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Biomedical subjects

D E Onions

Publications and source records attributed to D E Onions.

At least 19 recordsLinked to original sources
PubMed1998

Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity.

We have previously reported the cloning of the coding sequence for feline-specific interferon-gamma. Here, we describe the expression of this sequence in a baculovirus system and demonstrate the biological activity of the recombinant protein. The coding sequence for feline interferon was directionally cloned into the baculovirus transfer vector pAcCL29-1. Transfer vector and linearized wild-type AcMNPV (BacPAK6) were used to co-transfect Sf9 cells by calcium phosphate coprecipitation. Subsequently, wild-type and recombinant viruses were separated by plaque assay. Recombinant plaques were expanded and a master stock of virus is produced. Production of biologically active interferon-gamma from infected Sf9 cells was demonstrated using a standard cytopathic effect reduction assay, utilising vesicular stomatitis virus (VSV), and an MHC class II induction assay.

Animals↗
PubMed1998

DNA vaccination affords significant protection against feline immunodeficiency virus infection without inducing detectable antiviral antibodies.

To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion in pol (FIVDeltaRT). In a first experiment, FIVDeltaRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-gamma) DNA. The DNA was administered in four 100-microg doses at 0, 10, and 23 weeks. Immunization with FIVDeltaRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVDeltaRT vaccinates than in the controls. Immunization with FIVDeltaRT in conjunction with IFN-gamma gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVDeltaRT plus IFN-gamma and IFN-gamma alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines.

Amino Acid Sequence↗
PubMed1997

Proviral insertions induce the expression of bone-specific isoforms of PEBP2alphaA (CBFA1): evidence for a new myc collaborating oncogene.

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2alphaA (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2alphaA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2alphaA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

Amino Acid Sequence↗
PubMed1997

Skewed T-cell receptor Vbeta8.2 expression in transgenic CD2-myc induced thymic lymphoma: a role for antigen stimulation in tumour development?

Transgenic mice expressing the c-myc proto-oncogene under the control of the CD2-dominant control region show stochastic development of mainly clonal thymic lymphoma with long latency, indicating that cooperative events are needed for the development of the fully malignant phenotype. Previous studies have suggested that T-cell receptor-associated signals can contribute to tumour development. We have therefore used this transgenic model of T-cell transformation to determine whether antigen-specific responses could constitute an epigenetic event in lymphomagenesis. The T-cell receptor (TcR) repertoires of lymphoma clones were analysed with a panel of monoclonal antibodies (Abs) recognizing TcR Vbeta chains. The Vbeta repertoire of tumour clones arising in these mice was non-random with overrepresentation of Vbeta8.2 TcR species. The majority of Vbeta8.2+ clones were of a mature CD3+ CD8 single-positive (SP) phenotype. The biased TcR usage, together with a mature cell phenotype is consistent with the hypothesis that TcR-mediated signals cooperate with activated myc during T-cell transformation.

Animals↗
PubMed1997

Complete DNA sequence of canine adenovirus type 1.

The complete DNA sequence of a field strain of canine adenovirus type 1 was determined by sequencing random fragments of viral DNA cloned into pBluescript. The virus has a genome of 30536 bp flanked by two identical 161 bp inverted terminal repeats. Thirty ORFs have been identified, based on genomic location or sequence identity with published adenoviruses. These are arranged into similar discrete regions found in the human adenoviruses. ORFs in the late region show greatest identity with published human adenovirus sequences, whereas the E3 and E4 ORFs show little or none.

Adenoviridae↗
PubMed1997

Cloning and sequencing of equine transforming growth factor-beta 1 (TGF beta-1) cDNA.

Transforming growth factor beta (TGF-beta) belongs to a family of peptide growth factors which control critical stages of cell proliferation and differentiation. We report the cloning and sequencing of the cDNA for TGF-beta type 1 isoform of the horse. The predicted mature equine TGF beta-1 peptide is 112 amino acids in length and exhibits 99% identity to mature human TGF beta-1.

Amino Acid Sequence↗
PubMed1996

Detection of canine herpesvirus 1 in a wide range of tissues using the polymerase chain reaction.

Canine herpesvirus 1 (CHV-1), a member of the alphaherpesvirus sub-family, is known to cause fatal infections in litters of puppies and may also be involved in infertility, abortion, and stillbirths in adult dogs. The purpose of this study was to determine the presence of CHV-1 DNA using the polymerase chain reaction (PCR) in twelve key sites that have been associated with latency for the other herpesviruses. A 605 base pair portion of the viral glycoprotein B (gB) gene was amplified using degenerate primers, cloned, and sequenced. Conventional 20 mer primers were designed using this sequence information to amplify a 120 bp fragment of gB situated between the original degenerate primers. The specificity of amplification was confirmed by Southern Blot hybridisation using an internal oligonucleotide probe. DNA was extracted from tissue samples taken from twelve dogs at post mortem and from twenty-four blood samples. Nine out of twelve dogs showed evidence of infection with CHV-1; the tissues most commonly affected were lumbo-sacral ganglia (5/12 dogs), tonsil (5/12), parotid salivary gland (4/9), and liver (4/9). No positive results were detected within the twenty-four blood samples. These results indicate that exposure to CHV-1 may be much more common than previously suggested.

Animals↗
PubMed1996

Apparent bypass of negative selection in CD8+ tumours in CD2-myc transgenic mice.

A role for antigen stimulation in lymphoid neoplasia has been postulated and is supported by indirect evidence that suggests that the interaction of antigen with both T cells and B cells may constitute an epigenetic event that can contribute to tumour induction or tumour progression. Using myc-bearing transgenic mice that develop mainly clonal T-cell lymphomas we have investigated the possibility that endogenous antigen-mediated clonal deletion might be overridden in tumorigenesis. CD2-myc transgenic mice were backcrossed on to a CBA/Ca background to ensure Mtv-mediated deletion of V beta 11-expressing T cells in the resultant offspring. Lymphomas arising from these mice were subsequently screened for V beta 11 expression. There was a clear correlation between the age at which mice developed neoplasia and the tumour phenotype. Mice with CD4- CD8+ tumours succumbed to thymic lymphoma at a significantly younger age than mice developing CD4+ CD8+ tumours. A small number of tumours consisted of the 'forbidden' V beta 11 phenotype, showing that cells vulnerable to transformation could escape negative selection. The majority of the V beta 11-positive tumours were CD4- CD8+ and were only observed in mice showing clinical evidence of tumour development at a relatively young age. The phenotype of these cells and the age at which tumours arose suggests that T cells escaping tolerance may be susceptible to transformation.

Animals↗
PubMed1996

Moloney murine leukemia virus-induced lymphomas in p53-deficient mice: overlapping pathways in tumor development?

The effect of Moloney murine leukemia virus (MoMLV) infection was examined in mice lacking a functional p53 gene. Virus-infected p53-/- mice developed tumors significantly faster than uninfected p53-/- or virus-infected p53+/+ littermates. However, the degree of synergy between MoMLV and the p53 null genotype was weaker than the synergy between either of these and c-myc transgenes. A similar range of T-cell tumor phenotypes was represented in all p53 genotype groups, including p53-/- mice, which developed thymic lymphomas as the most common of several neoplastic diseases. Lack of p53 was associated with higher rates of metastasis and the ready establishment of tumors in tissue culture. Loss of the wild-type allele was a common feature of tumors in p53+/- mice and was complete in tumor cells in vitro, but this appeared to occur by a mechanism other than proviral insertion at the wild-type allele. A lower average MoMLV proviral copy number was observed in tumors of the p53 null and heterozygote groups, suggesting that the absence of a functional p53 gene reduced the number of steps required to complete the malignant phenotype. Mink cell focus-forming virus-like proviruses were detected in tumors of all infected mice but were relatively rare in p53 null mice. Analysis of c-myc, pim-1, and pal-1 showed that these loci were occupied by proviruses in some cases but at similar frequencies in p53 wild-type and null mice. In conclusion, while inactivation of p53 in the germ line predisposes mice to tumors similar in phenotype to those induced by MoMLV, it appears that virus-induced tumors generally occur without p53 loss. We speculate that a bcl-2-like function carried or induced by MoMLV may underlie this p53-independent pathway.

Alleles↗
PubMed1996

Complete nucleotide sequence and transcriptional analysis of snakehead fish retrovirus.

The complete genome of the snakehead fish retrovirus has been cloned and sequenced, and its transcriptional profile in cell culture has been determined. The 11.2-kb provirus displays a complex expression pattern capable of encoding accessory proteins and is unique in the predicted location of the env initiation codon and signal peptide upstream of gag and the common splice donor site. The virus is distinguishable from all known retrovirus groups by the presence of an arginine tRNA primer binding site. The coding regions are highly divergent and show a number of unusual characteristics, including a large Gag coiled-coil region, a Pol domain of unknown function, and a long, lentiviral-like, Env cytoplasmic domain. Phylogenetic analysis of the Pol sequence emphasizes the divergent nature of the virus from the avian and mammalian retroviruses. The snakehead virus is also distinct from a previously characterized complex fish retrovirus, suggesting that discrete groups of these viruses have yet to be identified in the lower vertebrates.

Amino Acid Sequence↗
PubMed1996

The cloning and sequencing of cDNAs encoding two isoforms of feline stem cell factor.

cDNA clones encoding two isoforms of feline stem cell factor (fSCF) have been isolated using RT-PCR and their sequences determined. The cDNAs encode a predicted full length fSCF protein of 274 amino-acids and a shorter isoform of 246 amino acids. Feline SCF shows a high degree of homology to the SCFs of other species at both the nucleic acid and protein level.

Amino Acid Sequence↗
PubMed1995

Synergy between a human c-myc transgene and p53 null genotype in murine thymic lymphomas: contrasting effects of homozygous and heterozygous p53 loss.

Activation of the c-myc oncogene and functional loss of the p53 tumour suppressor gene are among the most frequently recorded genetic lesions in neoplasia but their combined effect has not previously been investigated. By breeding together mice transgenic for human c-myc (CD2-myc) and mice carrying an inactive p53 allele (p53-/-) we found that these genetic lesions act synergistically in vivo. Offspring carrying the CD2-myc transgene and the homozygous p53 null mutation (p53-/-/CD2-myc) were viable but developed thymic lymphomas with dramatically increased frequency and reduced latency compared to both parental groups. The tumour phenotype was similar to that previously recorded for CD2-myc mice (predominantly CD3+, CD4+8+) but tumour clonal complexity and metastasis was significantly greater in the p53-/-/CD2-myc mice. In contrast, no significant increase in tumour incidence was seen in p53+/-/CD2-myc vs p53+/+/CD2-myc mice over a 6 month observation period. However, the loss of wild type p53 in a proportion of tumour cells in p53+/-/CD2-myc lymphomas suggests that wild type allele loss can occur as a late progression step rather than an initiating step in these tumours. We suggest that p53 loss of function may collaborate with the CD2-myc transgene at more than one stage in thymic lymphoma development.

Animals↗
PubMed1995

Case clustering, Epstein-Barr virus Reed-Sternberg cell status and herpes virus serology in Hodgkin's disease: results of a case-control study.

The Leukaemia Research Fund Data Collection Study (DCS) is a specialist registry of leukaemias and lymphomas. The present study involves 494 cases of Hodgkin's disease (HD) registered with the DCS between 1985 and 1989. This entire data set has been tested for localised spatial clustering using an established nearest neighbour method with 18% of all cases in young people classified as clustered (P < 0.05). No clustering was found in older cases. Subsamples were selected from the registered cases for a pilot study in which case clustering, herpes virus antibody titres and Epstein-Barr virus (EBV) presence within the Reed-Sternberg (RS) cells (EBV-RS status) were investigated together. Firstly, a case-control study of HD in young people or nodular sclerosing (NS) subtype (39 HD cases and 26 healthy controls) found significant elevation of antibody titres to EBV-viral capsid antigen (VCA), EBV-early antigen (EA) and human herpes virus 6 (HHV-6) in HD cases compared with controls. EBV viral genome was present in 5 cases and 4 of these were in clusters of HD in young people. Elevation of antibody titres to the EBV antigens was not associated with case clustering or EBV-RS status. Antibody titres to HHV-6 differed significantly between EBV-RS+ and EBV-RS- cases (P = 0.04). Geometric mean titres for HHV-6 for EBV-RS+ and EBV-RS- cases were 11.5 and 73.7, respectively, with the former lower than the control value of 20.5. Secondly, a cluster study included all other cases (n = 14) in clusters containing known EBV-RS+ cases. 3 further cases were EBV-RS+ positive but no cluster consisted entirely of positive cases. Overall, 5/16 clustered, 2/12 peripheral and 1/25 random cases in these studies were EBV-RS+ (P = 0.017). The interpretation of these results in terms of shared aetiological exposures of cases within clusters and the roles of EBV and HHV-6 is discussed, and hypotheses for testing in future studies proposed.

Adolescent↗
PubMed1995

Nucleotide and predicted peptide sequence of feline interferon-gamma (IFN-gamma).

Interferon gamma is a pleiotropic cytokine which is now recognised as an important modulator of the immune response. We now report the cloning and sequencing of feline interferon gamma cDNA which was generated by the polymerase chain reaction. At the nucleotide level feline ifn-gamma shares 78% and 63% homology with human and murine cDNA equivalents. At the amino acid level the feline IFN-gamma shares 63% and 43% homology with human and murine homologs respectively.

Amino Acid Sequence↗
PubMed1995

The isolation and sequence of canine interleukin-2.

A cDNA corresponding to canine IL-2 has been isolated and sequenced. The cDNA was synthesised using RT-PCR, with oligonucleotide primers designed from conserved regions of published IL-2 sequences. The cDNA encodes a predicted full length IL-2 protein of 155 amino-acids. At the nucleic acid level, the canine cDNA shows 92, 88, 88, 82 and 74% homology to published sequences of feline, human, equine, bovine and murine IL-2, respectively. The derived protein shows 90, 86, 85, 76 and 75% similarity to feline, human, equine, murine and bovine IL-2 homologs.

Amino Acid Sequence↗
PubMed1994

An improved cosmid vector for the cloning of equine herpesvirus DNA.

We have modified the commercial cosmid vector, triple helix vector (THV), such that I-Sce-I restriction endonuclease sites flank the cloning site. I-Sce-I is a rare-cutting endonuclease which recognizes an 18-bp sequence. It does not restrict the genome of either of the equine herpesvirus 1 or 4 (EHV-1 and EHV-4) strains we have cosmid cloned. Thus, cosmid-cloned EHV fragments can be excised intact from the vector by I-Sce-I digestion, facilitating production of large overlapping EHV fragments for use in transfections to produce recombinant virus.

Base Sequence↗
PubMed1994

Sequences of the ribonucleotide reductase-encoding genes of equine herpesvirus 4.

The equine herpesvirus 4 (EHV-4) genes encoding the two subunits of the enzyme ribonucleotide reductase (RR) were cloned and their nucleotide (nt) sequences determined. The large subunit (RR1) is predicted to comprise 789 amino acids (aa), which compares with lengths of 790, 775 and 1137 aa for the RR1 proteins encoded by equine herpesvirus 1 (EHV-1) gene 21, varicella zoster virus (VZV) gene 19 and herpes simplex virus type 1 (HSV-1) UL39, respectively. In common with VZV RR1, the EHV-4 RR1 protein lacks the N-terminal domain of HSV-1 RR1 which possesses protein kinase activity. EHV-4 RR1 demonstrates identities of 88, 52 and 29% with the RR1 proteins of EHV-1, VZV and HSV-1, respectively. The small subunit (RR2) is predicted to be 320 aa in length, which compares with lengths of 321, 306 and 340 aa for the RR2 proteins encoded by EHV-1 gene 20, VZV gene 18 and HSV-1 UL40, respectively. The EHV-4 RR2 protein exhibits identities of 90, 60 and 55% with the RR2 proteins of EHV-1, VZV and HSV-1, respectively.

Amino Acid Sequence↗