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Biomedical subjects

R J Simpson

Publications and source records attributed to R J Simpson.

At least 181 records · Page 10Linked to original sources
PubMed1994

Budesonide and terfenadine, separately and in combination, in the treatment of hay fever.

BACKGROUND: While hay fever is a very common experience, its treatment in primary care setting has been little reported in controlled studies. OBJECTIVE: This study sought to evaluate the patient's assessment of efficacy of an intranasal steroid spray (budesonide) alone or in combination with an antihistamine (terfenadine) against terfenadine alone or placebo alone. METHODS: A double-blind parallel group, placebo-controlled trial design was used, comparing the four groups. Each group used an active or placebo spray and active or placebo tablets. Symptom scores were recorded daily in diaries over a 21-day period. RESULTS: Overall assessment of efficacy by the 106 patients was significantly greater (P < .05) for budesonide versus terfenadine or placebo alone. There was a 40% placebo response. Budesonide was more effective than terfenadine for all individual symptom scores, particularly nasal blockage, against which terfenadine was ineffective. Adverse effects were mild and transient for all groups. CONCLUSIONS: Budesonide alone is a highly effective treatment for hay fever with few side effects.

Adolescent↗
PubMed1994

Controlled comparison of the characteristics of patients with panic disorder.

BACKGROUND: Increased general practice attendance rates have been associated with the diagnosis of mental illness but panic disorder has not been specifically investigated in this respect. In addition, studies have failed adequately to assess type and frequency of secondary care referral and patterns of psychotropic prescription in patients with panic disorder in relation to matched controls. AIM: This study set out to compare subjects with panic disorder with age and sex matched controls on measures of general practice consultation rate; psychotropic and non-psychotropic drug use; referral to secondary care, laboratory and radiological tests and hospital admissions; history of illness and complaints; and psychiatric comorbidity. METHOD: The study was carried out in nine practices in the Forth Valley area. One hundred patients with panic disorder, previously diagnosed using DSM III-R criteria, were identified and matched by age and sex with controls. Data were collected by review of general practice case notes. RESULTS: Subjects with panic disorder had significantly higher rates of general practice consultation over the 10 year period prior to DSM III-R diagnosis of panic disorder than controls. Subjects with panic disorder had also been prescribed a significantly greater number of psychotropic and non-psychotropic medications over this period, had had more secondary care investigations and had higher rates of mainly minor illness and related complaints than controls. High comorbidity of panic disorder with depression which had been diagnosed over the 10 year period prior to DSM III-R diagnosis of panic disorder was found. CONCLUSION: The results of this study describe a population of subjects with panic disorder who are long-term heavy users of primary care services. The results also suggest an association between panic disorder and both minor illness and psychiatric comorbidity over the 10 year period prior to DSM III-R diagnosis of panic disorder.

Adult↗
PubMed1993

Specific covalent modification of the tryptophan residues in murine interleukin-6. Effect on biological activity and conformational stability.

Modification of recombinant murine interleukin-6 (mIL-6) with the tryptophan-specific reagent 2-nitrophenylsulfenyl chloride under mild acidic conditions, 0.1 M sodium acetate, pH 3.5, yielded a derivative containing 2.02 mol 2-nitrophenylsulfenyl tryptophan/mol protein. The sites of modification were identified as Trp36 and Trp160. No detectable side reactions occurred on other amino acids in the molecule, as indicated by the combination of endoproteinase Asp-N peptide mapping, Edman degradation and electrospray mass spectrometry. Sulfenylation of the two tryptophan residues in mIL-6 caused a 50% reduction in both the biological activity in the murine-hybridoma-growth-factor assay using 7TD1 cells and receptor-binding affinity to mIL-6 receptors. Sulfenylation of mIL-6 did not significantly affect the overall conformation of the protein as measured by farultraviolet circular dichroism and binding to the neutralizing anti-mIL-6 mAb 6B4. The sulfenylated protein was, however, significantly less stable [delta delta G(H2O) = 3.98 kJ/mol] than unmodified mIL-6 as measured by urea-gradient gel electrophoresis.

Amino Acid Sequence↗
PubMed1993

Crystal matrix protein is related to human prothrombin.

Crystal matrix protein (CMP) is the principal protein found in calcium oxalate (CaOx) crystals precipitated from whole human urine. It is a potent inhibitor of crystal aggregation and may therefore be important in the aetiology of kidney stone disease. CMP was isolated from CaOx crystals by EDTA dissolution and purified by Sephacryl S-200 column chromatography and reversed-phase high pressure liquid chromatography. Edman degradation revealed 81.8% sequence identity of the 11 N-terminal amino acids of CMP with the N-terminus of human prothrombin, which contains 10 gamma-carboxyglutamic acid residues in the first 32 amino acids. The apparent relationship between CMP and prothrombin was confirmed when an antibody to human prothrombin reacted with CMP upon Western blotting of sodium dodecyl sulphate polyacrylamide gels of the protein.

Amino Acid Sequence↗
PubMed1993

Proteins of the Golgi apparatus. Purification to homogeneity, N-terminal sequence, and unusually large Stokes radius of the membrane-bound form of UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase from rat liver.

The Golgi marker enzyme, UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase (beta 1-4GalT) was purified 44300-fold in its intact, membrane-bound form from rat liver membranes. The protein was isolated from detergent extracts as a high-M(r) form, having a Stokes radius approximating a globular protein of M(r) 440,000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an M(r) near 51,000, and does not have intermolecular disulfide cross-links. N-terminal sequencing of the enzyme demonstrated that it contains an N-terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5' end of the mRNA [Russo, R. N., Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N-terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low-M(r) forms which were fully catalytically active and which, upon sequencing, were missing a 66-amino-acid stretch from the N-terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with M(r) near 50,000. The N-terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non-protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

Amino Acid Sequence↗
PubMed1993

Structural characterisation of native and recombinant forms of the neurotrophic cytokine MK.

The retinoic acid (RA)-inducible midkine (MK) gene encodes a heparin-binding protein which can induce neurite outgrowth in cultured mammalian embryonic brain cells. This cytokine shares 65% amino acid sequence identity with another RA-inducible cytokine, pleiotropin (PTN). Both proteins contain 10 conserved cysteine residues, all of which appear to be disulphide linked. MK and PTN are also rich in lysine and arginine residues rendering them susceptible to proteolysis during purification, and making large-scale preparation of these molecules inherently difficult. Recombinant MK has been expressed as a fusion protein using a pGEX vector transfected into E. coli. To enable refolding of MK, the fusion protein was stored in solution at 4 degrees C for 14 days in the presence of dithiothreitol (DTT). Thrombin cleavage of the fusion protein, post storage, typically generated 5 mg of MK per litre of bacterial pellet. To establish the structural integrity of the recombinant product, we have analysed the refolding kinetics and compared the disulphide bond assignment of recombinant MK with that of native MK and native PTN. The synergistic use of micropreparative HPLC, to separate and recover in small eluant volumes enzymatically derived peptide fragments, with matrix assisted laser desorption mass spectrometry (MALD-MS) and N-terminal sequence analysis has allowed the unambiguous identification of the disulphide bonded fragments of native and recombinant MK. The disulphide bond assignment of MK is C12-C36, C20-C45, C27-C49, C59-C91 and C69-C101, and is equivalent to that of PTN.

Amino Acid Sequence↗
PubMed1993

The cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (FKBP59).

Using a rapid single-step affinity chromatography procedure we have isolated the unactivated estrogen receptor from bovine uterus. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analyses for protein extracts recovered from affinity chromatography of receptor cytosols, either preincubated or untreated with estradiol, suggest a component structure for the intact oligomeric receptor which includes hsp90, hsp70, p59, a 40-kDa cyclophilin-related protein, and an uncharacterized 22-kDa protein species. We have chemically determined the amino acid sequences of eight peptides derived from the 40-kDa component and now report the cloning and primary sequence of a cDNA encoding this protein, which is designated estrogen receptor-binding cyclophilin (ERBC). Homology analyses confirm that ERBC is a new member of the cyclophilin family and contains a C-terminal domain with significant sequence homology to an internal region of p59, a binding protein for the immunosuppressant FK506 (FKBP59). This conserved region includes a 3-unit tetratricopeptide repeat domain bounded at the C terminus by a putative calmodulin binding site. We propose that the tetratricopeptide repeat domain mediates the protein interaction properties of ERBC and p59. Both immunophilins may have important roles in receptor assembly and may represent a new category of ligand- and calcium-dependent modulators of protein function.

Amino Acid Isomerases↗
PubMed1993

Epidermal growth factor induces serine phosphorylation of stathmin in a human colon carcinoma cell line (LIM 1215).

Changes in protein phosphorylation in the human colon carcinoma cell line LIM 1215 after stimulation with epidermal growth factor (EGF) have been analyzed by two-dimensional gel electrophoresis and phosphoamino acid analysis. In addition to a number of tyrosine-phosphorylated proteins, a family of small proteins (M(r) 19,000-20,000) is maximally phosphorylated on serine within 5 min of EGF stimulation. One member of the family has been purified by a combination of two-dimensional electrophoresis and reversed-phase high performance liquid chromatography and identified by amino acid sequence analysis as stathmin. Although phosphorylation of stathmin has been reported previously in leukemia cells and following stimulation of hemopoietic or lymphoid cells with several mitogenic agents, this is the first report of stathmin phosphorylation in response to EGF.

Amino Acid Sequence↗
PubMed1993

Partition and distribution coefficients of solutes and drugs in brush border membrane vesicles.

Partition and distribution coefficients (log P, log D) into rat small intestinal brush border membrane (BBM) were measured for a variety of ionizable and non-ionizable drugs and solutes using a novel technique. The log P values were compared with those determined with model solvents, octanol and propylene glycol dipelargonate (PGDP). Non-ionizable solutes with log P values up to 3.0 showed that octanol was a better model for partition into the BBM than PGDP. With one exception, BBM partition coefficients of greater than 3 were not observed, even for solutes with log P values in model solvents that were greater than 5. Liposomes prepared from BBM lipids, or synthetic lipid mixtures of similar composition to BBM, demonstrated similar trends in partition coefficients to the intact BBM. Two cationic drugs, Atenolol and Xamoterol were investigated for partition into BBM lipid liposomes. An apparent enhancement of log D with respect to octanol was attributed to a "surfactant-like" orientation in the membrane and an interaction of the ionized drug with anionic phospholipid head groups. The anionic drug Proxicromil shows the expected decrease in log D with increasing pH, at low NaCl concentrations. Changes in electrophoretic mobility of liposomes after incorporation of Proxicromil into them were consistent with the negative charge of the ionized drug being at the membrane surface. It was concluded that Proxicromil also associates with membranes in a "surfactant-like" orientation and that increased extraction with increasing NaCl concentrations is a result of ionic strength effects. Partition of solutes into BBM vesicles is more complex than into organic solvents and probably represents an important step in overall intestinal permeation of solutes.

1-Octanol↗
PubMed1993

Stratification of sudden death risk in patients receiving long-term amiodarone treatment for sustained ventricular tachycardia or ventricular fibrillation.

One hundred twenty-two patients treated chronically with amiodarone for sustained ventricular tachycardia or ventricular fibrillation after failing conventional antiarrhythmic therapy were analyzed to determine which factors were predictive of sudden cardiac death during follow-up. The mean left ventricular ejection fraction in the study group was 0.32, and 87% of the patients had coronary artery disease with a prior myocardial infarction. During a median follow-up of 19.5 months, 30 patients died suddenly. The only variable that was predictive of sudden death was left ventricular ejection fraction. Twenty-nine of the 84 patients with ejection fractions < 0.40 died suddenly, compared with 1 of 35 patients with ejection fractions > or = 0.40. The actuarial probability of sudden death at 5 years was 49% when the ejection fraction was < 0.40, and 5% when the ejection fraction was > or = 0.40 (p = 0.0004). These results indicate that patients treated with amiodarone for sustained ventricular tachycardia or ventricular fibrillation whose ejection fractions are > or = 0.40 are at low risk for sudden death. Patients with ejection fractions < 0.40 remain at high risk for sudden death, and should be considered for additional or alternative therapy.

Actuarial Analysis↗
PubMed1993

Identification of a chemotactic domain of the pro-inflammatory S100 protein CP-10.

We previously reported the purification and partial amino acid sequence of a novel murine cytokine designated CP-10, which has chemotactic activity for murine polymorphonuclear cells (PMN) and macrophages. The complete cDNA encoding an 88-amino acid polypeptide has been isolated and the sequence is presented here. Transient transfection of CP-10 cDNA into CV-1 cells confirmed the chemotactic activity of rCP-10 for murine PMN. CP-10 has sequence homology with members of the S100 family of Ca(2+)-binding proteins with pronounced amino acid sequence similarities within the putative N- and C-terminal Ca(2+)-binding sites, but differences within their connecting hinge and C-terminal regions. We have confirmed the hypothesis of Kligman and Hilt that functional specificity of individual members of the S100 protein family may reside in the hinge region. A synthetic peptide corresponding to the hinge region of CP-10 (CP-10(42-55) was compared with native CP-10 in chemotaxis and skin test assays. Native CP-10 had potent activity for phagocytic cells, but not lymphocytes, in vitro (optimal activity, 10(-11) to 10(-13) M) and elicited a sustained recruitment of neutrophils and mononuclear cells over 24 h in vivo. The hinge-region peptide had strong chemotactic activity for murine phagocytic cells (optimal activity, 10(-10) - 10(-11) M) but elicited only a transient infiltration of neutrophils over 4 to 8 h after intradermal injection. Results indicate that although the hinge region contributes significantly to the functional specificity of the S100 protein CP-10, sustained cellular recruitment typical of a delayed type hypersensitivity response is apparently dependent on the structural integrity of the protein.

Amino Acid Sequence↗
PubMed1993

Identification of a region of beta 2-glycoprotein I critical for lipid binding and anti-cardiolipin antibody cofactor activity.

beta 2-Glycoprotein I (beta 2-GPI), a phospholipid-binding plasma protein, is an absolute requirement (cofactor) for the binding of autoimmune-type anti-cardiolipin (aCL) antibodies to cardiolipin (CL). The nature of this cofactor activity and the specific regions of the molecule involved have not yet been determined. We have identified a preparation of beta 2-GPI that lacks aCL antibody cofactor activity. Analysis of the structural differences between the active and inactive forms enabled identification of the region of beta 2-GPI critically important for aCL cofactor activity. The active form of beta 2-GPI bound CL and displayed cofactor activity down to 1 microgram/ml. The inactive form failed to bind CL and possessed no cofactor activity even at concentrations up to 94 micrograms/ml, indicating that the ability of beta 2-GPI to bind lipids is an absolute requirement for aCL cofactor activity. Both forms possessed identical N-terminal sequences and were recognized as essentially immunoreactively identical by polyclonal antisera to beta 2-GPI. However, the inactive form has undergone proteolytic cleavage and exists primarily as a "clipped" molecule, the polypeptide chain being cleaved between Lys-317 and Thr-318 (a potential thrombin cleavage site), with the two cleaved segments linked as a disulfide-bonded complex. This indicates that the C-terminal region is critically important for beta 2-GPI to bind lipid and for aCL cofactor activity. The clipped form of beta 2-GPI would not be suitable for use as aCL cofactor and its use may have led some investigators to conclude incorrectly that beta 2-GPI does not interact with aCL antibodies.

Amino Acid Sequence↗
PubMed1993

The disulfide bond arrangement of leukemia inhibitory factor: homology to oncostatin M and structural implications.

Murine leukemia inhibitory factor (LIF) (the fully active recombinant form produced in E. coli) was digested in the unreduced state with trypsin and Staphylococcal V8 protease in 0.05% sodium dodecyl sulfate. Disulfide-bonded peptides were identified by altered mobility on reverse-phase high-performance liquid chromatography in the presence or absence of dithiothreitol and subjected to amino acid sequencing. Peptides containing more than one disulfide bond were subjected to further proteolysis and disulfide-bonded subfragments identified and sequenced. The three disulfide bonds are CYS13-CYS135, CYS19-CYS132 and CYS61-164 and the first and third of these are clearly homologous to the two disulfide bonds in oncostatin M. The spatial organization of the cysteine residues in the predicted four alpha-helical bundle structure of LIF (Bazan, Neuron 7,197;1991) is compatible with these disulfide assignments.

Amino Acid Sequence↗
PubMed1993

Tissue iron loading and histopathological changes in hypotransferrinaemic mice.

Tissue iron loading in hypotransferrinaemic (hpx/hpx) mice was investigated as a model for genetic (primary) haemochromatosis. Iron loading of liver preceded that in the pancreas and heart. One-year-old hpx/hpx mice showed iron staining in exocrine pancreas, liver parenchymal cells, and cardiac and intestinal smooth muscle cells. Iron-loaded macrophages were observed in all these tissues. Islets of Langerhans, biliary epithelial cells, and spleen were iron-free. The pancreas was fibrotic with massive macrophage infiltration and loss of secretory epithelium. Liver showed evidence of chronic inflammatory infiltration with increased collagen fibres in the parenchymal region but no cirrhosis. Serum aspartate aminotransferase activity and plasma glucose were increased in hpx/hpx compared with wild-type mice. Heavy iron loading with haemosiderin deposition in the liver could be demonstrated in hpx/hpx mice from 6 weeks of age. Heterozygous hypotransferrinaemic mice showed minor increases in liver iron stores at 6-12 weeks, but not at 1 year of age. Serum ferritin levels in heterozygous mice were also increased at 6-8 weeks of age. It was concluded that 1-year-old hpx/hpx mice showed evidence of liver and pancreatic damage secondary to tissue iron overload. The iron loading pattern and tissue damage showed some features which were distinct from those observed in haemochromatosis.

Animals↗
PubMed1993

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.

Amino Acid Sequence↗
PubMed1993

Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.

Amino Acid Sequence↗
PubMed1993

Physician-based diet counseling for cholesterol reduction: current practices, determinants, and strategies for improvement.

BACKGROUND: Physicians face increasing pressure to counsel their hypercholesterolemic patients about diet. To design effective physician-based treatment programs, a better understanding of current dietary counseling practice and its determinants is needed. METHODS: Using a survey previously tested for reliability and validity, we examined the relationship of dietary knowledge, attitudes, beliefs, organizational barriers, and treatment practices for cholesterol management among 60 resident and attending physicians practicing in the general medicine clinic of a university medical center that serves primarily rural and disadvantaged patients. The survey was administered in October of 1988 prior to the release of the National Cholesterol Education Program Guidelines. RESULTS: The response rate was 100%. Ninety-two percent of physicians surveyed believe that dietary treatment effectively lowers cholesterol and 68% feel responsible for providing such therapy. However, most (72%) feel ill-prepared to give diet counseling, lack confidence in their ability to help patients make meaningful dietary changes (95%), and cite organizational barriers, such as limited time (72%) or inadequate educational materials (47%). Physicians were more likely to report behaviorally focused diet counseling practices if they felt prepared to counsel (r = 0.42, P < 0.001), were confident in their counseling skills (r = 0.39, P < 0.01), or reported personally following a prudent diet (r = 0.36, P < 0.01). We discuss the implications of these findings and how they should guide the design of physician-based dietary interventions for cholesterol reduction.

Counseling↗