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Biomedical subjects

C Cunningham

Publications and source records attributed to C Cunningham.

At least 109 records · Page 6Linked to original sources
PubMed1998

Characterization of the protein encoded by gene UL49A of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) gene UL49A potentially encodes a primary translation product of 91 residues with a signal sequence at the N terminus and a membrane anchor domain near the C terminus. Mutants were generated in this gene and utilized to characterize the encoded protein on SDS-PAGE as a 6.7 kDa species which fractionated with infected cell membranes, was a relatively abundant virion component, and was not detectably O-glycosylated. The protein was identified by microsequencing as a 68 residue polypeptide formed by removal of 23 residues from the N terminus of the primary translation product. Cleavage of the signal sequence was also demonstrated by in vitro transcription and translation in the presence of microsomal membranes. The UL49A protein was efficiently solubilized along with envelope proteins by treatment of virions with a non-ionic detergent but only in the presence of a reducing agent, suggesting that it may be an envelope protein that is disulphide-linked to the tegument. It is apparent from mutational analysis that the 10 amino acid residues at the C terminus are not essential for synthesis of the protein, signal sequence cleavage, targeting to membranes and virions, linkage to the tegument and growth of virus in cell culture.

Amino Acid Sequence↗
PubMed1998

Rainbow trout cytokine and cytokine receptor genes.

Transforming growth factor-beta (TGF-beta) and interleukin-1 beta (IL-1 beta) have been cloned from rainbow trout (Oncorhynchus mykiss) in our laboratory. The trout TGF-beta is typical of members of the cysteine knot cytokine family, and has highest homology to TGF-beta 1, TGF-beta 4 and TGF-beta 5. The gene organisation is different to other TGF-beta genes despite the presence of seven exons. Trout IL-1 beta has less homology to known IL-1 beta s (49-56% amino acid similarity) but shows clear conservation of the secondary structure of the "mature peptide". An interleukin-converting enzyme cut site is not apparent however, and it remains to be determined whether fish IL-1 beta must be cleaved for biological activity. The trout IL-1 beta gene consists of six exons, one less than in mammals, with the missing exon at the 5' end of the gene. Two trout chemokine receptors have also been isolated in our laboratory, having high homology to CXC-R4 and CC-R7 (67% and 56% nucleotide identity respectively). With a view to studying the biological effects of fish cytokines we have also cloned the inducible nitric oxide (iNOS) and cyclooxygenase (COX-2) genes in trout. Both have high homology to known mammalian and chicken genes, and have been shown to be inducible in trout.

Amino Acid Sequence↗
PubMed1998

The genome sequence of herpes simplex virus type 2.

The genomic DNA sequence of herpes simplex virus type 2 (HSV-2) strain HG52 was determined as 154,746 bp with a G+C content of 70.4%. A total of 74 genes encoding distinct proteins was identified; three of these were each present in two copies, within major repeat elements of the genome. The HSV-2 gene set corresponds closely with that of HSV-1, and the HSV-2 sequence prompted several local revisions to the published HSV-1 sequence (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988). No compelling evidence for the existence of any additional protein-coding genes in HSV-2 was identified.

Amino Acid Sequence↗
PubMed1998

The herpes simplex virus type 1 U(L)17 gene encodes virion tegument proteins that are required for cleavage and packaging of viral DNA.

Previous studies have suggested that the U(L)17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U(L)17 open reading frame [HSV-1(deltaU(L)17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5' end of the U(L)17 open reading frame [HSV-1(U(L)17-stop)] were plaque purified on engineered cell lines containing the U(L)17 gene. A virus derived from HSV-1(U(L)17-stop) but containing a restored U(L)17 gene was also constructed and was designated HSV-1(U(L)17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(deltaU(L)17) nor HSV-1(U(L)17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(deltaU(L)17) compared to wild-type virus show no detectable differences. These data indicate that the U(L)17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U(L)17 gene product, an anti-U(L)17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent Mr 77,000 and weakly with a protein of apparent Mr 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U(L)17 protein. We therefore conclude that the U(L)17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.

Animals↗
PubMed1998

Emmetropisation, axial length, and corneal topography in teenagers with Down's syndrome.

AIM: To study the refractive status and corneal topography in Down's syndrome. METHOD: A matched cohort subgroup of 50 individuals with Down's syndrome in the Manchester area aged 15-22 years was studied by refraction, corneal topography, A-scan biometry, slit lamp examination, and orthoptic examination. RESULTS: (1) A linear relation was found between axial length and spherical equivalent refraction. There was no statistical relation between keratometry and the axial length. (2) 80% of the group had a hyperopic refraction (mean +2.46 D, range +0.5 to +7.5 D); 18% were myopic (mean -2.75 D, range -0.5 to -8.0 D); and 2% were emmetropic (within plus or minus 0.5 D of zero). The overall mean spherical equivalent refraction was +1.43 (SD 2.86) D. 63% of eyes could see 6/12 or better and 66% of the individuals had a binocular vision of 6/12 or better. (3) Corneal topography was generally of a regular "bow tie" pattern, but there was a high incidence of oblique cylinders. Mean cylinder strength was 1.14 (1.15) D. (4) The prevalence of overt keratoconus was 2%. 6% had corneal topography with inferior steepening which may be a preclinical keratoconic process. CONCLUSIONS: In this cohort of late teenagers with Down's syndrome, emmetropisation has failed to occur in most individuals. In a similar aged group of non-disabled individuals one would expect about 83% emmetropic (plus or minus 0.25 D), 13% myopic, and 4% hyperopic. The wide spread of oblique cylinders and the small proportion of with the rule astigmatism is probably related to this failure of emmetropisation. The prevalence of 2% keratoconus in Down's syndrome compares with that found by other authors of between 5.5 and 15%. The 6% with inferior steepening on topography will be followed up over the next few years to see if there is any development of clinical keratoconus. Hence we will see if corneal topography is useful as a screening tool for preclinical keratoconus in this high risk group.

Adolescent↗
PubMed1997

Actin-binding protein-280 binds the stress-activated protein kinase (SAPK) activator SEK-1 and is required for tumor necrosis factor-alpha activation of SAPK in melanoma cells.

SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segment of ABP-280, upstream of the ABP carboxyl-terminal dimerization domain. Activation of SEK-1 in situ increases the SEK-1 activity bound to ABP-280 without changing the amount of SEK-1 polypeptide bound. The influence of ABP-280 on SAPK regulation was evaluated in human melanoma cells that lack ABP-280 expression, and in stable transformants of these cells expressing wild type ABP, or an actin-binding but dimerization-deficient mutant ABP (ABPDeltaCT109). ABP-280-deficient cells show an activation of SAPK in response to most stimuli that is comparable to that seen in ABP-280-replete cells; ABP-280-deficient cells, however, fail to show the brisk tumor necrosis factor-alpha (TNF-alpha) activation of SAPK seen in ABP-replete cells and have an 80% reduction in SAPK activation by lysophosphatidic acid. Expression of the dimerization-deficient mutant ABP-280 fails to correct the defective SAPK response to lysophosphatidic acid, but essentially normalizes the TNF-alpha activation of SAPK. Thus, a lack of ABP-280 in melanoma cells causes a defect in the regulation of SAPK that is selective for TNF-alpha and is attributable to the lack of ABP-280 polypeptide itself rather than to the disordered actin cytoskeleton that results therefrom. ABP-280 participates in TNF-alpha signal transduction to SAPKs, in part through the binding of SEK-1.

Actins↗
PubMed1997

Natural antibodies that react with V-region peptide epitopes of DNA-binding antibodies are made by mice with systemic lupus erythematosus as disease develops.

Cross-reactive idiotypes (CRI) have been detected on anti-DNA autoantibodies associated with lesions typical of systemic lupus erythematosus. In order to analyse the antigenic make up of idiotypes on anti-DNA monoclonal antibodies (mAb) V-88 (IgG1 kappa) and F-423 (IgG3 kappa), derived respectively from an adult (NZB x NZW)F1 and a fetal MRL/Mp-lpr/lpr mouse, a set of overlapping hexapeptides representing the VH and VL regions of mAb V-88 and F-423 were synthesized and reacted with a range of sera in pepscan enzyme-linked immunosorbent assays (ELISA) taken from normal and lupus mouse strains. Serum pools were collected both from normal BALB/c and lupus MRL/Mp-lpr/lpr and (NZB x NZW)F1 mice at 10, 20 and 30 weeks of age and analysed for the presence of spontaneously produced anti-V-region peptide IgM and IgG antibodies. IgM antibodies from both the lupus mice reacted with the same V-region epitopes, and although some epitopes mapped to similar locations in the two mAb, the maps for V-88 and F-423 were not identical. In MRL/Mp-lpr/lpr mice, as lupus disease progressed there was a switch from IgM antibodies to IgG anti-peptide antibodies whose specificity for the peptide antigens coincided with but was better defined than that of the IgM antibodies. The identified idiotopes were located in both complementary determining regions (CDR) and framework region (FR) regions, indicating that some contribute to CRI shared by other related antibodies, while others were unique to either mAb V-88 or F-423. In conclusion, we have dissected and identified a mosaic of antibody V-region idiotopes that contribute to the idiotype of an anti-DNA autoantibody and against which autoantibodies are made naturally in lupus disease.

Amino Acid Sequence↗
PubMed1997

Behavioral, situational, and temporal effects of treatment of ADHD with methylphenidate.

OBJECTIVE: To determine the behavioral, situational, and temporal effects of 4 months of methylphenidate (MPH) treatment for attention-deficit hyperactivity disorder (ADHD). METHOD: Ninety-one children with ADHD were randomly assigned to receive either MPH (titrated to a target dose of 0.7 mg/kg twice a day) or a placebo. Treatment effects were investigated with measures sensitive to various behaviors (core and associated symptoms), situations (home and school), time periods (morning and afternoon, after reaching the target dose, and after 4 months of treatment), and side effects. RESULTS: MPH treatment improved symptoms of ADHD and oppositional behavior at school, both in the morning and afternoon, but not at home. Side effects (increase in physiological and effective symptoms, lack of weight gain) were significantly more frequent with MPH than with placebo treatment. Benefit was evident after titration, but the onset of some side effects was delayed. Side effects were reported by parents but not by teachers. CONCLUSIONS: Positive effects of MPH on behavior are evident in the classroom, but with MPH given twice daily, parents do not report that MPH improves behavior at home. Greater impact on home behavior may require three times daily MPH and combined treatments.

Affect↗
PubMed1997

The U(L)15 gene of herpes simplex virus type 1 contains within its second exon a novel open reading frame that is translated in frame with the U(L)15 gene product.

The U(L)15 gene of herpes simplex virus type 1 is composed of two exons. A mutation previously shown to preclude viral DNA cleavage and packaging at the nonpermissive temperature was identified as a change from a highly conserved serine to proline at codon 653. Separate viral mutants that contained stop codons inserted into exon I of U(L)15 (designated S648) or an insertion of the Escherichia coli lacZ gene into a truncated U(L)15 exon II [designated HSV-1(delta U(L)15ExII)] were constructed. Recombinant viruses derived from S648 and HSV-1(delta U(L)15ExII) and containing restored U(L)15 genes were constructed and designated S648R and HSV-1(delta U(L)15ExIIR), respectively. Unlike HSV-1(delta U(L)15ExIIR) and S648R, the viruses containing mutant U(L)15 genes failed to cleave and package viral DNA when propagated on noncomplementing cells. As revealed by electron microscopy, large numbers of enveloped capsids lacking viral DNA accumulated within the cytoplasm of cells infected with either S648 or HSV-1(delta U(L)15ExII) but not in cells infected with HSV-1(delta U(L)15ExIIR) or S648R. Thus, one function of the U(L)15 gene is to effectively prevent immature particles lacking DNA from exiting the nucleus by envelopment at the inner lamella of the nuclear membrane. Cells infected with HSV-1(delta U(L)15ExII) did not express the 75,000- or 35,000-apparent-Mr proteins previously shown to be products of the U(L)15 open reading frame, whereas the 35,000-apparent-Mr protein was readily detectable in cells infected with S648. We conclude that at least the 75,000-Mr protein is required for viral DNA cleavage and packaging and hypothesize that the 35,000-Mr protein is derived from translation of a novel mRNA located partially or completely within the second exon of U(L)15.

Animals↗
PubMed1997

N-acetyltransferase 2 genotype in colorectal cancer and selective gene retention in cancers with chromosome 8p deletions.

BACKGROUND: Genetic polymorphisms in N-acetyltransferase (NAT2) can change the normally fast acetylation of substrates to slow acetylation, and have been associated with the development of some cancers. The NAT2 locus may also suffer dysregulation during cancer progression, as the gene resides on chromosome 8p22, a region which is frequently deleted in colorectal cancer. SUBJECTS AND METHODS: A polymerase chain reaction based method was used to determine NAT2 genotype in 275 patients with colon cancer and 343 normal control DNAs. Within the cancer group, 65 cases known to contain deletions in chromosome 8p were examined for loss of heterozygosity at the NAT2 locus. RESULTS: Overall, there was no statistical difference in frequency or distribution of NAT2 alleles and genotype between colon cancer and control groups. There was a significant association between the slow acetylation genotype and early age of onset. NAT2 genotype did not vary with other clinical features of colon cancer, which included Dukes's stage, site of tumour, and sex. Of 48 informative cases, only three (6%) showed loss of heterozygosity, indicating that the NAT2 locus is not commonly deleted in colorectal cancer. This suggests that NAT2 is retained during the process of allele loss possibly because of its proximity to a gene necessary for cell viability. CONCLUSIONS: NAT2 does not play a major role in colorectal cancer risk, but may influence risk in some age groups. The nature of the loss of heterozygosity at the chromosome 8p site is complex and is worthy of further study.

Acetylation↗
PubMed1997

Absence of human herpes virus 8 DNA sequences in large granular lymphocyte (LGL) leukemia.

The etiology of large granular lymphocyte (LGL) leukemia is uncertain. Recently, a Kaposi's sarcoma-associated herpes virus, denoted as human herpes virus 8 (HHV-8), has been identified. Some data suggest that HHV-8 and Epstein-Barr virus (EBV) may interact to induce malignant transformation. Infection with EBV has been implicated in the pathogenesis of some cases of LGL leukemia. Therefore, we performed PCR analyses for HHV-8 detection in samples from nineteen patients with LGL leukemia; three of these samples contained the EBV genome. We could not detect HHV-8 sequences in any of these patients. Therefore, HHV-8 infection is not involved in the pathogenesis of T-LGL leukemia.

DNA, Viral↗
PubMed1996

Microsatellite instability and the role of hMSH2 in sporadic colorectalcancer.

Microsatellite instability (MSI) occurs in most tumours from patients with hereditary non-polyposis colorectal cancer (HNPCC) and in around 17% of sporadic colorectal cancers. Germline defects in mismatch repair (MMR) genes are responsible for the majority of large HNPCC families, with hMSH2 accounting for at least 50%. MMR gene defects also occur in a small proportion of sporadic colorectal tumours with MSI. Here we report a systematic analysis of mismatch repair deficiency in 215 Scottish patients with sporadic colorectal tumours. We found that 16.4% of tumours exhibited MSI; survival analysis by Cox proportional hazards method showed a substantial survival advantage for patients with tumours showing MSI, independent of other prognostic factors. Tumours with MSI were screened for hMSH2 mutations and although 61% were found to have alterations, of these only 1/24 was exonic. The majority of these changes were reductions in length at intronic mononucleotide tracts and we postulate that these alterations are the result of a genetic defect elsewhere, although they may compromise hMSH2 function as a second step in tumourigenesis. Our findings indicate that instability confers an improved prognosis in colorectal cancer and, despite the fact that these two groups of tumours share similar biological characteristics, the genetic basis of HNPCC and sporadic colorectal cancer with MSI is different.

Base Sequence↗
PubMed1996

Detailed physical and deletion mapping of 8p with isolation of YAC clones from tumour suppressor loci involved in colorectal cancer.

Loss of heterozygosity (LOH) of markers at chromosome 8p is frequently noted in many different tumour types, including colorectal cancer. Numerous investigations indicate the presence of more than tumour suppressor gene (TSG) located on 8p. In this study, we describe a detailed LOH map in colorectal cancer and relate this to physical mapping data from reduced radiation 8p hybrids, yeast artificial chromosome (YAC) co-localisation of markers and fluorescence in situ hybridisation data. These data indicate the presence of two regions harbouring putative TSG's between the polymorphic markers for the LPL gene-D8S298 (approximately 4 Mb) and the markers D8S136-D8S137 (approximately 8 Mb). Yeast Artificial Chromosomes (YAC) have been isolated from these regions of interest to aid the localisation of the putative TSG's.

Chromosome Mapping↗
PubMed1996

Isolation and characterization of herpes simplex virus type 1 mutants defective in the UL6 gene.

Previous studies have shown that the protein encoded by herpes simplex virus type 1 (HSV-1) gene UL6 is required for processing and packaging of replicated viral DNA and is a minor component of virions and capsids. In this report, we describe the construction of UL6- HSV-1 mutants with a disrupted UL6 gene using complementing cells and show that they fail to synthesize the UL6 protein or produce infectious virus in noncomplementing cells. The mutants synthesized but failed to process and encapsidate viral DNA and accumulated only immature capsids which lacked the UL6 protein. Immunofluorescence analysis showed that the UL6 protein, when expressed transiently in transfected cells in the absence of other HSV-1 proteins, is localized exclusively to the nucleus. We also investigated an HSV-1 mutant with a defect in gene UL33, the product of which is also thought to be involved in viral DNA processing and packaging. The phenotype of this mutant on noncomplementing cells with regard to failure to process and encapsidate viral DNA, accumulation of immature capsids, and inability to produce infectious virus was the same as that of UL6- viruses. This mutant, however, produced capsids containing the UL6 protein, indicating that association of the UL6 protein with the capsid is independent of the UL33 protein.

Animals↗
PubMed1996

Molecular genetic basis of colorectal cancer susceptibility.

The past decade has seen considerable advances in understanding of the molecular processes involved in the development of colorectal cancer. With an increased awareness of genetic aspects of the disease there have already been significant changes in clinical management. This is exemplified by familial adenomatous polyposis, where identification of mutations in the adenomatous polyposis coli (APC) gene in affected individuals can be used directly to reduce the requirement for clinical screening in at-risk relatives. In other more common but less well defined heritable forms of colorectal cancer, testing to identify individuals for early diagnosis and treatment will soon become routine practice. This review does not set out to discuss all aspects of the molecular genetics of colorectal cancer but concentrates on the roles of the APC gene and the recently discovered DNA mismatch repair genes in colorectal cancer. The identification of these genes and their functional significance in the neoplastic process is discussed, and the relevance of such discoveries to future research and clinical management explored.

Colorectal Neoplasms↗
PubMed1996

Extracolonic features of familial adenomatous polyposis in patients with sporadic colorectal cancer.

We have investigated the occurrence of attenuated extracolonic manifestations (AEMs) of familial adenomatous polyposis (FAP) in patients with non-polyposis colorectal cancer. In a prospective case-control study, we observed that significantly more colorectal cancer patients exhibited AEM than did age and sex-matched controls (19.5% vs 7.5%, P < 0.004). However patients with AEMs do not have occult FAP, as we found no heterozygous adenomatous polyposis coli (APC) gene mutations despite extensive analysis of constitutional DNA. Genome-wide DNA replication errors (RERs) occur in a proportion of colorectal cancers, particularly right-sided lesions and in almost all tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients. As AEMs have been reported in familial colon cancer cases, we investigated the relationship of AEMs to tumour RER phenotype. There was indeed an excess of AEMs in patients with right-sided tumours (30.2% of 53 patients vs 14.7% of 116 patients, P < 0.03) and in those with RER tumours (3 out of 12 patients with RER tumours vs none out of 21 patients with non-RER tumours, P < 0.05). Two patients with AEM were from HNPCC families compared with none of those without AEM (P < 0.05). The association of AEMs with colorectal cancer is intriguing, and we speculate that it may be a manifestation of mutational mosaicism of the APC gene, perhaps associated with a constitutional defect in DNA mismatch pair.

Adenomatous Polyposis Coli↗