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Biomedical subjects

A Whitehouse

Publications and source records attributed to A Whitehouse.

At least 37 records · Page 2Linked to original sources
PubMed1999

The activation domain of herpesvirus saimiri R protein interacts with the TATA-binding protein.

The herpesvirus saimiri open reading frame (ORF) 50 produces two transcripts. The first is spliced, contains a single intron, and is detected at early times during the productive cycle, whereas the second is expressed later and is produced from a promoter within the second exon. Analysis of their gene products has shown that they function as sequence specific transactivators. In this report, we demonstrate that the carboxy terminus of ORF 50b contains an activation domain which is essential for transactivation. This domain contains positionally conserved hydrophobic residues found in a number of activation domains, including the herpes simplex virus VP16 and the Epstein-Barr virus R proteins. Mutational analysis of this domain demonstrates that these conserved hydrophobic residues are essential for ORF 50 transactivation capability. Furthermore, this domain is required for the interaction between the ORF 50 proteins and the basal transcription factor TATA-binding protein.

Amino Acid Sequence↗
PubMed1999

Assessment of Herpesvirus saimiri as a potential human gene therapy vector.

Herpesvirus saimiri has characteristics that make it amenable to development as a gene therapy vector. The viral genome is thought to be capable of accommodating large quantities of heterologous DNA while the virus itself can infect many different cell types. Virus infection has been shown in many cases to be persistent by virtue of episomal maintenance in the target cell. In this article we examine the ability of nonselectable recombinant viruses expressing the beta-galactosidase gene product to infect a variety of human cells and demonstrate that this virus could be developed as an alternative hematopoietic stem cell gene therapy vector. In contrast to earlier observations, we demonstrate by a number of methods that the virus has the ability to replicate in many human cell types, suggesting the need for the development of a disabled virus for use as a gene therapy vector.

Blotting, Southern↗
PubMed1998

Structural and evolutionary characterization of the human sorbitol dehydrogenase gene duplication.

We have established that two very closely homologous human sorbitol dehydrogenase sequences lie within 0.5 Mb on Chromosome 15. We have defined the relative orientation of SORD1 and SORD2 genes with respect to both the centromere and each other and established their exact chromosome location. In addition, we have identified polymorphic variants in the locus, which may be useful, in association studies to predict predisposition to clinical problems resulting from decreased conversion of cellular sorbitol to fructose. To define the evolutionary relationship of these human genes, SORD from the marmoset was also sequenced for comparison. Marmoset SORD, which appears to be a single gene in this species, shows significantly less homology with either SORD1 or SORD2 than they do with each other, suggesting that the human homologs represent a recent gene duplication event. A hypothesis is presented to explain the retention of the redundant SORD2 sequence in the human genome.

Amino Acid Sequence↗
PubMed1998

The immediate-early gene product encoded by open reading frame 57 of herpesvirus saimiri modulates gene expression at a posttranscriptional level.

The herpesvirus saimiri (HVS) immediate-early gene product encoded by open reading frame (ORF) 57 shares limited amino acid homology with HSV-1 ICP27 and Epstein-Barr virus BMLF1, both regulatory proteins. The ORF 57 gene has been proposed to be spliced based on the genome sequence, and here we confirm the intron-exon structure of the gene. We also demonstrate that a cDNA construct of the ORF 57 gene product represses the transactivating capability of the ORF 50a gene product (which is produced from a spliced transcript), but activates that of ORF 50b (an unspliced transcript). Further analyses with cotransfection experiments show that ORF 57 can either activate or repress expression from a range of both early and late HVS promoters, depending on the target gene. These results indicate that repression of gene expression mediated by the ORF 57 gene product is dependent on the presence of an intron within the target gene encoding region. Furthermore, Northern blot analysis demonstrates that the levels of mRNA transcribed from genes not containing an intron are not significantly affected in the presence of the ORF 57 gene product. This suggests that it regulates gene expression through a posttranscriptional mechanism.

Animals↗
PubMed1998

The open reading frame (ORF) 50a gene product regulates ORF 57 gene expression in herpesvirus saimiri.

We have previously demonstrated that open reading frame (ORF) 50 and ORF 57 encode transcriptional regulating genes in herpesvirus saimiri. ORF 50, a homolog of Epstein-Barr virus R protein, is a sequence-specific transactivator, whereas ORF 57 acts posttranscriptionally. In this report, we demonstrate that the ORF 57 gene is regulated by the ORF 50a gene product. We show that the ORF 57 gene is expressed at basal levels early in the virus replication cycle and that thereafter it is transactivated by the ORF 50a gene product, due to an increase in RNA levels. As it has been shown that the ORF 57 gene product downregulates ORF 50a due to the presence of its intron, these combined observations identify a feedback mechanism modulating gene expression in herpesvirus saimiri, whereby ORF 50a transcription is downregulated by the ORF 57 gene product, a gene which it specifically transactivates. Furthermore, we propose that the intron-containing ORF 57 gene downregulates itself by the same mechanism as that for ORF 50a, as both genes are downregulated at similar times during the replication cycle.

Animals↗
PubMed1998

Medical education in the USA--adult-friendly?

In the United States of America, the Area Health Education Center (AHEC) system of training residents has allowed high-quality postgraduate education to flourish. This paper describes the evolution of the AHECs in the context of medical education over the past 50 years. The arrangements for programme administration and design, resident assessment and appraisal, training of trainers in educational methods, and the accreditation of training programmes are discussed. The fast-evolving UK postgraduate education scene can learn some useful lessons from the US system.

Accreditation↗
PubMed1998

DNA mismatch repair genes and their association with colorectal cancer (Review).

Mismatch repair genes are involved in increasing the fidelity of replication by specific repair of DNA polymerase incorporation errors. In Escherichia coli, the best studied mismatch repair (MMR) pathway is the methyl-directed long patch repair system which is mediated by three gene products; MutS, MutL and MutH. These are conserved in higher eukaryotes. Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC). Alterations in the coding regions of MMR genes result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair.

Colorectal Neoplasms, Hereditary Nonpolyposis↗
PubMed1997

Analysis of the mismatch and insertion/deletion binding properties of Thermus thermophilus, HB8, MutS.

The methyl-directed long patch repair pathway in Escherichia coli is involved in increasing the fidelity of replication specific repair of DNA polymerase incorporation errors. This pathway is mediated by three gene products, MutS, MutL, and MutH, which are conserved in higher eukaryotes. Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC). A MutS homologue has recently been identified in the extremely thermophilic bacterium, Thermus thermophilus. Here we describe analysis of the binding properties of this protein, which has indicated it can identify all specific base mismatches as well as one, two and three base pair insertion/deletion mutations. We therefore believe this protein may be generally useful for applications involving mismatch detection.

Adenosine Triphosphatases↗
PubMed1997

Mapping the minimal domain of hMSH-2 sufficient for binding mismatched oligonucleotides.

The human MSH-2 gene product is a member of a highly conserved family of proteins involved in post-replication mismatch repair. Germline mutations in this gene have been implicated in hereditary non-polyposis colorectal cancer (HNPCC). Alterations in the coding region of the hMSH-2 gene result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair. We have previously shown that a region of high homology between MutS proteins of different species containing a nucleotide binding domain, is sufficient to bind DNA containing specific mismatched residues. In order to determine the minimal domain of hMSH-2 necessary for binding mismatch-containing oligonucleotides, deletion analysis of the C-terminal region was performed. We have constructed a 5' and 3' deletion series, expressed each deletion as a bacterial fusion protein and assessed it for ATPase activity and its ability to identify mismatch containing DNA. Here we demonstrate that a 585 bp fragment encoding 195 amino acids within the C-terminal domain of hMSH-2 is sufficient to bind to DNA containing mismatches.

Adenosine Triphosphatases↗
PubMed1997

Identification of a cis-acting element within the herpesvirus saimiri ORF 6 promoter that is responsive to the HVS.R transactivator.

We have previously demonstrated that two distinct transcripts are produced from ORF 50, the major transcriptional activating gene of herpesvirus saimiri. The products of these transcripts trans-activate the delayed-early ORF 6 promoter, though to different degrees. Deletion analysis demonstrated that the ORF 50 responsive elements are contained in a 132 bp fragment situated 127-259 bp from the transcription initiation site within the ORF 6 promoter. This fragment conferred ORF 50-responsiveness on an enhancerless simian virus 40. Gel retardation analysis further mapped the responsive elements to a 38 bp fragment.

Base Sequence↗
PubMed1997

The herpesvirus saimiri ORF50 gene, encoding a transcriptional activator homologous to the Epstein-Barr virus R protein, is transcribed from two distinct promoters of different temporal phases.

The mRNA species encoding the herpesvirus saimiri (HVS) homolog of the Epstein-Barr virus R transcriptional activator (termed ORF50) have been identified and used to determine transcriptional start sites within the gene. The first transcript is spliced and starts from a promoter within ORF49 containing a single intron; the second is produced from a promoter within the second exon and is in the same reading frame. The spliced transcript is detected at early times during productive virus replication in OMK cells, whereas the nonspliced transcript is detected later. The spliced transcript is fivefold-more potent in activating the delayed-early ORF6 promoter; the function of the nonspliced transcript is unclear. Thus, the role of this protein in activating herpesvirus saimiri from the latent state may differ significantly from that of the Epstein-Barr virus R protein.

Animals↗
PubMed1997

Warwickshire consultants' 'training the trainers' course.

To train junior hospital doctors more quickly and effectively as envisaged by the Calman reforms, consultants will need to develop their adult education skills. This paper describes a course set up and attended by a mixed group of Warwickshire consultants to improve their understanding of these skills. The course organisation and content is described and the 'learner-centred education' model, educational supervision techniques, giving feedback on performance, goal setting, learning contracts and other topics covered are explained. A 12-month interval questionnaire evaluation by attendants shows that the principles taught in the course were being widely applied a year later. Thus, at a modest cost, consultants can receive a valuable basic training in adult education.

Consultants↗
PubMed1996

Mutational analysis of the nucleotide binding domain of the mismatch repair enzyme hMSH-2.

The genes involved in postreplicative DNA mismatch repair are a highly conserved family of proteins. In humans, germline mutations in these genes (hMSH-2, hMLH-1, hPMS-1, and hPMS-2) have been implicated in hereditary nonpolyposis colorectal cancer (HNPCC). We have previously shown that a region of high homology between the members of this class of proteins in different species contains a type A nucleotide binding site consensus sequence which has ATPase activity and is sufficient to bind DNA containing specific mismatched residues (1). To identify residues which are necessary for this activity, we have created a range of mutants containing amino acid substitutions within the nucleotide binding domain of hMSH-2. These mutants have been expressed and assessed for ATPase activity and their ability to identify mismatch-containing DNA. Here we demonstrate that a variant protein which has the conserved residue Lys 675 within the nucleotide binding consensus sequence altered to an alanine has severely impaired ATPase activity and is unable to bind DNA containing specific mismatched residues.

Adenosine Triphosphatases↗
PubMed1996

A carboxy terminal domain of the hMSH-2 gene product is sufficient for binding specific mismatched oligonucleotides.

The human MSH-2 gene product is a member of a highly conserved family of proteins which are involved in post-replication mismatch repair. hMSH-2 is homologous to Escherichia coli (E. coli) MutS and Sacchromyces cerevisiae MSH-1 and MSH-2 proteins, which recognise heteroduplex DNA at the sites of all single base mismatches and deletions or insertions up to 4 base pairs. hMSH-2 is one of the hereditary non-polyposis colorectal cancer (HNPCC) tumor suppressor genes, and maps to human chromosome 2p16. Alterations in the coding region of the hMSH-2 gene result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair. It has been shown that purified hMSH-2 binds specifically to nucleotide mismatches in double-stranded DNA. Here we demonstrate that a region of high homology between the members of this class of proteins contains a type A nucleotide binding site consensus sequence which has ATPase activity and is sufficient to bind DNA containing specific mismatched residues.

Adenosine Triphosphatases↗
PubMed1996

CD44 exon variant 6 epitope and hyaluronate synthase are expressed on HT29 human colorectal carcinoma cells in a SCID mouse model of metastasis formation.

The factors which lead to the formation of metastases are generally poorly understood; however the expression of a particular variant of the cell adhesion molecule CD44 may be important in facilitating metastasis formation in colon cancer. The aim of the present study was to investigate the expression of CD44 exon v 6 (CD44v6), hyaluronate (one of its ligands), and hyaluronate synthase, in a clinically relevant animal model of metastatic colon carcinoma. HT29 human colon carcinoma cells were injected subcutaneously between the scapulae of severe combined immunodeficient (SCID) mice and left for 3 weeks (by which time the tumours had produced metastases in the lungs). Morphological observations at the tumour-host interface were consistent with the dissociation of neoplastic cells from the primary tumours, and the ability of these cells to migrate through the extracellular matrix facilitating metastasis formation. Immunohistochemically detectable hyaluronate synthase expression was increased in vivo compared with the parent cell line in vitro. CD44v6 expression and hyaluronate were increased around single cells at the periphery of tumours compared with the central regions. CD44v6 and hyaluronate snythase expression were co-expressed in the same cells. Indeed, the present study is the first to demonstrate hyaluronate synthase expression by an epithelial cell type.

Animals↗
PubMed1995

The relationship between hassles, uplifts and irritable bowel syndrome: a preliminary study.

In a preliminary study, 30 sufferers of irritable bowel syndrome filled in daily symptom sheets and the combined Hassles and Uplifts questionnaire for five weeks. Initially, analyses were carried out for each symptom separately. Results showed that ratings on the hassles and symptoms questionnaires completed in the same week were more highly associated than ratings for hassles and symptoms in different weeks. No one symptom in any week was significantly associated with the following week's hassles. Similarly, hassles in any one week were not associated with the following week's symptoms. However, total symptoms were found to be significantly associated with hassles in the following weeks, whereas the association between hassles and total symptoms in the following weeks was not significant. The study suggests that there is a cumulative effect of symptoms such that an increase in the severity of the combined effect of symptoms is associated with an increase in severity of stress in the next week. Increased hassles do not appear to exacerbate symptoms. There was no evidence of an association between uplifts and IB symptoms.

Adult↗
PubMed1993

Growth hormone and physiological responses to clonidine in depression.

Clonidine (1.3 micrograms/kg) was administered to 62 control and 55 depressed patients free of psychoactive drugs for at least 7 days and fasted overnight. Growth hormone (GH), pulse, blood pressure and sedation were measured every 15 min for 1 h before and 2 h after clonidine infusion. GH response did not differ significantly between control and depressed subjects overall or when divided by sex. The systolic hypotensive and sedative responses were blunted in depressed subjects compared with controls; these effects appeared to be secondary to residual antidepressant drugs since the differences were only significant for those depressed subjects with short drug-free intervals. No differences between depressed subjects and controls were seen in diastolic hypotensive or bradycardic responses and no differences in GH, cardiovascular or sedative responses were found between endogenous and non-endogenous depressed subjects.

Adult↗