The P-glycoprotein repertoire of the equine parasitic nematode Parascaris univalens.

P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the major site of expression but individual gene expression patterns were not conserved between the two nematodes. In P. univalens, PunPgp-9, PunPgp-11.1 and PunPgp-16.2 consistently exhibited the highest expression level in two independent transcriptome data sets. Using RNA-Seq, no significant upregulation of any Pgp was detected following in vitro incubation of adult P. univalens with ivermectin suggesting that drug-induced upregulation is not the mechanism of Pgp-mediated ML resistance. Expression and functional analyses of PunPgp-2 and PunPgp-9 in Saccharomyces cerevisiae provide evidence for an interaction with ketoconazole and ivermectin, but not thiabendazole. Overall, this study established reliable reference gene models with significantly improved annotation for the P. univalens Pgp repertoire and provides a foundation for a better understanding of Pgp-mediated anthelmintic resistance.

Genomic Analyses Reveal the Influence of Geographic Origin, Migration, and Hybridization on Modern Dog Breed Development.

There are nearly 400 modern domestic dog breeds with a unique histories and genetic profiles. To track the genetic signatures of breed development, we have assembled the most diverse dataset of dog breeds, reflecting their extensive phenotypic variation and heritage. Combining genetic distance, migration, and genome-wide haplotype sharing analyses, we uncover geographic patterns of development and independent origins of common traits. Our analyses reveal the hybrid history of breeds and elucidate the effects of immigration, revealing for the first time a suggestion of New World dog within some modern breeds. Finally, we used cladistics and haplotype sharing to show that some common traits have arisen more than once in the history of the dog. These analyses characterize the complexities of breed development, resolving longstanding questions regarding individual breed origination, the effect of migration on geographically distinct breeds, and, by inference, transfer of trait and disease alleles among dog breeds.

Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin.

P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp.

An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog.

BACKGROUND: ABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models. METHODS: Transcriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and Drosophila melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses. RESULTS: A protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers. CONCLUSION: Findings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.

Actin polymerization controls the activation of multidrug efflux at fertilization by translocation and fine-scale positioning of ABCB1 on microvilli.

Fertilization changes the structure and function of the cell surface. In sea urchins, these changes include polymerization of cortical actin and a coincident, switch-like increase in the activity of the multidrug efflux transporter ABCB1a. However, it is not clear how cortical reorganization leads to changes in membrane transport physiology. In this study, we used three-dimensional superresolution fluorescence microscopy to resolve the fine-scale movements of the transporter along polymerizing actin filaments, and we show that efflux activity is established after ABCB1a translocates to the tips of the microvilli. Inhibition of actin polymerization or bundle formation prevents tip localization, resulting in the patching of ABCB1a at the cell surface and decreased efflux activity. In contrast, enhanced actin polymerization promotes tip localization. Finally, interference with Rab11, a regulator of apical recycling, inhibits activation of efflux activity in embryos. Together our results show that actin-mediated, short-range traffic and positioning of transporters at the cell surface regulates multidrug efflux activity and highlight the multifaceted roles of microvilli in the spatial distribution of membrane proteins.

Molecular cloning and characterisation of a novel P-glycoprotein in the salmon louse Lepeophtheirus salmonis.

The salmon louse, Lepeophtheirus salmonis, is a crustacean ectoparasite of salmonid fish. At present, sea louse control on salmon farms relies heavily upon chemical treatments. Drug efflux transport, mediated by ABC transporters such as P-glycoprotein (Pgp), represents a major mechanism for drug resistance in parasites. We report here the molecular cloning of a new Pgp from the salmon louse, called SL-PGY1. A partial Pgp sequence was obtained by searching sea louse ESTs, and extended by rapid amplification of cDNA ends (RACE). The open reading frame of SL-PGY1 encodes a protein of 1438 amino acids that possesses typical structural traits of P-glycoproteins, and shows a high degree of sequence homology to invertebrate and vertebrate P-glycoproteins. In the absence of drug exposure, SL-PGY1 mRNA expression levels did not differ between a drug-susceptible strain of L. salmonis and a strain showing a ~7-fold decrease in sensitivity against emamectin benzoate, the active component of the in-feed sea louse treatment SLICE (Merck Animal Health). Aqueous exposure of the hyposensitive salmon louse strain to emamectin benzoate (24h, 410 µg/L) provoked a 2.9-fold upregulation of SL-PGY1. Adult male lice of both strains showed a greater abundance of SL-PGY1 mRNA than adult females.

Use of shape similarities for the classification of P-glycoprotein substrates and nonsubstrates.

BACKGROUND: The multidrug transporter P-glycoprotein (P-gp) ATP-binding cassette B1 (ABCB1) is one of the key proteins influencing bioavailability and uptake of drugs in the brain. In addition, it is one of the main factors contributing to multidrug resistance in tumor therapy. Due to its promiscuous substrate recognition, prediction of substrate properties for the multidrug transporter P-gp represents a challenging task. RESULTS: Here, we present data on three classification methods of ABCB1 substrates and nonsubstrates based on 2D and 3D shape similarity calculations with special emphasis on the use of the similarity-based relationship approach. The results indicate that a reference set structurally similar to the data set performs superiorly to those selected on the basis of maximum diversity and suggests Random Forest as the most suitable classification method for this data set. CONCLUSION: This study suggests 2D descriptors representing 3D features best suited to the classification of P-gp substrates and nonsubstrates.

Relationship between drugs and functional activity of various mammalian P-glycoproteins (ABCB1).

P-glycoprotein (Pgp, ABCB1) is an efflux transporter for a variety of amphipathic agents that can affect the pharmacokinetics of drugs. In order to extrapolate transport and pharmacokinetic data of the drug candidates obtained from in vitro and animal models to those in humans, it is important to understand the functional differences of Pgps from various mammalian species including human, monkey, dog, rat, and mouse. Here, we review differences/similarities in the properties of Pgp from numerous mammalian species commonly used in preclinical studies and discuss their relevance to the pharmacokinetics of potential drug molecules.

Inhibitors of p-glycoprotein--lead identification and optimisation.

The membrane bound drug efflux pump P-glycoprotein (P-gp) transports a wide variety of functionally and structurally diverse cytotoxic drugs out of tumour cells. Overexpression of P-glycoprotein is one of the predominant mechanisms responsible for development of multiple drug resistance in tumour therapy. Thus, inhibition of P-gp represents a promising approach for treatment of multidrug resistant tumours. This review highlights concepts for identification and optimization of new inhibitors of P-glycoprotein.

Definition of the domain boundaries is critical to the expression of the nucleotide-binding domains of P-glycoprotein.

Heterologous expression of domains of eukaryotic proteins is frequently associated with formation of inclusion bodies, consisting of aggregated mis-folded protein. This phenomenon has proved a significant barrier to the characterization of domains of eukaryotic ATP binding cassette (ABC) transporters. We hypothesized that the solubility of heterologously expressed nucleotide binding domains (NBDs) of ABC transporters is dependent on the definition of the domain boundaries. In this paper we have defined a core NBD, and tested the effect of extensions to and deletions of this core domain on protein expression. Of 10 NBDs constructed, only one was expressed as a soluble protein in Escherichia coli, with expression of the remaining NBDs being associated with inclusion body formation. The soluble NBD protein we have obtained corresponds to residues 386-632 of P-glycoprotein and represents an optimally defined domain. The NBD has been isolated and purified to 95% homogeneity by a two-step purification protocol, involving affinity chromatography and gel filtration. Although showing no detectable ATP hydrolysis, the protein retains specific ATP binding and has a secondary structure compatible with X-ray crystallographic data on bacterial NBDs. We have interpreted our results in terms of homology models, which suggest that the N-terminal NBD of P-glycoprotein can be produced as a stable, correctly folded, isolate domain with judicious design of the expression construct.

SMR-type multidrug resistance pumps.

Multidrug resistance (MDR) efflux pumps in pathogenic microorganisms nullify the effects of antimicrobial drugs used in medicine. We have conducted phylogenetic analyses showing that these efflux pumps are associated with five superfamilies of transport systems. One of these, the drug/metabolite transporter (DMT) superfamily includes a family of small multidrug resistance (SMR)-conferring proteins that are discussed in detail in this review. A single microorganism such as Bacillus subtilis may possess multiple homologs of this family, and these homologs are believed to form both homo-oligomeric or hetero-oligomeric pumps, some of which export cationic drugs. The characteristics of some of these systems and the genes that encode them are described, with emphasis on the eight homologs encoded within the B subtilis genome. Anomalies and unanswered questions that provide impetus for future studies are presented.

Molecular properties of bacterial multidrug transporters.

One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells.

Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.

Anticuerpos anti-beta, glicoproteína I y niveles plasmáticos de la proteína de unión a C4b / Antibodies against beta glycoprotein I and C4b binding protein plasmatic levels

Autoantibodies directed to beta2 glycoprotein I (a beta 2 GPI) are frequently found in patients with antiphospholipid antibodies (aPL). They are more strongly associated with clinical manifestations of the antiphospholipid syndrome then a PL. It has been shown that beta2 GPI and C4b bibding protein (C4bBP) share certain homology. In a previous study we have shown that anticardiolipin antibodies were associated with a plasma decrease of C4bBP. The aim of the present study was to evaluate in 131 patients with a PL whether the decrease in C4bBP is related to the presence of abeta2 GPI. Lower C4bBP levels (mean +- SD) in the group of patients having abeta2 GPI (n=57) were observed when compared with the normal group (n=44), (74.3 porciento +-28.1 vs 94.6 porciento +-20.9,p<0.005).This difference was more significant consideing the IgG isotype. The group of patients with positive abeta2GPI-igG (n=41) had lower values of C4bBP (70.1 porciento +- 26.8) than both the normal group (p<0.005) and the group of patients with negative abeta2 IgG (n=90, 86.0 porciento +- 30.5 porciento, p<0.05). C4bBP deficiency (level <70 porciento) was also morefrequent in the group abeta 2GPI-IgG (+) (63.4 porciento) then in the group abeta2GPI-IgG 8-) (34.4 porciento, p<0.005). Moreover, patients with a PL and previews venous thrombosis (n=32) showed lower C4bBP values (75.1 porciento +- 27.9) compared with the normal group (p<0.05). As this time, the mechanisms responsibles for the C4bBP decrease are not known. Our findings on the close relationship between abnormalitiesin the C4bBP/protein S system and the presence of abeta2GPI could explain the major thrombotic risk in patients havingthese autoantibodies

Anticuerpos anti-beta, glicoproteína I y niveles plasmáticos de la proteína de unión a C4b / Antibodies against beta glycoprotein I and C4b binding protein plasmatic levels

Autoantibodies directed to beta2 glycoprotein I (a beta 2 GPI) are frequently found in patients with antiphospholipid antibodies (aPL). They are more strongly associated with clinical manifestations of the antiphospholipid syndrome then a PL. It has been shown that beta2 GPI and C4b bibding protein (C4bBP) share certain homology. In a previous study we have shown that anticardiolipin antibodies were associated with a plasma decrease of C4bBP. The aim of the present study was to evaluate in 131 patients with a PL whether the decrease in C4bBP is related to the presence of abeta2 GPI. Lower C4bBP levels (mean +- SD) in the group of patients having abeta2 GPI (n=57) were observed when compared with the normal group (n=44), (74.3 porciento +-28.1 vs 94.6 porciento +-20.9,p<0.005).This difference was more significant consideing the IgG isotype. The group of patients with positive abeta2GPI-igG (n=41) had lower values of C4bBP (70.1 porciento +- 26.8) than both the normal group (p<0.005) and the group of patients with negative abeta2 IgG (n=90, 86.0 porciento +- 30.5 porciento, p<0.05). C4bBP deficiency (level <70 porciento) was also morefrequent in the group abeta 2GPI-IgG (+) (63.4 porciento) then in the group abeta2GPI-IgG 8-) (34.4 porciento, p<0.005). Moreover, patients with a PL and previews venous thrombosis (n=32) showed lower C4bBP values (75.1 porciento +- 27.9) compared with the normal group (p<0.05). As this time, the mechanisms responsibles for the C4bBP decrease are not known. Our findings on the close relationship between abnormalitiesin the C4bBP/protein S system and the presence of abeta2GPI could explain the major thrombotic risk in patients havingthese autoantibodies (AU)

Genetic dissection of the function of mammalian P-glycoproteins.

Mammalian P-glycoproteins are plasma membrane proteins belonging to the superfamily of ATP-binding cassette transporters. They were discovered as drug pumps in multidrug-resistant cancer cells, but are also present in many normal tissues. Genetic approaches have helped to dissect the physiological functions and mode of action of P-glycoproteins. Disruption of both genes for the drug-transporting P-glycoproteins in mice has no effect on the normal sheltered life of these mice, but renders them hypersensitive to many drugs. P-glycoprotein appears to be especially important in protecting the brain and in limiting uptake of hydrophobic drugs from the gut. Recent experiments with polarized cells support the idea that drug-transporting P-glycoproteins act by flipping drugs from the inner to the outer leaflet of the plasma membrane.

Proton-dependent multidrug efflux systems.

Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also discuss whether the normal physiological role of the multidrug efflux systems is to protect the cell from toxic compounds or whether they fulfil primary functions unrelated to drug resistance and only efflux multiple drugs fortuitously or opportunistically.