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Identification and characterization of primate P-glycoprotein.

Yimam M - J Pharmacol Pharmacother (2015 Jul-Sep)

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, University of Washington, Washington, USA.

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Sir, Achieving the therapeutic goal of human immunodeficiency virus (HIV) infection is complex in nature due to the potential drug–drug interactions, modulations in the cytochrome P450 enzyme and/or drug transporters such as P-glycoprotein... The sequence of the P-glycoprotein encoding gene, MDR1, is well characterized in humans; however, no complete sequence has been reported for the MDR1 gene in non-human primates particularly of the most versatile noble model such as Macaca nemestrinas... Existing human MDR1 full-length sense (F6) and antisense (R11) primers were selected and subsequently, each tissue was tested as shown in the gel electrophoresis [Figure 1] where two different sizes, approximately 2kb and 3.8kb, fragments of P-glycoprotein were observed for brain and kidney... Reproducibility for the presence of the fragments was confirmed in additional run as in Figure 2... After analyzing the electropherogram for its nucleotide signals and purity, sequence text files were blasted with the M. fascicularies MDR1 coding region sequence (accession #AF537134) and M. nemestrinas MDR1 sequence was constructed by aligning both the forward and reverse primers... A total of 3843 bases were found for all the macaques (n = 3)... Further amino acid blast analysis indicated the presence of changes in amino acids at 185 and 1277 positions... While the changes in nucleotide at positions 540, and 544/5 are the most frequently observed polymorphisms in the human MDR1gene, the single nucleotide polymorphism (SNP) at position 3829 A → G could be the significant variant between the two species (Homo sapiens and M. nemestrinas) leading to an amino acid change at position1277 Thr → Ala which could in turn affect the fate of an experimental drug PK/PD... Significant data have been reported on the most widely studied SNPs in MDR1 such as C3435CT and its association with Lopinavir/Ritonavir monotherapy failure in HIV-1 patients, G2677T polymorphism in susceptibility of myeloid leukemia, C1236T SNP in HIV-1 positive children causing significant reduction in Lopinavir plasma concentration affecting the virological response to highly active antiretroviral therapy are some of the direct impacts of polymorphisms influencing the pharmacodynamics and pharmacokinetics outcome of a therapy... P-glycoprotein may limit penetration of PIs into several therapeutically relevant compartments and thus diminishing the chance of achieving a curative treatment regimen... As a result, identifying and characterizing the presence of P-glycoprotein, an ATP-dependent multidrug efflux membrane pump with extensive substrate specificity, could guide in designing a target specific therapeutic regimen in patients favoring a good pharmacological outcome specifically for those organs that provide potential HIV sanctuary sites in the body... However, further elucidation for sequence confirmation of the mini-P-glycoprotein in the brain and/or kidney and functional analysis of A3829G are a necessity.

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Primate MDR1 cDNA gel electrophoresis from Liver. cDNA extracted and purified by Qiagene kit was amplified by fail safe PCR
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Figure 3: Primate MDR1 cDNA gel electrophoresis from Liver. cDNA extracted and purified by Qiagene kit was amplified by fail safe PCR

Mentions: Primate MDR1cDNA Sequence for M. nemestrinas was constructed for sequence homology analysis with the human MDR1. Both forward and reverse primers were designed as pF11- AGT GTC CAG GTC GGA GCA AAG CGC CAG TGA A and pR11- TTC ACT GGC GCT TTG CTC CAG CCT GGA CAC T, based on M. fascicularies MDR1 cDNA sequence and acquired from Invitrogen. 3.2 pmol primers and 150 ng purified PCR gel (Qiagen kit) [Figure 3] product were used for sequencing (ABI Prism, Model 3100, Version 3.7). After analyzing the electropherogram for its nucleotide signals and purity, sequence text files were blasted with the M. fascicularies MDR1 coding region sequence (accession #AF537134) and M. nemestrinas MDR1 sequence was constructed by aligning both the forward and reverse primers. A total of 3843 bases were found for all the macaques (n = 3). Sequence analyses of human MDR1 and M. nemestrinas pMDR1 coding region from liver cDNA were then conducted and there were found more than 99% sequence homology [Table 1] with four nucleotide alterations at position 540, 544/5, and 3829. Further amino acid blast analysis indicated the presence of changes in amino acids at 185 and 1277 positions. While the changes in nucleotide at positions 540, and 544/5 are the most frequently observed polymorphisms in the human MDR1gene,[5] the single nucleotide polymorphism (SNP) at position 3829 A → G could be the significant variant between the two species (Homo sapiens and M. nemestrinas) leading to an amino acid change at position1277 Thr → Ala which could in turn affect the fate of an experimental drug PK/PD.


Identification and characterization of primate P-glycoprotein.

Yimam M - J Pharmacol Pharmacother (2015 Jul-Sep)

Primate MDR1 cDNA gel electrophoresis from Liver. cDNA extracted and purified by Qiagene kit was amplified by fail safe PCR
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4544137&req=5

Figure 3: Primate MDR1 cDNA gel electrophoresis from Liver. cDNA extracted and purified by Qiagene kit was amplified by fail safe PCR
Mentions: Primate MDR1cDNA Sequence for M. nemestrinas was constructed for sequence homology analysis with the human MDR1. Both forward and reverse primers were designed as pF11- AGT GTC CAG GTC GGA GCA AAG CGC CAG TGA A and pR11- TTC ACT GGC GCT TTG CTC CAG CCT GGA CAC T, based on M. fascicularies MDR1 cDNA sequence and acquired from Invitrogen. 3.2 pmol primers and 150 ng purified PCR gel (Qiagen kit) [Figure 3] product were used for sequencing (ABI Prism, Model 3100, Version 3.7). After analyzing the electropherogram for its nucleotide signals and purity, sequence text files were blasted with the M. fascicularies MDR1 coding region sequence (accession #AF537134) and M. nemestrinas MDR1 sequence was constructed by aligning both the forward and reverse primers. A total of 3843 bases were found for all the macaques (n = 3). Sequence analyses of human MDR1 and M. nemestrinas pMDR1 coding region from liver cDNA were then conducted and there were found more than 99% sequence homology [Table 1] with four nucleotide alterations at position 540, 544/5, and 3829. Further amino acid blast analysis indicated the presence of changes in amino acids at 185 and 1277 positions. While the changes in nucleotide at positions 540, and 544/5 are the most frequently observed polymorphisms in the human MDR1gene,[5] the single nucleotide polymorphism (SNP) at position 3829 A → G could be the significant variant between the two species (Homo sapiens and M. nemestrinas) leading to an amino acid change at position1277 Thr → Ala which could in turn affect the fate of an experimental drug PK/PD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, University of Washington, Washington, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Sir, Achieving the therapeutic goal of human immunodeficiency virus (HIV) infection is complex in nature due to the potential drug–drug interactions, modulations in the cytochrome P450 enzyme and/or drug transporters such as P-glycoprotein... The sequence of the P-glycoprotein encoding gene, MDR1, is well characterized in humans; however, no complete sequence has been reported for the MDR1 gene in non-human primates particularly of the most versatile noble model such as Macaca nemestrinas... Existing human MDR1 full-length sense (F6) and antisense (R11) primers were selected and subsequently, each tissue was tested as shown in the gel electrophoresis [Figure 1] where two different sizes, approximately 2kb and 3.8kb, fragments of P-glycoprotein were observed for brain and kidney... Reproducibility for the presence of the fragments was confirmed in additional run as in Figure 2... After analyzing the electropherogram for its nucleotide signals and purity, sequence text files were blasted with the M. fascicularies MDR1 coding region sequence (accession #AF537134) and M. nemestrinas MDR1 sequence was constructed by aligning both the forward and reverse primers... A total of 3843 bases were found for all the macaques (n = 3)... Further amino acid blast analysis indicated the presence of changes in amino acids at 185 and 1277 positions... While the changes in nucleotide at positions 540, and 544/5 are the most frequently observed polymorphisms in the human MDR1gene, the single nucleotide polymorphism (SNP) at position 3829 A → G could be the significant variant between the two species (Homo sapiens and M. nemestrinas) leading to an amino acid change at position1277 Thr → Ala which could in turn affect the fate of an experimental drug PK/PD... Significant data have been reported on the most widely studied SNPs in MDR1 such as C3435CT and its association with Lopinavir/Ritonavir monotherapy failure in HIV-1 patients, G2677T polymorphism in susceptibility of myeloid leukemia, C1236T SNP in HIV-1 positive children causing significant reduction in Lopinavir plasma concentration affecting the virological response to highly active antiretroviral therapy are some of the direct impacts of polymorphisms influencing the pharmacodynamics and pharmacokinetics outcome of a therapy... P-glycoprotein may limit penetration of PIs into several therapeutically relevant compartments and thus diminishing the chance of achieving a curative treatment regimen... As a result, identifying and characterizing the presence of P-glycoprotein, an ATP-dependent multidrug efflux membrane pump with extensive substrate specificity, could guide in designing a target specific therapeutic regimen in patients favoring a good pharmacological outcome specifically for those organs that provide potential HIV sanctuary sites in the body... However, further elucidation for sequence confirmation of the mini-P-glycoprotein in the brain and/or kidney and functional analysis of A3829G are a necessity.

No MeSH data available.