Dataset Information

Cloning and characterization of the rat multidrug resistance-associated protein 1.

ABSTRACT: Multidrug resistance-associated protein 1 (MRP1) was originally shown to confer resistance of human tumor cells to a broad range of natural product anticancer drugs. MRP1 has also been shown to mediate efflux transport of glutathione and glucuronide conjugates of drugs and endogenous substrates. An ortholog of MRP1 in the mouse has been cloned and characterized. Significant functional differences between murine and human MRP1 have been noted. Since drug disposition and pharmacology studies often are conducted in rats, there is a need to clone and characterize the rat ortholog of MRP1. We isolated a rat MRP1 (rMRP1) cDNA from rat brain astrocytes, characterized its coding sequences, and verified the transport activity of the protein expressed in MRP1 cDNA-transfected Madin-Darby canine kidney (MDCK) cells. Our results showed that rMRP1 has a coding sequence of 4599 bp, which predicts a polypeptide of 1533 amino acids with an apparent molecular weight of 190 kd by Western immunoblot analysis. rMRP1-transfected MDCK cells are capable of efflux transport of a fluorescent MRP1 marker - calcein - that is inhibitable by known MRP1 inhibitors, indomethacin, and MK571. Sequence analysis indicates that rMRP1 is more closely related to mouse MRP1 than human MRP1.

SUBMITTER: Yang Z

PROVIDER: S-EPMC2751354 | biostudies-other | 2002

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Cloning and characterization of the rat multidrug resistance-associated protein 1.

Yang Ziping Z Li Cheryl S W CS Shen Danny D DD Ho Rodney J Y RJ

AAPS pharmSci 20020101 3

Multidrug resistance-associated protein 1 (MRP1) was originally shown to confer resistance of human tumor cells to a broad range of natural product anticancer drugs. MRP1 has also been shown to mediate efflux transport of glutathione and glucuronide conjugates of drugs and endogenous substrates. An ortholog of MRP1 in the mouse has been cloned and characterized. Significant functional differences between murine and human MRP1 have been noted. Since drug disposition and pharmacology studies often ...[more]

PMID: 12423064

Similar Datasets

Project description:The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance.The pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium spp. MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels of pbmrp in blood stages.The pbmrp gene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from the Plasmodium spp. analysed. Expression of pbmrp was detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone.In this study, the pbmrp gene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to the Plasmodium y. yoelii and Plasmodium knowlesi mrp, and the Plasmodium vivax and Plasmodium falciparum mrp2 genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation of pbmrp from chromosome 13/14 to chromosome 8 was detected in RC.

Project description:BackgroundSperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented.MethodsComputational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay.ResultsIn this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10-60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E.ConclusionThe abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.

Project description:ATP-binding cassette (ABC)-transporters protect tissues by pumping their substrates out of the cells in many physiological barriers, such as the blood-brain barrier, intestine, liver, and kidney. These substrates include various endogenous metabolites, but, in addition, ABC transporters recognize a wide range of compounds, therefore affecting the disposition and elimination of clinically used drugs and their metabolites. Although numerous ABC-transporter inhibitors are known, the underlying mechanism of inhibition is not well characterized. The aim of this study is to deepen our understanding of transporter inhibition by studying the molecular basis of ligand recognition. In the current work, we compared the effect of 44 compounds on the active transport mediated by three ABC transporters: breast cancer resistance protein (BCRP and ABCG2), multidrug-resistance associated protein (MRP2 and ABCC2), and P-glycoprotein (P-gp and ABCB1). Eight compounds were strong inhibitors of all three transporters, while the activity of 36 compounds was transporter-specific. Of the tested compounds, 39, 25, and 11 were considered as strong inhibitors, while 1, 4, and 11 compounds were inactive against BCRP, MRP2, and P-gp, respectively. In addition, six transport-enhancing stimulators were observed for P-gp. In order to understand the observed selectivity, we compared the surface properties of binding cavities in the transporters and performed structure-activity analysis and computational docking of the compounds to known binding sites in the transmembrane domains and nucleotide-binding domains. Based on the results, the studied compounds are more likely to interact with the transmembrane domain than the nucleotide-binding domain. Additionally, the surface properties of the substrate binding site in the transmembrane domains of the three transporters were in line with the observed selectivity. Because of the high activity toward BCRP, we lacked the dynamic range needed to draw conclusions on favorable interactions; however, we identified amino acids in both P-gp and MRP2 that appear to be important for ligand recognition.

Dataset Information

Cloning and characterization of the rat multidrug resistance-associated protein 1.

Publications

Cloning and characterization of the rat multidrug resistance-associated protein 1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets