are consistent with the virus exerting temporal control on protein synthesis by a method analogous to that of the HIV Rev protein.

Animals↗
PubMed1994

Progressive retinal atrophy: a model for retinitis pigmentosa in companion animals.

The generalised progressive atrophies (PRA's) are a heterogeneous group of inherited retinopathies in dogs and cats. They show marked similarities to the retinitis pigmentosas (RP) in man, as well as to a number of inherited retinal degenerations of laboratory rodents. Of the various recessively inherited forms of PRA in the dog, that in the Irish setter is due to a nonsense mutation in the gene encoding cGMP-PDE-beta subunit. Mutations at this locus are also known to cause a proportion of human RP cases. We are interested in applying gene therapy to these diseases.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Expression and characterization of bioactive recombinant ovine TNF-alpha: some species specificity in cytotoxic response to TNF.

We have expressed and partially purified recombinant ovine tumour necrosis factor alpha (rovTNF-alpha) using a yeast Ty, virus like particle, expression system. RovTNF-alpha is at least as active as recombinant human TNF-alpha (rhTNF-alpha) in two different bio-assays performed on ovine material, whilst approximately 1000-fold more rovrTNF-alpha than rhTNF-alpha is required to induce the same level of cytotoxicity in TNF-sensitive murine cell lines L929 and WEHI 164 clone 13. When cytotoxic assays are performed on the porcine TNF sensitive cell line PK(15)-1512, rovTNF-alpha shows about 2 logs greater activity than on murine cells, whilst rhTNF-alpha is about 1 log more active. A monoclonal antibody, raised against rovTNF-alpha, has been used to demonstrate the presence of nanogram amounts of an appropriately sized glycoprotein to be native ovine TNF-alpha in supernants of LPS stimulated ovine alveolar macrophages. These samples show no detectable cytotoxicity to L929 cells, although they show activity attributable to TNF-alpha (through neutralization by a polyclonal antiserum raised to rovTNF-alpha) in an assay on ovine material. The relative lack of activity on murine cells helps to explain previous reports of inability to assay native ovine TNF-alpha using these cells, in spite of their routine use to assay TNF-alpha from several other species. The sequence features in ovine TNF-alpha which might reduce its affinity for the murine TNF type 1 receptor are discussed.

Amino Acid Sequence↗
PubMed1993

Confirmation of the rod cGMP phosphodiesterase beta subunit (PDE beta) nonsense mutation in affected rcd-1 Irish setters in the UK and development of a diagnostic test.

Rod/cone dysplasia type one (rcd-1) is an early onset inherited retinal dystrophy segregating in the Irish setter breed. It is classed as one of the autosomal recessive canine generalised Progressive Retinal Atrophies (PRA). The disease results in complete loss of photoreceptors by approximately one year of age. Levels of retinal cGMP are markedly elevated and of abnormal distribution in rod photoreceptors. Rod phosphodiesterase activity is absent and mRNA encoding the beta subunit (PDE beta) of the holoenzyme is uniquely reduced in predegenerate retinae. Using retinae from normal, unrelated adult dogs we have PCR-amplified and sequenced the cDNA for PDE beta. The cDNA is almost identical to that recently described for the Irish setter in the USA apart from two translationally silent single nucleotide changes. Using carrier and affected setters from a UK breeding colony we have screened genomic DNA and can confirm the G to A transition in rcd-1 affected dogs at position 2420, creating an amber mutation in codon 807. However, PRA-affected Tibetan terriers and miniature longhaired dachshunds are normal at this locus, underlining the genetic heterogeneity of this disease group. In addition we have developed a rapid, PCR-based diagnostic test for this mutation that will differentiate normal dogs from asymptomatic carriers.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Characteristics of the T cell-mediated immune response to maedi-visna virus.

Virus-specific T cell-mediated immunity was investigated in healthy sheep persistently infected with the ruminant lentivirus maedi-visna. Visna-specific lymphocyte proliferation was demonstrated in response to both purified virions and recombinant p25, the major core protein of maedi-visna virus. The responding T cell population in this assay was mainly of the CD4+ phenotype, although in some individuals CD8+ T cells were also shown to respond to visna antigen in this system.

Animals↗