Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between approaches. Drug Metab

This paper is available online at http://www.dmd.org ABSTRACT: Relative activity factors (RAFs) and immunoquantified levels of cytochrome P450 (CYP) isoforms both have been proposed as scaling factors for the prediction of hepatic drug metabolism from studies using cDNA-expressed CYPs. However, a systematic comparison of the two approaches, including possible mechanisms underlying differences, is not available. In this study, RAFs determined for CYPs 1A2, 2B6, 2C19, 2D6, and 3A4 in 12 human livers using lymphoblast-expressed enzymes were compared to immunoquantified protein levels. 2C19, 2D6, and 3A4 RAFs were similar to immunoquantified enzyme levels. In contrast, 1A2 RAFs were 5-to 20-fold higher than CYP1A2 content, and the RAF:content ratio was positively correlated with the molar ratio of NADPH:CYP oxidoreductase (OR) to CYP1A2. The OR:CYP1A2 ratio in lymphoblast microsomes was 92-fold lower than in human liver microsomes. Reconstitution experiments demonstrated a 10-to 20-fold lower activity at OR:CYP1A2 ratios similar to those in lymphoblasts, compared with those in human livers. CYP2B6-containing lymphoblast microsomes had 29-and 13-fold lower OR:CYP and cytochrome b 5 :CYP ratios, respectively, than did liver microsomes and yielded RAFs that were 6-fold higher than CYP2B6 content. Use of metabolic rates from cDNA-expressed CYPs containing nonphysiologic concentrations of electron-transfer proteins (relative to human liver microsomes) in conjunction with hepatic CYP contents may lead to incorrect predictions of liver microsomal rates and relative contributions of individual isoforms. Scaling factors used in bridging the gap between expression systems and liver microsomes should not only incorporate relative hepatic abundance of individual CYPs but also account for differences in activity per unit enzyme in the two systems. The cloning and heterologous expression of the drug-metabolizing human CYPs 1 has resulted in the commercial availability of cDNAexpressed CYPs for use as reagents for in vitro quantitative phenotyping, and bridging the gap between cDNA-expressed cytochromes and human liver microsomes has been the subject of several recent reviews The turnover number of CYPs in human liver microsomes is affected by several factors that are biochemically distinct from the CYP enzyme itself. Examples include the accessory electron-transfer proteins NADPH cytochrome P450-oxidoreductase (OR) and cytochrome b 5 , membrane lipid composition, and ionic strength of the in vitro incubation matrix The relative contribution of each isoform can then be determined as follows: Enzyme kinetic studies using cDNA-expressed human CYPs are being increasingly used to define the functions v i (s) for whatever individual CYPs biotransform a drug. Such analyses indicate the relative affinities and capacities of the CYPs contributing to a biotransformation. However, the relative contribution of each enzyme cannot be estimated unless the results incorporate the scaling factors (A i ). Although immunoquantified levels of CYP isoforms in human liver microsomes RAFs are determined for specific CYPs by comparing the rate of an isoform-specific index reaction at saturating substrate concentrations in human liver microsomes (V max for liver microsomes) to the rate of the same reaction catalyzed by the specific cDNA-expressed CYP under identical conditions (V max for cDNA-expressed enzyme): Mean V max for isoform-specific reaction in human liver microsomes V max for isoform-specific reaction by cDNA expressed isoform When the numerator is expressed in picomoles metabolite per milligram of protein per minute and the denominator as picomoles of metabolite per picomole of CYP per minute, the units of RAF are picomoles of CYP per milligram of protein, which can be compared with the immunoquantified CYP contents. RAFs have been used as estimates of A i in reaction phenotyping Materials and Methods Human Liver Microsomes and Heterologously Expressed CYP Isoforms. Liver samples, obtained from the International Institute for the Advancement of Medicine (Exton, PA) or the Liver Tissue Procurement and Distribution Service (University of Minnesota), were from 12 different transplant donors (L1-L12) with no known liver disease. The donor population (median age 27 years, range 3-50 years; 5 females and 7 males) was balanced with respect to age, sex, race, smoking habits, and alcohol consumption. The tissue was partitioned and kept at Ϫ80°C until the time of microsome preparation as described previously Microsomes from cDNA-transfected human lymphoblastoid cells expressing CYP 1A2, 2B6, 2C19, 2D6, or 3A4 (Crespi, 1995) were purchased from Gentest Corporation (Woburn, MA), aliquoted and stored at Ϫ80°C, and thawed on ice before use. Lymphoblast-expressed CYPs 3A4 and 2D6 used in the study were coexpressed with OR, whereas the activities of lymphoblast- expressed CYPs 1A2, 2B6, and 2C19 were supported by endogenous levels of reductase native to the host cell line. Microsomal protein concentrations and CYP content were provided by the manufacturer. Antibodies and Quantitative Western Blotting. Concentrations of CYP1A2, 2B6, 2C19, 2D6, and 3A4/5 in human liver microsomal preparations from livers L1 to L12 were determined by quantitative Western blotting In Vitro Metabolic Incubations and Metabolite Analysis. Incubations of index substrates with human liver microsomes and lymphoblas

A Systematic Nomenclature for the <i>Drosophila </i>Ventral Nerve Cord

The ventral nerve cord (VNC) of Drosophila is an important model system for understanding how nervous systems generate locomotion. In this issue of Neuron, Court et al. define the structures of the adult VNC to provide an anatomical framework for analyzing the functional organization of the VNC.Drosophila melanogaster is an established model for neuroscience research with relevance in biology and medicine. Until recently, research on the Drosophila brain was hindered by the lack of a complete and uniform nomenclature. Recognizing this, Ito et al. (2014) produced an authoritative nomenclature for the adult insect brain, using Drosophila as the reference. Here, we extend this nomenclature to the adult thoracic and abdominal neuromeres, the ventral nerve cord (VNC), to provide an anatomical description of this major component of the Drosophila nervous system. The VNC is the locus for the reception and integration of sensory information and involved in generating most of the locomotor actions that underlie fly behaviors. The aim is to create a nomenclature, definitions, and spatial boundaries for the Drosophila VNC that are consistent with other insects. The work establishes an anatomical framework that provides a powerful tool for analyzing the functional organization of the VNC

Modest but Variable Effect of Rifampin on Steady-State Plasma Pharmacokinetics of Efavirenz in Healthy African-American and Caucasian Volunteers

ABSTRACT Efavirenz-based antiretroviral regimen is preferred during rifampin-containing tuberculosis therapy. However, current pharmacokinetic data are insufficient to guide optimized concurrent dosing. This study aimed to better characterize the effects of rifampin on efavirenz pharmacokinetics. Subjects were randomized to receive 600 mg efavirenz/day or 600 mg efavirenz with 600 mg rifampin/day for 8 days, with plasma samples collected for pharmacokinetic analysis over 24 h on day 8. Treatments were then crossed over after at least a 2-week washout period, and procedures were repeated. Efavirenz concentrations were determined by high-performance liquid chromatography (HPLC), and pharmacokinetic parameters were estimated by noncompartmental analysis. Efavirenz pharmacokinetic differences between treatment periods were evaluated by paired t test. The coefficients of variation in efavirenz plasma AUC 0-24 (area under the concentration-time curve from 0 to 24 h) were 50% and 56% in the absence and presence of rifampin, respectively. Of the 11 evaluable subjects (6 white, 5 black; 6 women, 5 men), the geometric mean AUC 0-24 ratio on/off rifampin (90% confidence interval) was 0.82 (0.72, 0.92), with individual AUC 0-24 ratios varying from 0.55 to 1.18. Five subjects had a 24-hour efavirenz concentration ( C 24 ) of <1,000 ng/ml on rifampin. They were more likely to have received a lower dose in milligrams/kilogram of body weight and to have lower efavirenz AUC 0-24 values in the basal state. Although rifampin resulted in a modest reduction in efavirenz plasma exposure in subjects as a whole, there was high variability in responses between subjects, suggesting that efavirenz dose adjustment with rifampin may need to be individualized. Body weight and genetic factors will be important covariates in dosing algorithms

Candida albicans AGE3, the Ortholog of the S. cerevisiae ARF-GAP-Encoding Gene GCS1, Is Required for Hyphal Growth and Drug Resistance

BACKGROUND: Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Delta mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Delta cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Delta cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Delta mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p. CONCLUSIONS/SIGNIFICANCE: The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Delta cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Delta cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Delta cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it represents a promising antifungal drug target

Empirical Legal Studies Before 1940: A Bibliographic Essay

The modern empirical legal studies movement has well-known antecedents in the law and society and law and economics traditions of the latter half of the 20th century. Less well known is the body of empirical research on legal phenomena from the period prior to World War II. This paper is an extensive bibliographic essay that surveys the English language empirical legal research from approximately 1940 and earlier. The essay is arranged around the themes in the research: criminal justice, civil justice (general studies of civil litigation, auto accident litigation and compensation, divorce, small claims, jurisdiction and procedure, civil juries), debt and bankruptcy, banking, appellate courts, legal needs, legal profession (including legal education), and judicial staffing and selection. Accompanying the essay is an extensive bibliography of research articles, books, and reports

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]