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Biomedical subjects

E Ferguson

Publications and source records attributed to E Ferguson.

At least 19 recordsLinked to original sources
PubMed1999

Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase.

Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix [Stivers, J. T., et al. (1999) Biochemistry 38, 952]. A general acid-base mechanism has been proposed whereby His187 facilitates leaving group departure by protonating the O2 of uracil and Asp64 activates a water molecule for nucleophilic attack at C1' of the deoxyribose. Detailed kinetic studies on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respectively, with little effect on substrate or product binding. The pH dependence of k(cat) for wild-type and H187Q UDG indicates that an unprotonated group in the enzyme-substrate complex (pK(a) = 6.2 +/- 0.2) is required for catalysis. This unprotonated group has a small DeltaH of ionization (-0.4 +/- 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting that it corresponds to the general base Asp64. The pH dependence of k(cat) for wild-type, H187Q, and D64N UDG shows no evidence for an essential protonated group over the pH range of 5.5-10. Hence, the pK(a) of His187 must be outside this pH range if it serves as an electrophilic catalyst. These results support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge on uracil O2 in the transition state. In the following paper of this issue we establish by crystallography and heteronuclear NMR spectroscopy that the imidazole of His187 is neutral during the catalytic cycle of UDG.

Asparagine↗
PubMed1999

Characterization of the adduct formed from the reaction between homocysteine thiolactone and low-density lipoprotein: antioxidant implications.

Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol/mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactive nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spin labeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2(+)-mediated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.

Antioxidants↗
PubMed1998

Hypochondriacal concerns, symptom reporting and secondary gain mechanisms.

Hypochondriacal concerns (HCs) and somatic symptom reporting (SSR) are associated. However, HCs are believed to be linked to the ego-defence coping strategy of avoiding help and SSR is believed to be linked to secondary gain. One hundred and twenty undergraduate students completed measures of HCs, SSR and the Desire for Control (DC) Scale. Subscales of the DC scale were used as indices of help avoidance and secondary gain. Both hierarchical multiple linear regression and LISREL structural modelling were used to control for the confound between HCs and SSR and explore the links with help avoidance and secondary gain. HCs were found to be primarily associated with an increased desire to strive for independence (avoid help) and levels of SSR were associated with the desire to have others make decisions (secondary gain).

Adaptation, Psychological↗
PubMed1998

Generation and initial characterization of a novel polyclonal antibody directed against homocysteine thiolactone-modified low density lipoprotein.

Elevated plasma homocysteine (homocysteinemia) are presumed to be responsible for the development of coronary artery disease, however, the precise etiology is unclear. We examined the possibility that the adduct formed from the reaction between homocysteine thiolactone, a metabolic product of homocysteine, and apolipoprotein B-100 lysyl residues of low density lipoprotein (LDL) was immunogenic. New Zealand White rabbits were immunized with this adduct at 6-week intervals. Antisera collected following the 3rd immunization was assayed for antibody titers using solid phase ELISA techniques. Titers (defined as the inverse of the greatest serum dilution in which there was a significant difference (P < 0.05) between the percentage antibody bound from the antiserum and the pre-immune serum) were approximately 10(5). In competition-based ELISAs, homocysteine thiolactone-treated LDL competed for binding with the antiserum, as the 50% inhibitory concentration was approximately 10 microg/ml. Neither homocysteine, homocystine (homocysteine disulfide), nor Cu2-oxidized LDL competed for binding. LDL in which lysyl residues were derivatized by acetylation or methylation were not recognized by the antiserum. Homocysteine thiolactone-treated plasma competed for binding to the antiserum, whereas native plasma did not. All lipoprotein fractions from the homocysteine thiolactone-treated plasma competed for binding to the antiserum. We conclude that homocysteine thiolactone-modified LDL is highly immunogenic and specific for homocysteine thiolactone-modified lysines. The potential for using this antibody as a diagnostic tool for measuring plasma homocysteine concentrations and the implications for understanding diseases induced by homocysteinemia are discussed.

Antibody Specificity↗
PubMed1997

The mechanism of apolipoprotein B-100 thiol depletion during oxidative modification of low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL) is recognized to be a key step in atherogenesis. Previous studies show that LDL contains low-molecular-weight antioxidants such as vitamin E, beta-carotene, and ubiquinol, which can retard oxidative modification. In this report, we have evaluated the antioxidant potential of apolipoprotein B-100 (apo-B) thiols during LDL oxidation. Both apo-B thiols and vitamin E were depleted concomitantly during the lag phase of Cu(2+)-mediated LDL oxidation. The rate of thiol depletion was significantly inhibited by the lipophilic spin trap N-tert-butyl-alpha-phenylnitrone (PBN) but not by the water-soluble spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Blocking apo-B thiols with sulfhydryl modifying agents increased the oxidizability of LDL. As with Cu2+, peroxynitrite also caused depletion of apo-B thiols, and again thiol depletion was inhibited by PBN but not by POBN. A PBN/lipid-derived radical adduct was observed by the electron spin resonance technique during oxidation of LDL with peroxynitrite. We conclude that apo-B thiol depletion is mediated by lipid peroxidation, prior to the onset of the propagation phase of LDL oxidation. The implications of apo-B thiols an intrinsic antioxidants of LDL are discussed.

Antioxidants↗
PubMed1997

Stress and blood donation: effects of music and previous donation experience.

Making a blood donation, especially for first-time donors, can be a stressful experience. These feelings of stress may inhibit donors from returning. This paper applies stress theory to this particular problem. The effects of a stress management intervention (the provision of music) and previous donor experience were examined in relation to pre- and post-donation mood, environmental appraisals and coping behaviour. Results indicated that the provision of music had detrimental effects on environmental appraisals for those who have donated up to two times previously, but beneficial effects for those who had donated three times before. These effects were, to an extent, moderated by coping processes but not perceived control. It is recommended that the provision of music is not used as a stress management technique in the context of blood donation.

Adaptation, Psychological↗
PubMed1997

Nurses' evaluations of sources of information about HIV and AIDS.

Reviews of the literature indicate that nurses feel ill-informed about HIV/AIDS and that poor knowledge is associated with anxiety and negative attitudes towards infected patients and their care. Although some studies have sought to identify the sources of HIV/AIDS information available to nurses, few have attempted to understand how nurses evaluate such sources. In this study in 1992, 15 sources of HIV/AIDS information were identified during group discussions with nursing staff and nurse tutors. 277 nursing staff evaluated each of the sources in terms of perceived frequency (how often the source is used) and six items chosen to assess the usability and usefulness of each source (e.g. how informative the source is, how easy it is to understand). The results indicate that in-service training, basic training and professional colleagues are the sources evaluated most highly while posters and advertisements, television and radio and popular newspapers are the most frequently used sources of information. Trades unions' journals and pamphlets are the least frequently used sources of information and receive only modest evaluations. 20% of respondents report never having received any training regarding HIV and AIDS. Implications for the future provision of HIV/AIDS information and directions for further research are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on transcription factor NF-kappa B.

In the present studies, we examined the effect of flavonoids on the endothelial cell expression of adhesion molecules, an early step in inflammation and atherogenesis. Addition of tumor necrosis factor-alpha (TNF) to human aortic endothelial cells (HAECs) led to the induction of vascular cell adhesion molecule-1 (VCAM-1) expression and enhancement in expression of intercellular adhesion molecule-1 (ICAM-1). A flavonoid, 2-(3-amino-phenyl)-8-methoxy-chromene-4-one (PD 098063), markedly inhibited TNF-induced VCAM-1 cell-surface expression in a concentration-dependent fashion with half-maximal inhibition at 19 mumol/L but had no effect on ICAM-1 expression. Another structurally distinct flavonoid, 2-phenyl-chromene-4-one, similarly selectively decreased VCAM-1 expression. The inhibition in cell-surface expression of VCAM-1 by PD 098063 correlated with decreases in steady-state mRNA levels, but there was no effect on ICAM-1 mRNA levels. The decrease in VCAM-1 mRNA levels was not due to changes in mRNA stability but rather resulted from a reduction in the rate of transcription of the gene. However, electrophoretic mobility shift assays using nuclear extracts from TNF-induced HAECs treated with PD 098063 failed to show a decrease in the activation of NF-kappa B, indicating that inhibition of activation of this transcription factor may not be its mode of action. Similarly, PD 098063 did not affect chloramphenicol acetyltransferase reporter gene activity in TNF-inducible minimal VCAM-1 promoter constructs containing two NF-kappa B sites, suggesting that the compound does not affect the transactivation driven by these sites. We conclude that this compound selectively blocks agonist-induced VCAM-1 protein and gene expression in HAECs by NF-kappa B-independent mechanism(s).

Cells, Cultured↗
PubMed1995

Identification of a novel 85-kDa lipoprotein lipase binding protein on human aortic endothelial cell surface.

Lipoprotein lipase (LPL), bound to the luminal surface of vascular endothelium catalyzes lipoprotein triglyceride hydrolysis. Studies were performed to identify human aortic endothelial (HAEC) cell-surface proteins having high affinity for LPL. LPL-sepharose affinity chromatography of [35S]O4 labeled HAEC proteins identified a 220-kDa proteoglycan. Ligand blotting of HAEC plasma membrane proteins with LPL revealed two specific binding proteins of MW 116 kDa and 85 kDa, respectively, which were not released from the cell-surface by heparin treatment. Since the 220-kDa and 116-kDa proteins have been reported previously in bovine endothelial cells, we focused on the 85-kDa protein. The 85-kDa protein was not labelled by incubation of the cells with [35S]O4, suggesting that it is not a sulfated proteoglycan. Treatment of HAEC with tunicamycin markedly decreased detection of the 85-kDa protein, suggesting that it is likely a glycoprotein synthesized by HAEC. We conclude that HAEC cell surface has three specific LPL binding proteins, a 220-kDa proteoglycan, a 116-kDa protein and a novel 85-kDa protein.

Aorta↗
PubMed1995

Inhibition of tumor necrosis factor induced human aortic endothelial cell adhesion molecule gene expression by an alkoxybenzo[b]thiophene-2-carboxamide.

The effects of a novel anti-inflammatory agent, 5-methoxy-3-(1-methyl-ethoxy)benzo[b]thiophene-2-carboxamide-1-oxide (PD 144795) on adhesion molecule expression in tumor necrosis factor (TNF) stimulated human aortic endothelial cells (HAEC) were examined. PD 144795 treatment markedly inhibited the TNF-induced cell expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein and mRNA. Gel shift assays using nuclear extracts from HAEC treated with PD 144795 failed to show a decrease in the activation of NFkB by this compound, whereas pyrrolidine dithiocarbamate (PDTC), an antioxidant, markedly inhibited the activation of this transcription factor. Thus, PD 144795 inhibits agonist-stimulated VCAM-1 and ICAM-1 expression likely via an NFkB independent mechanism, distinct from that of PDTC. Such agents may provide a novel approach for control of adhesion molecule gene expression in inflammation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1995

Phosphonates and phosphinates: novel leaving groups for benzisothiazolone inhibitors of human leukocyte elastase.

A novel class of alkyl and aryl phosphonate and phosphinate acid-based leaving groups has been developed for utilization in the synthesis of benzoisothiazolone (BIT) inhibitors of human leukocyte elastase (HLE). A number of BITs were synthesized with phosphonate and phosphinate acid-based leaving groups and were found to be potent inhibitors of HLE. Compound 3c with a diethyl phosphonate leaving group is the most potent inhibitor synthesized in this series with Ki* = 0.035 nM and ED50 = 2.0 mg/kg.

Humans↗
PubMed1995

Acyl-coenzyme A:cholesterol-acyltransferase (ACAT) inhibitors modulate monocyte adhesion to aortic endothelial cells.

Increased monocyte adhesion to aortic endothelium is observed in the pathogenesis of atherosclerosis. The role of endothelial acyl-coenzyme A:cholesterol-acyltransferase (ACAT) in the regulation of monocyte adhesion is not known. To examine the potential role of this enzyme in monocyte adhesion, a specific ACAT inhibitor, CI-976, was utilized. Although the basal adhesion of U937 monocytic cells to porcine aortic endothelial cells was low, treatment of the endothelial cells with lipopolysaccharide (LPS) markedly increased monocyte adhesion. Monocyte adhesion to LPS-treated endothelial cells was markedly inhibited by CI-976 treatment of the endothelial cells. Similarly, another ACAT inhibitor, PD 132301-2, whose structure is distinct from CI-976, also decreased monocyte adhesion. CI-976 treatment of endothelial cells also decreased endothelial cell ACAT activity. Since leukotriene B4 (LTB4) is known to promote leukocyte-endothelial cell adhesion, endothelial cell production of this leukotriene was examined after incubation with CI-976. CI-976 treatment markedly decreased LTB4 synthesis. Exogenous LTB4 addition to CI-976 treated cells reversed the effects of this compound on monocyte adhesion. These data demonstrate that ACAT inhibitors decrease monocyte adhesion to endothelial cells. Similar mechanisms may contribute to antiatherosclerotic effects of ACAT inhibitors in vivo.

Anilides↗
PubMed1995

Canine atopic disease: the prevalence of positive intradermal skin tests at two sites in the north and south of Great Britain.

Results of intradermal skin test responses to the same panel of 53 allergens were compared in 118 dogs with atopic disease presented at two geographical centres, Edinburgh (87 cases) and London (31 cases). The allergens most commonly positive at both centres were human dander and Dermatophagoides farinae, but positive tests to all of the allergens used occurred in at least one case. The mean number of allergens to which positive tests resulted in atopic dogs was 5.126 (Edinburgh) and 5.129 (London). The majority of animals were sensitive to allergens from more than one group. A significantly higher number of positive reactors to house dust allergen was observed at London than at Edinburgh (P < 0.05), while a significantly higher number of positive reactions to grass pollens was detected at Edinburgh than in London (P < 0.05). Sensitivity to Dermatophagoides pteronyssinus, in the absence of sensitivity to D. farinae, was uncommon and therefore both of these mite allergens should be incorporated in intradermal skin testing panels in Great Britain.

Animals↗
PubMed1995

A measure of knowledge and confidence in relation to HIV and AIDS: reliability and validity.

Knowledge of HIV and AIDS is widely seen as an important determinant of anxiety about such infection in health care workers. However, existing measures of this knowledge suffer from a number of methodological problems and few demonstrate adequate reliability and validity. This paper documents the development of a new measure detailing its reliability and validity. Knowledge, and also confidence in knowledge, were assessed across seven domains of relevant information (e.g. epidemiology, personal risk, symptomatology) in one non-expert group (non-medical undergraduate psychologist) and in three groups with greater expertise (nursing students and third year and final year medical students). The measure was shown to be reliable and to discriminate between the four groups in a manner consistent with the level of education received by each group and in terms of both the scale scores for the seven domains and the individual items. In general, final year medical students were shown to be more knowledgeable, and more confident in their knowledge, than the other groups. The non-medical undergraduates showed the lowest level of knowledge. In general, the groups appeared under-confident in their knowledge about HIV/AIDS. This was interpreted in terms of members of these groups exhibiting caution and the perceived fluctuating nature of the HIV knowledge base. Implications for the training of health care workers in relation to HIV/AIDS are discussed.

Adult↗
PubMed1995

Apolipoprotein B and E basic amino acid clusters influence low-density lipoprotein association with lipoprotein lipase anchored to the subendothelial matrix.

Lipoprotein accumulation in the subendothelial matrix is an important step in atherogenesis. We have previously shown that addition of lipoprotein lipase (LPL) markedly increased binding of apolipoprotein B (apoB)-containing lipoproteins to an endothelial cell-derived matrix, and this enhanced lipoprotein binding was inhibited by apoE. In the present studies we examined the role of various regions of apoB in the binding of LDL to LPL-containing endothelial cell matrix and the ability of various apoE domains to decrease lipoprotein retention. We studied three apoB epitope-specific monoclonal antibodies for their ability to block the binding of 125I-LDL to LPL-containing matrix. Of these, monoclonal antibody 4G3, which recognizes an arginine-containing epitope in apoB, was the most effective in reducing LDL binding. Chemical modification of LDL apoB lysines or arginines markedly reduced the ability of the lipoprotein to block the binding of 125I-LDL to LPL-containing matrix, suggesting that apoB positively charged amino acids are involved in the interaction. Furthermore, polyarginine or polylysine markedly decreased 125I-LDL binding to LPL-containing matrix, whereas polyleucine was ineffective. These data suggest that apoB positively charged regions are important in LDL binding. To explore the role of charge modifications on apoE by single arginine-cysteine interchanges, we examined the effects of the three major human apoE isoforms (apoE2, apoE3, and apoE4). ApoE3 was the most effective in decreasing 125I-LDL retention, followed by apoE4; apoE2 was the least effective. Similarly, apoE2-containing HDL was much less effective than apoE3-containing HDL in decreasing 125I-LDL retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1994

Susceptibility of human LDL to oxidative modification. Effects of variations in beta-carotene concentration and oxygen tension.

Epidemiological studies suggest that beta-carotene supplementation may decrease atherosclerotic events. Because beta-carotene is transported in low-density lipoprotein (LDL), one mechanism by which this protective effect may occur is through direct inhibition of LDL oxidation. Addition of beta-carotene to LDL in vitro inhibits the susceptibility of LDL to oxidation. In contrast, we have shown that feeding large doses of beta-carotene results in beta-carotene-enriched plasma LDL (16- to 35-fold), but such LDL does not show increased resistance to oxidation. Potential criticisms of our initial study relate to the unique antioxidant properties of beta-carotene. Beta-Carotene provides better quenching of some free radicals, such as singlet oxygen, than others. Additionally, since beta-carotene can easily autooxidize, forming metabolites that can also generate free radicals, it has been argued that at sufficient concentrations in vivo the antioxidant effect of beta-carotene may be diminished. It has thus been suggested that in our initial study we may have failed to properly assess the unique antioxidant effects of beta-carotene by our selection of oxidizing conditions and/or failed to achieve antioxidant activity because of excessive enrichment of LDL with beta-carotene. We now report the effects of feeding lower doses of beta-carotene on the susceptibility of LDL to oxidation and test whether any antioxidant activity of beta-carotene can be detected when different modes of initiating oxidation are used. Because the antioxidant activity of beta-carotene is reputedly most pronounced in low-oxygen environments, we also investigated its effect on LDL oxidation under conditions of reduced oxygen tension.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗