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R J Edwards

Publications and source records attributed to R J Edwards.

At least 37 records · Page 2Linked to original sources
PubMed2001

Cytochrome P450 3A expression in the human fetal liver: evidence that CYP3A5 is expressed in only a limited number of fetal livers.

CYP3A is the major cytochrome P450 subfamily constitutively expressed in the human liver. CYP3A4 is the predominant hepatic P450 form in adults and it is expressed at high but very variable levels among individuals. The fetal liver contains mainly CYP3A7, while the presence of the other CYP3A enzymes in fetal liver has remained controversial. In this study, the relative levels of CYP3A4, CYP3A5 and CYP3A7 expression were determined in a panel of 9-11 fetal livers with a similar gestation age (9-12 weeks) and compared to adult livers. CYP3A7 was found to be the major CYP3A form in all the fetal liver samples. The abundance of CYP3A7 varied more at the mRNA (77-fold variation) than at the protein level (4.8-fold variation). CYP3A5 mRNA was also detected in all of the fetal liver samples, but the average level was 700-fold lower than that of CYP3A7. CYP3A5 protein was detected by immunoblot analysis in only 1 fetal liver out of the 9 investigated, the level of expression being moderately high in this sample. CYP3A4 mRNA was detected in only a subset of the fetal liver samples and its level was the lowest of the CYP3A forms. This is the first study to demonstrate the polymorphic expression of CYP3A5 and the variability of CYP3A7 expression in fetal liver and suggests that significant interindividual differences in the metabolism of xenobiotics may already exist at the prenatal stage. These differences may contribute to individual pharmacological and/or toxicological responses in the fetus.

Aryl Hydrocarbon Hydroxylases↗
PubMed2001

Differential induction of rat hepatic cytochromes P450 3A1, 3A2, 2B1, 2B2, and 2E1 in response to pyridine treatment.

Pyridine (PY) effects on rat hepatic cytochromes P450 (CYP) 3A1 and 3A2 expression were examined at the levels of metabolic activity, protein, and mRNA and were compared with those of CYP2B1/2 and CYP2E1. CYP3A metabolic activity as well as CYP3A protein and mRNA levels increased following treatment of rats with PY. CYP3A1 and CYP3A2 were differentially affected by PY treatment in terms of induction levels, dose dependence, and stability of mRNA. CYP3A1 mRNA levels maximally increased ~42-fold after PY treatment, whereas CYP3A2 mRNA level increased ~4-fold. Moreover, CYP3A1 mRNA levels decreased more rapidly than those of CYP3A2 as determined following inhibition of transcription with actinomycin D or cordycepin. Treatment of rats with PY resulted in a dose-dependent increase in CYP3A1, CYP3A2, and CYP2B1/2B2 protein levels. In contrast to the effects of PY treatment on CYP3A1 and 2B, CYP2E1 protein levels increased in the absence of a concomitant increase in CYP2E1 mRNA levels. Treatment of rats with PY at 200 mg/kg/day for 3 days increased both protein and mRNA levels of CYP3A2, whereas treatment with higher than 200 mg/kg/day for 3 days increased CYP3A2 protein levels without an increase in CYP3A2 mRNA levels. These data demonstrated that PY regulates the various CYPs examined in this study at different levels of expression and that PY regulates CYP3A1 expression through transcriptional activation and CYP3A2 expression through transcriptional and post-transcriptional activation at a low- and high-dose PY treatment, respectively.

Animals↗
PubMed2001

An assessment of human liver-derived in vitro systems to predict the in vivo metabolism and clearance of almokalant.

The ability of various human derived in vitro systems to predict various aspects of the in vivo metabolism and kinetics of almokalant have been investigated in a multicenter collaborative study. Although almokalant has been withdrawn from further clinical development, its metabolic and pharmacokinetic properties have been well characterized. Studies with precision-cut liver slices, primary hepatocyte cultures, and hepatic microsomal fractions fortified with UDP-glucuronic acid all suggested that almokalant is mainly glucuronidated to the stereoisomers M18a and M18b, which is in good agreement with the results in vivo. Both in vivo and in vitro studies indicate that the formation of M18b dominates over that of M18a, although the difference is more pronounced with the in vitro systems. Molecular modeling, cDNA-expressed enzyme analysis, correlation analysis, and inhibition studies did not clearly indicate which P450 enzymes catalyze the oxidative pathways, which may indicate a problem in identifying responsible enzymes for minor metabolic routes by in vitro methods. All of the in vitro systems underpredicted the metabolic clearance of almokalant, which has previously been reported to be a general problem for drugs that are cleared by P450-dependent metabolism. Although few studies on in vivo prediction of primarily glucuronidated drugs have appeared, in vitro models may consistently underpredict in vivo metabolic clearance. We conclude that in vitro systems, which monitor phase II metabolism, would be beneficial for prediction of the in vivo metabolism, although all of the candidate liver-derived systems studied here, within their intrinsic limitations, provided useful information for predicting metabolic routes and rates.

Anti-Arrhythmia Agents↗
PubMed2001

Endoscopic methods of hydrocephalus treatment.

This article discusses the use of the endoscope in treatment of hydrocephalus without shunting. The selection of candidates for third ventriculostomy and choroid plexus coagulation are described together with the overall published rates of success with each technique. A summary of the nature and frequency of complications of third ventriculostomy are given after reviewing published data on over a thousand patients who have had the operation to date. The role of other endoscopic techniques, such as septal fenestration and decompression of obstructing cysts, also are discussed.

Adolescent↗
PubMed2000

Expression and distribution of CYP2C enzymes in rat basal ganglia.

The function and integrity of the basal ganglia is modulated by sex steroids whose activity may be controlled by P450 enzymes, such as members of the CYP2C subfamily. The expression of CYP2C enzymes in rat basal ganglia was examined by immunohistochemistry along with some of the factors that might control their expression. Whereas no CYP2C11 or CYP2C12 immunoreactivity was detected in the basal ganglia of either male or female rats, marked CYP2C13 immunoreactivity was evident in neurones of the subthalamic nucleus, substantia nigra, and interpeduncular nucleus. Strong CYP2C13 immunoreactivity was also expressed in the cortex, olfactory tubercle, hippocampus, dentate gyrus, hypothalamic nuclei, medial habenular nucleus, red nucleus, and medial forebrain bundle. Similar results were found in male and female rats. Following 6-hydroxydopamine lesioning of the nigro-striatal tract, tyrosine hydroxylase immunoreactivity was absent and CYP2C13 immunoreactivity was decreased markedly in the substantia nigra pars compacta, implying its presence in dopaminergic neurones. Modulation of sex steroids, using castrated rats, had no effect on the number of CYP2C13 positive neurones in the substantia nigra pars compacta. These results indicate that CYP2C13 protein is constitutively and widely expressed in rat brain. However, its expression is not sex-specific and is unaffected by castration. The role of CYP2C13 in brain is unknown but it may be involved in the generation of neurosteroids and catecholoestrogens.

Animals↗
PubMed2000

Expression of CYP1A1, CYP1B1 and CYP3A, and polycyclic aromatic hydrocarbon-DNA adduct formation in bronchoalveolar macrophages of smokers and non-smokers.

Variability in the expression of enzymes metabolizing carcinogens derived from cigarette smoke may contribute to individual susceptibility to pulmonary carcinogenesis. This study was designed to determine the effects of smoking and 3 major cytochrome P450 (CYP) enzymes, i.e., CYP1A1, CYP1B1 and CYP3A, which metabolize polycyclic aromatic hydrocarbons (PAH) on PAH-DNA adduct formation in the bronchoalveolar macrophages (BAM) of 31 smokers and 16 non-smokers. CYP protein levels were determined by immunoblotting and PAH-DNA adduct levels by the nuclease P1 enhanced (32)P-postlabeling method. The expression of specific CYP forms was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) from 10 additional samples. CYP3A protein, CYP3A5 by RT-PCR, was detected in the majority of samples from smokers and non-smokers. The levels of CYP3A appeared to be lower in active smokers than in ex-smokers (p = 0.10) or never smokers (p = 0.02). CYP1A1 was not detectable by either immunoblotting or RT-PCR. The expression of CYP1B1 was low or undetectable in most samples. The PAH-DNA adduct levels were higher (mean 1.57/10(8) nucleotides) in samples from smokers compared with non-smokers (mean 0.42/10(8) nucleotides, p < 0.001) and the number of adducts correlated with the number of cigarettes smoked daily (regression analysis, p < 0. 001). Higher levels of adducts were detected in samples from smokers with a high level of CYP3A compared with those with a low level (regression analysis, p = 0.002). As CYP3A5 is abundant in both lung epithelial cells and BAM, its association with adduct formation suggests that this CYP form may be important in the activation of cigarette smoke procarcinogens.

Adult↗
PubMed2000

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey.

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Animals↗
PubMed2000

The potential of acetaminophen as a prodrug in gene-directed enzyme prodrug therapy.

Acetaminophen is oxidized by human CYP1A2 to the cytotoxic metabolite N-acetylbenzoquinoneimine (NABQI). Incubation of cells transfected with human CYP1A2 (H1A2 MZ cells) with 4-20 mM acetaminophen for 6 hours at 37 degrees C caused extensive cytotoxicity (cell viability <10%). In contrast, nontransfected V79 MZ cells were unaffected (viability >95%). By mixing H1A2 MZ cells with V79 MZ cells in various proportions and incubating with 4 mM acetaminophen, it was shown that the NABQI released from H1A2 MZ cells also caused cytotoxicity of bystander cells. Thus, in a mixture containing 5% H1A2 MZ cells, exposure to 4 mM acetaminophen for 6 hours resulted in complete cell killing by 24 hours. A similar bystander effect was found by incubating the same proportion of CYP1A2-containing cells with ovarian tumor-derived SK-OV-3 cells or colon tumor-derived HCT116 cells. However, breast tumor-derived MDA-MB-361 cells displayed resistance to the cytotoxic effect of NABQI, and it was necessary to increase the proportion of H1A2 MZ cells to 50% to achieve complete cell killing. In conclusion, the use of acetaminophen as prodrug and CYP1A2 as an activating enzyme is a promising combination for gene-directed enzyme prodrug therapy.

Acetaminophen↗
PubMed2000

Management of tuberculomas of the craniovertebral junction.

Tuberculosis of the craniovertebral junction is extremely rare. However, recent evidence suggests that the incidence of this condition may be increasing in the United Kingdom. The diagnosis is often difficult despite advances in imaging using MRI. CT guided biopsy of lesions often yields inconclusive results. The transoral approach to the anterior craniovertebral junction provides excellent access to this region with a low operative morbidity and mortality, enabling biopsy of lesions and decompression of the neuraxis. Management of secondary atlantoaxial instability, regarding both timing and method of stabilization, is controversial. We report two cases of tuberculomas of the craniovertebral junction, that illustrate the role of transoral surgery in both diagnosis and treatment of this condition. Previous management strategies are reviewed and future recommendations are presented.

Adult↗
PubMed2000

Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes.

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes "preconditioned" with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N(G)-monomethyl-L-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K(+) channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.

Alkaloids↗
PubMed2000

Affinity and potency of proinhibitory antipeptide antibodies against CYP2D6 is enhanced using cyclic peptides as immunogens.

A series of antipeptide antibodies directed against CYP2D6 were produced by immunizing rabbits with peptides that were sterically unrestrained (linear) or conformationally restricted by cyclization. A variety of sites within the region comprising residues 254 to 290 of CYP2D6 were targeted. In immunoblotting studies, each of the antibodies against the linear and cyclic peptides recognized only a single immunoreactive band of 54 kDa in human liver microsomal fraction and bound to recombinant CYP2D6, but not recombinant CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, or CYP3A4. However, the relative intensity of immunoreactive bands was considerably stronger for those antibodies raised against cyclic peptides. Similarly, in an enzyme-linked immunosorbent assay, antibodies raised against cyclic peptides bound 10 to 100 times more strongly to recombinant CYP2D6 than antibodies raised against the corresponding linear peptides. None of the antibodies raised against linear peptides had any effect on debrisoquine 4-hydroxylase activity of human hepatic microsomal fraction; however, anticyclic peptide antibodies targeted against residues 254 to 273, 261 to 272, and 257 to 268 of CYP2D6 inhibited enzyme activity by a maximum of 60, 75, and 91%, respectively. In contrast, despite binding strongly to CYP2D6, an anticyclic peptide antibody directed against residues 278 to 290 did not inhibit enzyme activity. The epitope of the proinhibitory anticyclic peptide antibody directed against residues 257 to 268 of CYP2D6 included Thr-261 and Trp-262, and indicates a role for these residues in enzyme inhibition. In conclusion, immunization with peptides conformationally restricted by cyclization to mimic loop regions of CYP2D6 resulted in strongly binding antibodies that when targeted appropriately were able to inhibit CYP2D6-catalyzed activity.

Amino Acid Sequence↗
PubMed2000

Differential maintenance of cytochrome P450 enzymes in cultured precision-cut human liver slices.

The maintenance of the major hepatic cytochrome P450 (CYP) enzymes has been studied in precision-cut human liver slices cultured for up to 72 h in supplemented RPMI 1640 medium. The relative apoprotein levels of 11 CYP enzymes were determined using a panel of antipeptide antibodies. In addition, 7-ethoxyresorufin O-deethylase, tolbutamide methylhydroxylase, debrisoquine 4-hydroxylase, and testosterone 6beta-hydroxylase activities were determined as enzymatic markers for CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. There was a large variation in the rate of decline of different CYP levels with time in culture. Based on the rate of decrease, CYP enzymes could be separated into two groups, with CYP2C9, CYP2D6, CYP3A4, and CYP4A11 being relatively stable (half-lives between 70 and 104 h), compared with CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, and CYP3A5, which were relatively unstable (half-lives between 23 and 36 h). Enzyme activities decreased at rates similar to those of their corresponding apoproteins. There was also a large difference in the stability of individual CYP enzymes from different liver donors, particularly for the most rapidly declining CYP enzymes. Similar losses of CYP enzymes were found when human liver slices were cultured in supplemented Williams' medium E for 72 h, except that CYP2E1 apoprotein levels were better maintained. Because of the variable decreases of CYP enzymes, xenobiotic metabolism studies are best performed with freshly cut rather than cultured human liver slices.

Adolescent↗
PubMed1999

HIV-1-trans-activating (Tat) protein: both a target and a tool in therapeutic approaches.

Tat proteins (trans-activating proteins) are present in all known lentiviruses and are early RNA binding proteins that regulate transcription. Tat from the human immunodeficiency virus type-1 is a protein comprising 86 amino acids and encoded by 2 exons. The first 72 amino acids are encoded by exon 1 and exhibit full trans-activating activity. The second exon encodes a 14-amino-acid C-terminal sequence that is not required for trans-activation but does contain an RGD motif, which is important in binding to alphavbeta3 and alpha5beta1 integrins. Tat has an unusual property for a transcription factor; it can be released and enter cells freely, yet still retain its activity, enabling it to up-regulate a number of genes. Tat also has an angiogenic effect; it is a potent growth factor for Kaposi sarcoma-derived spindle cells, and, separately, it has been shown to bind to a specific receptor, Flk-1/KDR, on vascular smooth muscle cells, as well as to integrin-like receptors present on rat skeletal muscle cells and the lymphocyte cell line H9. It appears that the basic domain of tat is important, not only for translocation but also for nuclear localisation and trans-activation of cellular genes. As such, targeting of tat protein or, more simply, the basic domain provides great scope for therapeutic intervention in HIV-1 infection. There is also opportunity for tat to be used as a molecular tool; the protein can be manipulated to deliver non-permeable compounds into cells, an approach that already has been employed using ovalbumin, beta-galactosidase, horseradish peroxidase, and caspase-3.

Anti-HIV Agents↗
PubMed1999

Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Actins↗
PubMed1999

Nucleosomes bind to cell surface proteoglycans.

Material on the surface of activated T-cells was displaced following incubation with a sulfated polysaccharide, dextrin 2-sulfate (D2S), and purified by anion-exchange chromatography. This revealed a complex comprising histones H2A, H2B, H3, and H4 and DNA fragmented into 180-base pair units characteristic of mono-, di-, tri, and polynucleosomes, a pattern of fragmentation similar to that found in apoptotic cells. An antibody raised against the purified nucleosome preparation bound to the plasma membrane of activated T-cells confirming the surface location of nucleosomes. The interaction of sulfated polysaccharides with nucleosomes was investigated using a biotinylated derivative of D2S. It was found that sulfated polysaccharides bound to nucleosomes via the N termini of histones, especially H2A and H2B. Treatment of T-cells with either heparinase or heparitinase abolished nucleosome binding to plasma membranes. This suggests that nucleosomes are anchored to the surface of T-cells by heparan sulfate proteoglycans through an ionic interaction with the basic N-terminal residues in the histones. Furthermore, nucleosomes bound to the cell surface in this manner are then able to bind other sulfated polysaccharides, such as D2S, heparin, or dextran sulfate, through unoccupied histone N termini forming a complex comprising cell surface heparan sulfate proteoglycans, nucleosomes, and sulfated polysaccharides.

Animals↗