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The aim of this study was to examine the role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially with regard to multidrug resistant (MDR) tumour cells. It was found that at a high NADPH concentration (500 moll), the anthracenedione agent ametantrone, with an unmodified quinone structure, was susceptible to CPR-dependent reductive activation. In contrast, it was shown that compounds with modified quinone grouping (benzoperimidine BP1, anthrapyridone CO1 and pyrazolopyrimidoacridine PPAC2) did not undergo reductive activation by CPR. This suggests that the presence of a modified quinone function is the structural factor excluding reductive activation of antitumour anthraquinone derivatives and analogues by CPR. In the second part of the work, the ability of antitumour anthraquinone derivatives and analogues to inhibit the growth of the human promyelocytic, sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60VINC) or MRP1 (HL60DOX) was studied in the presence of exogenously added CPR. A significant increase in the activity of ametantrone with an unmodified quinone structure after its reductive conversion by CPR was observed against HL60 as well as HL60VINC and HL60DOX cells, whereas in the case of quinone-modified compounds (BP1, CO1 and PPAC2), the presence of the activation system had no effect on their activity against the sensitive and MDR tumour cells examined.

Central retinal artery occlusion is caused by a platelet-fibrin thrombus or embolic occlusion and is a stroke of the eye. Observational studies suggest that thrombolytics may restore ocular perfusion and visual function. We hypothesized that intravenous tissue-type plasminogen activator (tPA) administered within 24 hours of symptom onset might restore ocular perfusion and visual function.

Cytochrome P450 3A4, a major drug-metabolizing enzyme in man, is well known to show non-Michaelis-Menten steady-state kinetics for a number of substrates, indicating that more than one substrate can bind to the enzyme simultaneously, but it has proved difficult to obtain reliable estimates of exactly how many substrate molecules can bind. We have used a simple method involving studies of the effect of large inhibitors on the Hill coefficient to provide improved estimates of substrate stoichiometry from simple steady-state kinetics. Using a panel of eight inhibitors, we show that at least four molecules of the widely used CYP3A4 substrate 7-benzyloxyquinoline can bind simultaneously to the enzyme. Computational docking studies show that this is consistent with the recently reported crystal structures of the enzyme. In the case of midazolam, which shows simple Michaelis-Menten kinetics, the inhibitor effects demonstrate that two molecules must bind simultaneously, consistent with earlier evidence, whereas for diltiazem, the experiments provide no evidence for the binding of more than one molecule. The consequences of this "inhibitor-induced cooperativity" for the prediction of pharmacokinetics and drug-drug interactions are discussed.

Patients with cancer often take many different classes of drugs to treat the effects of their malignancy and the side effects of treatment, as well as their comorbidities. The potential for drug-drug interactions that may affect the efficacy of anticancer treatment is high, and a major source of such interactions is competition for the drug-metabolizing enzymes, cytochromes P450 (P450s). We have examined a series of 20 drugs commonly prescribed to cancer patients to look for potential interactions via CYP2D6. We used a homology model of CYP2D6, together with molecular docking techniques, to perform an in silico screen for binding to CYP2D6. Experimental IC50 values were determined for these compounds and compared with the model predictions to reveal a correlation with a regression coefficient of r2 = 0.61. Importantly, the docked conformation of the commonly prescribed antiemetic metoclopramide predicted a new site of metabolism that was further investigated through in vitro analysis with recombinant CYP2D6. An aromatic N-hydroxy metabolite of metoclopramide, consistent with predictions from our modeling studies, was identified by high-performance liquid chromatography/mass spectrometry. This metabolite was found to represent a major product of metabolism in human liver microsomes, and CYP2D6 was identified as the main P450 isoform responsible for catalyzing its formation. In view of the prevalence of interindividual variation in the CYP2D6 genotype and phenotype, we suggest that those experiencing adverse reactions with metoclopramide, e.g., extrapyramidal syndrome, are likely to have a particular CYP2D6 genotype/phenotype. This warrants further investigation.

Previous research has suggested that free-ranging mourning doves (Zenaida macroura) may ingest spent lead pellets, succumb to lead toxicosis, and die in a relatively short time (i.e., an acute lead toxicosis hypothesis). We tested this hypothesis by administering 157 captive mourning doves 2–24 lead pellets, monitoring pellet retention and short-term survival, and measuring related physiological characteristics. During the 19- to 21-day posttreatment period, 104 doves that received lead pellets died (deceased doves) and 53 survived (survivors); all 22 birds in a control group survived. Within 24-hr of treatment, blood lead levels increased almost twice as fast for deceased doves compared to survivors (F1,208= 55.49; P < 0.001). During the first week, heterophil:lymphocyte (H:L) ratios increased twice as fast for deceased doves than with survivors (F1,198= 23.14, P < 0.001). Posttreatment survival differed (?2= 37.4, P < 0.001) among the 5 groups of doves that retained different numbers of pellets, and survival ranged from 0.57 (95% CI: 0.44–0.74) for doves that retained =2 lead pellets 2 days posttreatment compared to 0.08 (95% CI: 0.022–0.31) for those doves that retained 13–19 lead pellets on 2 days posttreatment; significant differences existed among the 5 groups. After controlling for dove pretreatment body mass, each additional lead pellet increased the hazard of death by 18.0% (95% CI: 1.132–1.230, P < 0.001) and 25.7% (95% CI: 1.175–1.345, P < 0.001) for males and females, respectively. For each 1-g increase in pretreatment body mass, the hazard of death decreased 2.5% (P = 0.04) for males and 3.8% (P = 0.02) for females. Deceased doves had the highest lead levels in liver (49.20 ± 3.23 ppm) and kidney (258.16 ± 21.85 ppm) tissues, whereas controls showed the lowest levels (liver, 0.08 ± 0.041 ppm; kidney, 0.17 ± 0.10 ppm). For doves dosed with pellets, we observed simultaneous increases in blood lead levels and H:L ratios, whereas packed-cell volume (PCV) values declined. Our results support an acute lead toxicosis hypothesis. Although further research is necessary to investigate the magnitude of lead shot ingestion and toxicosis in mourning doves, we recommend that management agencies initiate development of a long-term strategic plan aimed at implementing a nontoxic shot regulation for mourning dove hunting.

Cytochrome P450 3A4 (CYP3A4) is the major enzyme responsible for phase I drug metabolism of many anticancer agents. It is also a major route for metabolism of many drugs used by patients to treat the symptoms caused by cancer and its treatment as well as their other illnesses, for example, cardiovascular disease. To assess the ability to inhibit CYP3A4 of drugs most commonly used by our patients during cancer therapy, we have made in silico predictions based on the crystal structures of CYP3A4. From this set of 33 common comedicated drugs, 10 were predicted to be inhibitors of CYP3A4, with the antidiarrheal drug loperamide predicted to be the most potent. There was significant correlation (r2 = 0.75–0.66) between predicted affinity and our measured IC50 values, and loperamide was confirmed as a potent inhibitor (IC50 of 0.050 ± 0.006 µM). Active site docking studies predicted an orientation of loperamide consistent with formation of the major (N-demethylated) metabolite, where it interacts with the phenylalanine cluster and Arg-212 and Glu-374; experimental evidence for the latter interaction comes from the ∼12-fold increase in KM for loperamide observed for the Glu-374-Gln mutant. The commonly prescribed drugs loperamide, amitriptyline, diltiazem, domperidone, lansoprazole, omeprazole, and simvastatin were identified by our in silico and in vitro screens as relatively potent inhibitors of CYP3A4 that have the potential to interact with cytotoxic agents to cause adverse effects, highlighting the likelihood of drug-drug interactions affecting chemotherapy treatment.

Human novel oxidoreductase 1 (NDOR1) is a diflavin reductase closely related to cytochrome P450 reductase (POR) and nitric oxide synthase (NOS), which are involved in the metabolism of antitumour agents. A variant cDNA sequence of NDOR1, NDOR1 p.518-519ins9 or NDOR1v1, has been deposited in GenBank (accession no. AK026089 and AY077845) that encodes an additional nine amino acids, which led us to investigate NDOR1polymorphism.

Visualization systems for tracking proteins are standard experimental tools in most areas of biological research apart from protein purification. Here, we have sought to plug this gap by producing red and yellow visual tags using the heme-binding domain of mosquito cytochrome b5 and the flavin mononucleotide (FMN)-binding domain of human P450 reductase. Tests with colorless glutathione-S-transferase (GST) show them to be simple and effective tools for visually identifying correctly folded protein and tracking protein molecules through protein expression and purification. Furthermore, the characteristic absorbance signatures of the colored tags can be used to quantify protein concentrations directly, which allows purification to be linked to colorimetric detection. This technology, which we call Rainbow Tagging, facilitates expression and downstream processing of recombinant proteins, paving the way for the development of automated high-throughput protein expression systems.

The competing interests of preventing recurrent ischemic optic neuropathy (ION) and minimizing medication side effects make corticosteroid dose reduction in giant cell arteritis (GCA) a difficult problem. The authors sought to determine whether any factors were predictive of recurrent ION.

Although the residues that determine the preference of CYP2D6 (cytochrome P450 2D6) for compounds containing a basic nitrogen are well characterized, the contribution of other active site residues to substrate binding and orientation is less well understood. Our structural model of CYP2D6 identifies the aromatic residue Phe120 as a likely major feature of the active site. To examine the role of Phe120, mutants of CYP2D6 in which this residue has been substituted by alanine, leucine, tyrosine, serine, histidine, tryptophan or methionine residues have been prepared in bacterial membranes co-expressing human cytochrome NADPH cytochrome P450 oxidoreductase. The mutants have been characterized using the prototypical bufuralol 1´ hydroxylase and dextromethorphan O- and N-demethylase activities of CYP2D6. Larger effects on Km values are observed for dextromethorphan O-demethylation than for bufuralol 1´ hydroxylation, indicating that the Phe120 side chain is more important in dextromethorphan than in bufuralol binding. A role for this side chain in determining the regiospecificity of substrate oxidation was indicated by changes in the relative rates of O- and N-demethylation of dextromethorphan and, notably, by the formation of 7-hydroxy dextromethrophan, a novel dextromethorphan metabolite, in mutants in which it had been substituted. Computational studies of dextromethorphan binding to the active site of the Phe120→Ala mutant were carried out to throw light on the way in which the removal of this side chain leads to different modes of ligand binding.

Several retrospective studies have suggested that immunotherapy, including prednisolone, azathioprine and thymectomy, reduces progression of ocular myasthenia gravis to generalized myasthenia gravis. This study examines the effect of immunotherapy on generalization rates in ocular myasthenia patients who are acetylcholine receptor (AChR) antibody-positive.

Human cytochrome P450 2C9 (CYP2C9) is important in the metabolism of non-steroidal anti-inflammatory compounds such as diclofenac, the antidiabetic agent tolbutamide and other clinically important drugs, many of which are weakly acidic. Multiple sequence alignment of CYPs identified CYP2C9 Asp293 as corresponding to Asp301 of CYP2D6, which has been suggested to play a role in the binding of basic substrates to the latter enzyme. Replacement of Asp293 with Ala (D293A) decreased activity by more than 90%, and led to an approx. 3- to 10-fold increase in Km values for the three test substrates tolbutamide, dextromethorphan and diclofenac. Conservative replacement of the carboxyl side chain in a Glu (D293E) mutant produced no significant changes in Km values and slight increases in kcat values. Changes in regiospecificity were observed for both the Ala and Glu substitutions; low levels of both dextromethorphan O- and N-demethylation were observed in the D293A mutant, whereas increased preference for O-demethylation was observed for the D293E mutant. Expression of constructs coding for Asn (D293N) and Gln (D293Q) substitutions failed to form a P450 correctly. Our analysis suggests a structural role for the carboxyl side chain of Asp293 in CYP2C9 substrate binding and catalysis. The conservation of an Asp residue in other CYP families in a position equivalent to Asp293 indicates a common mechanism for maintaining the active-site architecture.

AbstractInstitutions involved in public policy and technological innovation are concerned with processes that influence the behaviour of people. This paper reports on research in the New Zealand dairy industry that is seeking to develop improved process models for managing change in a range of policy, industry and business contexts. The Practice Interplay Model was used to evaluate mediating methods for negotiating environmental and production outcomes within the context of a farm dairy effluent management case study. Significant misalignments between research, farming, extension and policy practitioners' expectations and perceptions of factors required for effective farm dairy effluent management were found. The combination of interactive workshops linked to quantitative verification and testing and follow-up interviews enabled a depth of investigation that was rich in information yet cost-effective in terms of research resources. The process used identified opportunities to change the actions of system participants and alignment between them. It enabled strategies to be agreed that would continually improve and strengthen the process of change. Further development and application of the Practice Interplay Model to policy problems, including the ongoing development of tools and techniques used within the methodology, has the potential to break though into a new policy-development paradigm in New Zealand.

Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60–100 nmol/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH. Turnover values for bufuralol 1′-hydroxylation, metoprolol α-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation, using membranes expressing the fusion enzyme, were 5.6, 0.4, 0.72, and 6.19 min−1, respectively. These values were similar to E. coli membranes which coexpressed human CYP2D6 and CPR (CYP2D6/R). The Km and kcat values for bufuralol metabolism were estimated to be 10.2 μM and 4.1 min−1, respectively. The enzyme was purified using ion-exchange chromatography, affinity chromatography (2′-5′ ADP-Sepharose), and gel filtration. Estimated turnover rates for bufuralol 1′-hydroxylation, metoprolol α-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation were 1.2, 0.52, 0.79, and 0.76 min−1, respectively. Bufuralol 1′-hydroxylase activity by purified CYP2D6F was enhanced by phospholipids and added CPR. The CYP2D6F enzyme was able to stimulate CYP3A4 testosterone 6β-hydroxylase activity in a reconstitution system indicating that electron transfer may be largely intermolecular. The catalytically self-sufficient CYP2D6F enzyme will facilitate investigations of P450-CPR interactions and the development of new biocatalysts.

Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescencein situhybridization analysis. The amino acid sequence of the protein contained distinct FMN-, FAD-, and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochromec, with apparent Kmandkcatvalues of 21 μmand 1.3 s−1, respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.

The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2's. A striking feature of the cDNA is the high sequence conservation of the 5' and 3' untranslated regions in cDNAs coding for PLA2's from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2's in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2's.

The two functional domains of a cloned human fibroblast NADPH-cytochrome P450 reductase have been expressed inEscherichia coliand purified on the milligram scale for crystallization studies. One domain contains the cofactor FMN-binding site and the other contains the binding sites for cofactor FAD and substrate NADPH. Crystals of both domains have been obtained by the microbatch method. The crystals of the FMN domain belong to the monoclinic space group P21, with unit cell dimensions ofa= 39.3 Å,b= 51.5 Å,c= 47.8 Å, and β = 105.7° and have one molecule in the asymmetric unit. Diffraction data up to 2.3 Å were collected with a merging residual on intensity of 9.3%. The crystals of the FAD/NADPH domain belong to the ortho-rhombic space group P212121with unit cell dimensions ofa= 55.9 Å,b= 58.6 Å,c= 131.1 Å and have one molecule in the asymmetric unit. Diffraction data up to 2.6 Å were collected with a merging residual on intensity of 8.0%.

The FMN-binding domain of human NADPH-cytochrome P450 reductase,corresponding to exons 3-;7, has been expressed at high level in anactive form and labelled with 13C and 15N. Mostof the backbone and aliphatic side-chain 1H, 15Nand 13C resonances have been assigned using heteronucleardouble- and triple-resonance methods, together with a semiautomaticassignment strategy. The secondary structure as estimated from the chemicalshift index and NOE connectivities consists of six α-helices and fiveβ-strands. The global fold was deduced from the long-range NOEsunambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQCspectrum. The fold is of the alternating α/β type, with the fiveβ-strands arranged into a parallel β-sheet. The secondarystructure and global fold are very similar to those of the bacterialflavodoxins, but the FMN-binding domain has an extra short helix in place ofa loop, and an extra helix at the N-terminus (leading to the membrane anchordomain in the intact P450 reductase). The experimental constraints werecombined with homology modelling to obtain a structure of the FMN-bindingdomain satisfying the observed NOE constraints. Chemical shift comparisonsshowed that the effects of FMN binding and of FMN reduction are largelylocalised at the binding site.

Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including β-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 inSpodoptera frugiperda(Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05–0.20 nmol/mg cell protein; 0.05–0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000–1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10–20 pmol/mg cell protein. TheKmandKcatvalues for bufuralol metabolism were estimated as 4.7 μMand 12.23 min−1, respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the “late” basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced. Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme.

Several letters to the editor are presented which include being a mountain biker, bicycling in Southern California (SoCal), and the cyclists who do not wear helmets down the trail.

A new farming systems extension approach was developed as part of the dairy industry extension program in Victoria. This approach was the outcome of collaborative work undertaken by the Department of Primary Industries and The University of Melbourne, funded by Dairy Australia. The project was instigated in response to a growing concern that dairy farm businesses were confronting complex challenges which in turn challenged extension staff to use a systems approach when servicing these businesses. Challenges arose from the increasing need to adjust to changing terms of trade and also respond to the drought conditions. Together, this had farmers questioning their farming system. This paper focuses on one aspect of the project, the emergence of a decision-making framework that proved useful in understanding farm management issues, designing extension programs, and building staff capacity to support decisions around these issues. The decision-making framework is comprised of five levels that progressively encompass a more expansive scope of the farming system considerations. The decision levels are: 1. product choice decisions, 2. component decisions such as grazing management, 3. sub-system decisions, 4. farm system decisions and 5. beyond-farm decisions. Implications arising from the use of these levels relate to the focus of the extension approach, the associated methodology, and the skills required to deliver at different levels. An overview of contemporary developments in Victorian dairy extension strategy is provided, outlining the evolution from a single topic focused extension program such as grazing management 15 years ago, to now where a more integrated feedbase management approach is used. This shift in farmer needs over the past two decades has challenged the previously successful programmed learning approaches and called for a new extension approach. Three key lessons arising from this work are that the 'Levels of Decision Making' framework can improve: 1. the targeting of extension services to achieve desired outcomes. 2. the design of extension programs, activities and tools. 3. the planned approach to professional development of extension staff. [ABSTRACT FROM AUTHOR]

The article discusses the use of online career profiles and a skills passport scheme for New Zealand's dairy industry. Employers looking for new farm team personnel would be able to facilitate the search and processing of job applicants in the future through online career profiles that contain verified records of skills and credentials such as the dairy industry's Career Pathways tool. In relation to this, a skills passport scheme would be useful in linking careers with information on professional development and education.