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M Egerton

Publications and source records attributed to M Egerton.

At least 37 records · Page 2Linked to original sources
PubMed1995

Comparison of the sensitivity to change of long and short form pain measures.

Our objective was to assess the sensitivity to change over time of three methods of assessing pain. We conducted a comparison of the sensitivity to change of three pain assessment measures in a double blind, double dummy parallel group study evaluating the efficacy of one of two doses of oral bromfenac and one of two doses of sublingual buprenorphine. Our subject pool consisted of 75 patients following general surgical and orthopaedic procedures. Pain intensity was measured by the affective, sensory and evaluative dimensions of the McGill Pain Questionnaire together with total score and word score, a 10 cm Visual Analogue Scale and by a four word scale. Effect size calculations indicated considerable variation in the sensitivity of the instruments to change. However, despite variation in the size of effect indicated by different measures, there was consistent agreement as to the most effective treatment regimen. This study suggests that simple measures of pain are sensitive to change and are worthwhile indicators of the impact of an intervention upon acute pain, although some care is needed in interpreting results from such instruments. More complex forms of assessment, such as the calculation of the MPQ sub-scales may not add anything to such data.

Adult↗
PubMed1995

Yeast Gaa1p is required for attachment of a completed GPI anchor onto proteins.

Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI-anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA1, encoding an essential ER protein required for GPI anchoring. gaal mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA1 improves the ability of cells to attach anchors to a GPI-anchored protein with a mutant anchor attachment site. Therefore, Gaa1p is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase.

Amino Acid Sequence↗
PubMed1995

Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization.

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.

Adenylyl Cyclases↗
PubMed1994

Biochemical characterization of valosin-containing protein, a protein tyrosine kinase substrate in hematopoietic cells.

Engagement of the T cell antigen receptor (TCR) leads to activation of multiple tyrosine kinases and rapid tyrosine phosphorylation of intracellular protein substrates. A number of these substrates have been identified and they include TCR subunits, phospholipase C-gamma 1, p95vav, and ezrin. In a recent study we have demonstrated that VCP (valosin-containing protein) becomes tyrosine phosphorylated upon TCR cross-linking. Analysis of the predicted amino acid sequence of this protein indicates that it is a member of a family of oligomeric proteins containing duplicated domains with predicted ATPase activity. In the current study we determine the site of tyrosine phosphorylation in VCP, demonstrate that murine VCP indeed is an oligomeric ATPase, and show that the tyrosine phosphorylation of the protein has no effect on VCP ATPase activity. Recent evidence suggests that VCP associates with clathrin. A possible role of tyrosine phosphorylation in regulating this protein-protein interaction is discussed.

Adenosine Triphosphatases↗
PubMed1994

Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1).

Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE. Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference. The h6.1 cDNA was extended at the 5'-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids. The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3'-untranslated region, with the previously reported human PDE-IVA cDNA [Livi G. P., Kmetz P., Mchale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J. and Balcarek J. M. (1990) Mol. Cell Biol. 10, 2678-2686]. The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals. The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5'-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain. The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells. Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S. cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype. Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour. Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: subcellular distribution of a protein required for yeast protein secretion.

Strains of Saccharomyces cerevisiae harbouring temperature-sensitive mutations in the SEC1 and SEC5 genes exhibit an accumulation of post-Golgi secretory vesicles at 37 degrees C. We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements a sec1 mutation, and the other a sec5 mutation. Genetic test confirm that the sec1-complementing gene is indeed SEC1, and is essential for cell growth. Nucleotide sequence analysis reveals that the cloned SEC1 gene is the same as a previously sequenced sec1-complementing gene. The SEC1 sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa. Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent-resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cytoskeleton.

Cloning, Molecular↗
PubMed1993

Diabetes, glaucoma, sex, and cataract: analysis of combined data from two case control studies.

Data from two case control studies in Oxfordshire were combined and analysed. The combined study covered 1940 subjects, 723 cases, and 1217 controls, between the ages of 50 and 79 with a response rate of 97% for cases and 94% for controls. Diabetes was shown to be a powerful and highly significant risk factor for cataract with a relative risk of 5.04. More than 11% of cataracts in Oxfordshire are attributable to diabetes. The relative risk did not increase significantly with age within the range 50 to 79 years but was higher in females than in males. For females with diabetes the relative risk was 7.85 with 95% confidence interval from 4.30 to 14.3 compared with 3.42 with confidence interval from 2.05 to 5.71 for males with diabetes. Diabetes remained a powerful risk factor when other identified risk factors had been controlled for. No known mechanism for the development of diabetic complications provides an explanation for the excess risk in females. Combination of the two studies led to better estimates of the relative risk of glaucoma as a risk factor for cataract (3.96 with 95% confidence interval from 2.35 to 6.68). The relative risk appeared to be greater in women than in men but this difference was not statistically significant. There was no significant change in risk with age. Glaucoma is a powerful and independent risk factor for cataract in both sexes and may be responsible for 5% of all cataracts in our area.

Aged↗
PubMed1992

Cytokine-induced phosphorylation of pp100 in FDC-ER cells is at tyrosine residues.

Using FDC-P1 cells stably transfected with a murine erythropoietin receptor cDNA as a model, we recently have shown that erythropoietin (EPO), IL-3 and GM-CSF each induce the rapid phosphorylation of a common cytosolic target, i.e., a M(r) 100,000 phosphoprotein "pp100". Presently, we demonstrate that cytokine-induced phosphorylation of pp100 is primarily at tyrosine residues. This is shown by Western blotting with the anti-phosphotyrosine antibody PY20, and by the resistance of [32P]-pp100 to hydroxide-mediated hydrolysis of phosphates. These data, together with the recent observation by Linnekin et al. that pp100/p97 apparently associates directly with EPO receptors, suggest that pp100 may comprise an immediate common component in the signal transduction pathways of EPO, IL-3, GM-CSF and possibly other type I/II cytokine receptors. Additional analyses suggest that pp100 is distinct from a previously described M(r) 100,000 cytosolic target which is tyrosine phosphorylated in hematopoietic cells upon activation of T-cell receptors.

Animals↗
PubMed1992

Identification of ezrin as an 81-kDa tyrosine-phosphorylated protein in T cells.

We have used APT affinity purification to isolate tyrosine-phosphorylated proteins from MRL lpr/lpr (lpr) mouse T cells. One such protein is pp81 ezrin, previously identified as a tyrosine-phosphorylated protein in epidermal growth factor-stimulated A431 carcinoma cells. Biochemical analyses in A431 and gastric parietal cells have revealed ezrin to be a cytoskeleton-associated cytosolic protein. In Jurkat T cells, however, using similar methods we have shown ezrin to be a cytosolic protein with no measurable cytoskeletal association. We also observed no increases in ezrin tyrosine phosphorylation in TCR-stimulated Jurkat T cells, unless the cells were pretreated with protein tyrosine phosphatase inhibitors, suggesting that T cell ezrin tyrosine phosphorylation is tightly controlled by protein tyrosine phosphatases. The fraction of tyrosine phosphorylated ezrin in lpr T cells was 5 to 10 times that observed in Jurkat T cells, which along with constitutive TCR-zeta phosphorylation and pp60fyn overexpression, is a feature of the lpr defect.

Amino Acid Sequence↗
PubMed1992

VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control.

Adenosine Triphosphatases↗
PubMed1992

Characterization of the T cell antigen receptor--p60fyn protein tyrosine kinase association by chemical cross-linking.

Engagement of the TCR by specific antigen results in activation of a tyrosine kinase pathway. A candidate for the kinase responsible for the rapid tyrosine phosphorylation detected with T cell activation is p60fyn, a member of the src kinase family. In an earlier study [Samelson et al. (1990) Proc. Natl Acad. Sci. USA 87:4358] this enzyme was co-immunoprecipitated with the TCR from T cells solubilized in digitonin. In that study a sensitive in vitro kinase assay was used to detect the associated p60fyn. It was subsequently found that the reproducibility of the interaction depended on lot-to-lot variations in digitonin. To eliminate the possibility that the association of antigen receptor and kinase is an artifact of solubilization with ill-defined digitonin preparations, a cross-linking protocol was developed to stabilize the interaction between the TCR and p60fyn. T cells were permeabilized with tetanolysin and proteins were cross-linked with the water soluble chemical cross-linker, 3,3' dithiobis(sulfosuccinimidylpropionate). These experiments allowed the confirmation of the interaction between the TCR, p60fyn, and several additional proteins. The cross-linking studies also enabled the mapping of the interaction of p60fyn and associated proteins to the TCR zeta-chain. This technique should have a general use in stabilizing interactions between other receptors and molecules required for intracellular signaling.

Animals↗
PubMed1991

The kinetics of T cell antigen receptor expression by subgroups of CD4+8+ thymocytes: delineation of CD4+8+3(2+) thymocytes as post-selection intermediates leading to mature T cells.

Cortical thymocytes from adult mice, separated on the basis of coexpression of CD4 and CD8 or of binding of high levels of peanut agglutinin (PNA), were subdivided according to the level of expression of the T cell receptor (TCR)-CD3 complex. The incidence of dividing cells in the resultant subpopulations was determined by DNA staining. Precursor-product relationships and the timing of TCR-CD3 acquisition were studied using continuous in vivo [3H]TdR labeling and radioautography. The extent of intrathymic selection for TCR specificity in the subpopulations was determined from the incidence of cells bearing V beta 6 or V beta 17a in different mouse strains. The majority of dividing CD4+8+ blast cells expressed extremely low levels of TCR-CD3, indicating that TCR expression and specificity selection generally occurred after division ceased. The [3H]TdR-labeling studies indicated that postdivision TCR expression was rapid, and that those nondividing cortical thymocytes which had not expressed significant levels of TCR by day 1, remained extremely low or negative for their entire 3.6-d lifespan. Small cortical thymocytes which expressed moderate levels of TCR-CD3, were predominantly an unselected population with a lifespan of 3.8 d. A small subgroup of CD4+8+ PNA+ cortical thymocytes expressing high levels of TCR-CD3 was identified as a nondividing intermediate between the small cortical thymocytes expressing moderate levels of TCR and mature medullary thymocytes. These intermediates showed a 1-d lag in [3H]TdR labeling, then a 3.4-d transit time. The cell flux through this intermediate subpopulation was approximately 10(6) cells/d, similar to the rate of turnover of mature thymocytes; thus, although only 3-4% of thymocytes progressed to this intermediate state, once reaching it most then progressed to full maturity. In accordance with this, the incidence of the V beta selection markers within the intermediate subpopulation indicated that both positive and negative selection had already occurred. Selection for TCR specificity in the systems studied appeared to take place among CD4+8+ thymocytes expressing intermediate levels of TCR.

Animals↗
PubMed1991

CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus.

A continuous but low input of stem cells or 'prothymocytes' is necessary to maintain T-cell development in the adult thymus, but the colonizing cell has not been characterized. Precursors of T cells have been found in the minor CD4-8- population of thymocytes, but even the earliest cells of this population already have partially rearranged T-cell antigen receptor (TCR) genes. We now demonstrate that the thymus contains a minute population of lymphoid cells similar in some but not all respects to bone marrow-derived haemopoietic stem cells. This population has TCR genes in a germline state. It gives a slow but extensive reconstitution of both alpha beta and gamma delta lineages on transfer into an irradiated thymus, with kinetics indicating that it includes the earliest intrathymic precursor cells so far isolated. Surprisingly, these cells express low surface levels of the mature T-cell marker CD4.

Animals↗
PubMed1991

Multiple signal transduction pathways activated through the T cell receptor for antigen.

The T cell receptor for antigen (TCR) is a multichain complex on the surface of T lymphocytes which binds peptide antigen and transduces a transmembrane signal leading to IL-2 secretion. Engagement of the TCR leads to activation of a tyrosine phosphorylation pathway and a phospholipase C (PLC) pathway leading to activation of protein kinase C (PCK). Currently available data suggest that the primary event in signal transduction is tyrosine kinase activation, since when this pathway is inhibited, PLC activation is blocked and there is no production of IL-2. The nature of the tyrosine kinase which initiates the signaling cascade is currently unknown. The CD4/CD8 associated kinase p56lck clearly plays a role in tyrosine phosphorylation, but it is clearly not the only tyrosine kinase involved. Studies demonstrating physical association of p59lyn with the TCR implicate fyn as an important candidate for the TCR tyrosine kinase. The protein tyrosine phosphatase CD45 also plays a critical early role in signal transduction since in cells where it is deficient, neither tyrosine kinase activation nor later signaling events are seen. The importance of the PLC/PKC pathway is illustrated by the fact that activation of this pathway alone may lead to IL-2 production. However, there may also be other mechanisms which can generate an IL-2 response. Two proteins known to be involved in growth regulation--p21ras and c-raf--have now been shown to be downstream targets of the PLC/PKC pathway.

Animals↗
PubMed1990

Cell generation within human thymic subsets defined by selective expression of CD45 (T200) isoforms.

Although the thymus is the source of all mature peripheral T lymphocytes, the majority of thymocytes die intrathymically. Until recently, there has been no phenotypic marker to allow definition of the generative thymocyte lineage, thereby distinguishing those thymocytes committed to death from those which will eventually give rise to thymic emigrants. We believe that expression of the high-molecular-mass isoforms (p190, p205, and/or p220) of the leukocyte common antigen (CD45) distinguishes the thymic generative lineage from the vast majority of thymocytes expressing the low-molecular-mass isoform (p180) of CD45 and committed to die within the thymus. The thymocytes defined by their lack of CD45 p180, the low-molecular-mass isoform, comprise all thymocytes with clonogenic potential and include all major subsets defined by CD4 and CD8. We have proposed that a CD45 p180- lineage exists in the human thymus and that this lineage results in the production of mature thymocytes and thymic emigrants. The objective of the present study was to determine by DNA analysis whether the degree of cell cycling in subsets of human thymus, defined by selective expression of high-molecular-mass isoforms of CD45, was sufficient to account for the generation of thymic emigrants. Multicolor immunofluorescence analysis of surface markers and 7-amino actinomycin D as well as propidium iodide staining was used to measure the DNA content of thymic subsets. Negative depletion methods were used to isolate and characterize human thymocyte subsets defined by CD45 isoform, CD3, CD4, and CD8, and subsequently to determine the cell cycle status of the isolated subsets by flow-cytometric analysis of cellular DNA content. CD3-/lo thymocytes had a high number and CD1-/lo thymocytes a low number of cycling cells, consistent with murine data. CD45 p180- cells, as well as the CD4-(8-) and CD3-(4-)(8-) subsets, which express high molecular-weight CD45 isoforms, exhibited a significant number of cycling cells. Since CD45 p180- thymocytes exhibited a significant number of cycling cells, based on numerical arguments we conclude that this cycling thymocyte fraction is capable of generating the daily requirement of mature thymocytes and thymic emigrants.

Adolescent↗