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Biomedical subjects

R J Edwards

Publications and source records attributed to R J Edwards.

At least 127 records · Page 7Linked to original sources
PubMed1990

Thromboxane release by lymphokine-differentiated U937 human monocytic cells: response to platelet-activating factor (PAF) and chemotactic peptide (FMLP) but not to low affinity IGE-receptor (Fc epsilon RII/CD23) occupation.

The primary objective of this study was to explore if the CD23 antigen is a functional low affinity IgE receptor on macrophages for the release of thromboxane B2 (TXB2). The responsiveness of U937 monocytic cells and their macrophage-like inducible forms to platelet-activating factor (Paf), the chemotactic peptide fMLP, and low affinity IgE-receptor occupation was examined. Differentiation of U937 cells by phorbol myristate acetate (PMA) and a cancer cell line (HBT 5637) conditioned medium (5637-CM), but not INFg or IL4, resulted in a macrophage-like cell line which released TXB2. A high basal release of TXB2 with no significant response to Paf or fMLP challenge was seen following culture of cells with PMA. In 5637-CM-differentiated cells, Paf and fMLP induced a rapid release of TXB2, about 10 fold above basal activity. There was a slow Ca-independent response to short-term treatment with PMA and a rapid Ca-dependent response to the ionophore A23187. Both stimulants acted synergistically on TXB2 synthesis in 5637-CM differentiated cells. Although low affinity receptors for IgE (Fc epsilon RII/CD23) were induced by 5637-CM, no TXB2 was released in response to soluble or latex-bound IgE-antigen complexes or to anti-Fc epsilon RII/CD23-antibodies. IL4 and to a lesser extent INFg both induced Fc epsilon RII/CD23 receptor expression, but inhibited release of TXB2 in response to Paf, fMLP, or PMA. We conclude that the functional receptors for IgE on mature macrophages are most probably not Fc epsilon RII/CD23.

Antibodies, Anti-Idiotypic↗
PubMed1990

Species variation in the response of the cytochrome P-450-dependent monooxygenase system to inducers and inhibitors.

1. In the safety evaluation of drugs and other chemicals it is important to evaluate their possible inducing and inhibitory effects on the enzymes of drug metabolism. 2. While many similarities exist between species in their response to inducers and inhibitors, there are also important differences. Possible mechanisms of such variation are considered, with particular reference to the cytochrome P-450 system. 3. Differences in inhibition may be due to differences in inhibitory site of the enzyme involved, which is not always the active site of the enzyme, in competing pathways or in the pharmacokinetics of the inhibitor. 4. Differences in induction could be due to differences in the nature of the induction mechanism, in the isoenzyme induced, in tissue- or age-dependent regulation, in competing pathways for the substrate or its products, or in the pharmacokinetics of the inducing agent. 5. Examples of each of these possible differences are considered, often from our own work on the P450 IA subfamily, and results in animals are compared with those in humans, where possible. 6. At present, the differences between species in their response to inducers and inhibitors make extrapolation to humans from the results of animal studies difficult, so that ultimately such effects should be studied in the species of interest, humans.

Allosteric Regulation↗
PubMed1989

Identification and location of alpha-helices in mammalian cytochromes P450.

A model of the alpha-helical structure of mammalian cytochromes P450 is proposed. The location and sequence of alpha-helices in mammalian cytochromes P450 were predicted from their homology with those of cytochrome P450cam, and these sequences were generally confirmed as helical in nature by using a secondary structure prediction method. These analyses were applied to 26 sequences in 6 gene families of cytochrome P450. Mammalian cytochromes P450 consist of approximately 100 amino acid residues more than cytochrome P450cam. This difference was accounted for by three major areas of insertion: (1) at the N-terminus, (2) between helices C and D and between helices D and E, and (3) between helices J and K. Insertion 1 has been suggested by others as a membrane anchoring sequence, but the apparent insertions at 2 and 3 are novel observations; it is suggested that they may be involved in the binding of cytochrome P450 reductase. Only the mitochondrial cytochrome P450 family appeared to show a major variation from this pattern, as insertion 2 was absent, replaced by an insertion between helices G and H and between helices H and I. This may reflect the difference in electron donor proteins that bind to members of this cytochrome P450 family. Other than these differences the model of mammalian cytochromes P450 proposed maintains the general structure of cytochrome P450cam as determined by its alpha-helical composition.

Amino Acid Sequence↗
PubMed1989

Cross-reaction of antibodies to coupling groups used in the production of anti-peptide antibodies.

A polyclonal antibody raised against a peptide conjugated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to maleic anhydride-derivatised lysozyme showed substantial cross-reactivity with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised haemocyanin. This was due to antibodies produced against maleic anhydride-derivatised groups on lysozyme that reacted with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised groups on haemocyanin. This observation is important because it is common practice, in the production of anti-peptide antibodies, to use two conjugates. The same peptide is coupled to two different protein carriers by two different coupling methods. One conjugate is used for immunisation and the other for testing the serum. This method assumes that the only antigen common to the two conjugates is the peptide and this was not the case here. A method is described for screening sera which involves affinity purification of the anti-peptide antibody and comparison of binding to the immunogen with that to an appropriate control conjugate. This method avoids the problem of any cross-reaction to coupling groups or proteins.

Adsorption↗
PubMed1989

The Z-band lattice in skeletal muscle in rigor.

Previous work with tetanized and relaxed muscle has shown a correlation between active tension and the structure of the Z-band. This suggests that there is a correlation between the cross-bridge binding in the A-band and the structure of the Z-band. Using electron microscopy and optical diffraction we have examined this correlation in glycerinated muscle in rigor and in unstimulated intact muscle. We have found that the Z-bands of muscles in rigor always show the basketweave form, while those of the unstimulated muscles always show the small square form. The basketweave form found in rigor muscles is similar in form and dimension to that found in tetanized muscle. Thus it appears that the small square form of the Z-band is found in physiological states with little cross-bridge binding and the basketweave form is found in states with a high degree of cross-bridge binding.

Animals↗
PubMed1989

Immunosuppression by immunoglobulin deaggregation is not effective in reducing the anti-xenogeneic immunoglobulin response: experimental and clinical studies.

A major complication of in vivo monoclonal antibody therapy in patients with cancer is the host's immune response to the administered xenogeneic immunoglobulin. We have performed parallel clinical and experimental studies to investigate the possibility that deaggregation of the therapeutic monoclonal antibody might render it non-immunogenic, or even tolerogenic, as has been suggested in several animal studies. Deaggregation of xenogeneic immunoglobulin has been shown by others to induce non-responsiveness in some ('susceptible') but not in other ('resistant') strains of mice. We have used an improved deaggregation method of size exclusion chromatography connected to FPLC and have developed a sensitive ELISA detection system to determine whether highly purified human immunoglobulin G (hIgG) monomers could be tolerogenic even to 'resistant' mice. However, our data show that all preparations of hIgG are immunogenic to 'resistant' mice, and that although deaggregation does significantly reduce the anti-hIgG response to 'susceptible' strains, tolerance is not induced. Concomitant administration of cyclosporin A and deaggregated hIgG had a additive effect in reducing the murine anti-hIgG secondary response. In clinical studies of patients with ovarian cancer who received in vivo immunotherapy with either iodine-131 (not aggregated) or yttrium-90 (aggregated) HMFG1 mouse monoclonal antibody, no significant difference was found between the immune responses to aggregated and non-aggregated murine immunoglobulin G. Our data suggest that deaggregation alone is unlikely to be useful in controlling the human anti-murine immunoglobulin G response in our outbred patient population, although in combination with an immunosuppressant it may be more effective.

Animals↗
PubMed1989

Selective destruction of cytochrome P-450d and associated monooxygenase activity by carbon tetrachloride in the rat.

1. The effects of acute treatment of 3-methylcholanthrene (MC)-induced rats with carbon tetrachloride (CCl4) on the content and activity of the polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 (P-450c and P-450d) in liver and kidney have been determined. 2. Post-treatment of MC-induced rats with CCl4 in vivo decreased the specific content of total, spectrally determined, P-450 in both hepatic and renal microsomes, by 60% and 40%, respectively. CCl4 treatment destroyed almost all of the hepatic P-450d (specific content after 6 h, less than 2% of control), but had no effect on P-450c, which increased slightly over the 6 h, to 30% above control values. 3. Immunocytochemical measurements demonstrated greater loss of P-450d from the centrilobular and midzonal than from periportal regions of the liver. 4. Hepatic phenacetin O-deethylase, an activity catalysed specifically by P-450d in this tissue, was dramatically decreased following administration of CCl4 to MC-induced rats. Loss of monooxygenase activity was highly correlated with the decrease in P-450d content (r = 0.947, P less 0.001). Aryl hydrocarbon hydroxylase activity of liver, catalysed almost entirely by P-450c, was unchanged and neither activity was affected in kidney. 5. Treatment of MC-induced rats with CCl4 causes a selective loss of hepatic P-450d and associated monooxygenase activities. Phenacetin O-deethylation is catalysed specifically by P-450d in liver, but not in kidney. The mechanism for this destruction of P-450d may be suicide activation of CCl4, but the rate of such activation appears to be much lower than with P-450b. Alternatively, P-450d may be particularly sensitive, and P-450c particularly resistant, to the active metabolite of CCl4 diffusing from a distant site of formation.

Animals↗
PubMed1989

Descriptive analysis of medical attrition in U.S. Army aviation.

Although U.S. Army aviators are carefully screened at entry, disease develops in the aviator population with time. Improving the ability to predict and prevent such diseases necessitates proper analysis of their incidence. This information can provide the basis for future improvements in screening and prevention. A descriptive analysis of diseases for the U.S. Army aviation population is presented. The frequency of international Classification of Diseases (ICD) codes leading to disqualification from flying status is summarized and discussed. Suggestions for future screening criteria and for intervention practices are proposed.

Adolescent↗
PubMed1988

Antibodies to a synthetic peptide that react specifically with a common surface region on two hydrocarbon-inducible isoenzymes of cytochrome P-450 in the rat.

An antibody that reacts with two hydrocarbon-inducible isoenzymes of rat cytochrome P-450 (c and d) in the rat was produced by immunising with a synthetic peptide, Leu-Ile-Ser-Lys-Phe-Gln-Lys-Leu-Met, which has the same primary structure as that of a region of both of these isoenzymes. There was no crossreactivity with hydrocarbon-inducible isoenzymes in liver microsomes from rabbit, mouse or in man. Nor was there any crossreactivity detected with liver microsomes from uninduced rats, or rats induced with phenobarbitone or isonicotinic acid hydrazide. This is consistent with the primary structure of these isoenzymes in the regions aligned with amino acids 174-182 (the immunising peptide) in rat isoenzyme c and demonstrates the ability to produce antibodies of defined specificity against isoenzymes of cytochrome P-450 by using synthetic peptide. As the antibody preparation is able to bind to isoenzymes c and d in their native conformations, either as partially purified enzymes, or in microsomes, it is suggested that this region is present on the surface of these cytochromes P-450.

Animals↗
PubMed1988

A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O-deethylation of phenacetin and is inducible by cigarette smoking.

1. In previous studies (Boobis et al., 1985b) it was shown that a monoclonal antibody (MAb 3/4/2), raised against rat cytochrome P450 form c, reacts with an isoenzyme(s) of cytochrome P450 in human liver. It was predicted that the epitope with which this antibody reacts should be present on both isoenzymes of the P450IA gene sub-family (the orthologues of forms c and d) in man (Edwards et al., 1987). 2. This antibody was used to probe 45 different samples of human liver, by the technique of Western blotting. With one exception, all of the samples contained immunoreactive protein, a single band at Mr 54,000 (orthologous to rat form d), which ranged in content from less than 0.5 to 33.5 pmol mg-1 microsomal protein. The content of the human orthologue of form c was below 0.5 pmol mg-1, the limit of detection of the assay. 3. Thirteen of the samples were from patients of known smoking status. Immunoreactive P450 content was 3.5-fold higher, and phenacetin O-deethylase activity was four-fold higher, in the smokers than in the non-smokers. 4. There was a highly significant correlation between the amount of immunoreactive cytochrome P450 and the high affinity component of phenacetin O-deethylase activity in both smokers and non-smokers. 5. It is concluded that the high affinity component of phenacetin O-deethylase activity in man is catalysed by the orthologue of rat cytochrome P450d, and that this isoenzyme is inducible by cigarette smoking. 6. In a number of previous publications it has been suggested that there is an association between the poor metaboliser (PM) phenotype for debrisoquine and impaired phenacetin O-deethylation. In the present study it was shown that not all subjects PM for debrisoquine are poor metabolisers of phenacetin.

Animals↗
PubMed1987

Development of carboxypeptidase K (creatine kinase conversion factor) in man.

The developmental appearance of carboxypeptidase K in human plasma was determined by quantifying the creatine kinase (CK) isoforms present and by measuring the conversion of tetrathionate-blocked CK in vitro. Analysis of plasmas from second and third trimester fetuses, newborns and adults showed that isoforms were absent from second trimester fetuses, were present in similar amounts in third trimester fetuses and newborns, and had increased to about twice this amount in adults. Carboxypeptidase K activity was correspondingly absent from second trimester fetuses but present in the other three groups in statistically indistinguishable amounts. Carboxypeptidase N was present in all groups although activity was low in second trimester fetuses. Addition of cobalt ions activated both carboxypeptidases K and N in all samples and unmasked carboxypeptidase K in second trimester samples.

Adult↗
PubMed1987

Prostacyclin analogues reduce ADP-ribosylation of the alpha-subunit of the regulatory Gs-protein and diminish adenosine (A2) responsiveness of platelets.

Prostacyclin and adenosine activate adenylate cyclase in human platelet membranes and inhibit platelet aggregation. Results are presented which show that prolonged incubation of platelets with iloprost (a stable prostacyclin analogue) results in a reduction in the capacity for adenylate cyclase activation by the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA), NaF, guanyl-5'-yl imidodiphosphate or GTP. However, iloprost pretreatment resulted in no change in the binding of [3H]-NECA to platelet membranes. These results contrast with those obtained after pretreatment with 2-chloroadenosine which revealed no change in NaF or guanyl-5'-yl imidodiphosphate sensitivity of adenylate cyclase. Pretreatment with 2-chloroadenosine resulted in reduced NECA-dependent adenylate cyclase activation, and loss of [3H]-NECA binding sites. The heterologous desensitization of adenosine A2-receptors by iloprost is accompanied by a loss (greater than 80%) of a 45 kDa protein from the plasma membrane, as revealed by [32P]-ADP-ribosylation in the presence of cholera toxin. It is proposed that this example of heterologous desensitization is mediated by elimination of Gs alpha, a subunit of the stimulatory guanyl nucleotide-binding regulatory protein.

2-Chloroadenosine↗
PubMed1985

Fetal plasma carbonic anhydrase III in prenatal diagnosis of Duchenne muscular dystrophy.

Carbonic anhydrase III (CAIII), a skeletal-muscle-specific enzyme which is elevated in the plasma of Duchenne muscular dystrophy (DMD) patients, was measured by radioimmunoassay in fetal plasma in order to evaluate its application to prenatal diagnosis of DMD. Using fetoscopy, pure fetal blood samples were taken at 17-24 weeks gestation from 25 fetuses at risk for DMD and from 78 control fetuses. Care was taken in the handling and storage of all samples. Normal sons were born in eight cases at risk for DMD. The CAIII levels in the infants were not significantly different from those of the control infants. Pregnancies were terminated in the remaining 17 at-risk cases. The CAIII levels in the fetuses were significantly different (p = 0.0034) from those of the control fetuses, although the distributions overlapped. Based on prior maternal risk, seven affected fetuses were expected in the terminated group; five had CAIII levels at or above the 95th centile of the control range. It is suggested that measurement of CAIII achieves partial discrimination between affected fetuses and their normal at-risk brethren.

Carbonic Anhydrases↗