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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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Tissue expression of P-glycoprotein in primates. Total RNA was extracted from 50mg of liver, brain, kidney and intestine using stratagen RNA extraction protocol and amplified by superscript II one step RT-PCR protocol from Invitrogen
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Figure 1: Tissue expression of P-glycoprotein in primates. Total RNA was extracted from 50mg of liver, brain, kidney and intestine using stratagen RNA extraction protocol and amplified by superscript II one step RT-PCR protocol from Invitrogen

Mentions: To demonstrate the assumption, specimens from freshly frozen liver, brain, kidney, and intestine of M. nemestrinas (n = 3) were screened for the expression of P-glycoprotein. Tissues were provided by the Washington Regional Primate Research Center at the University of Washington. tRNA was extracted from 50 mg of each tissues by stratagen RNA extraction according to the manufacturers protocol and amplified by superscript II one step RT-PCR protocol from Invitrogen. 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. All the forward and reverse primers were acquired from Invitrogen (Life technologies, Grand Island, NY). These findings suggest that in addition to the full length P-glycoprotein, there could be a possibility of existence of a smaller size transporter at least in the brain and kidney of the nemestrinas. Substantiating these findings, the existence of another form of P-glycoprotein with shorter length (mini p-glycoprotein) in murine leukemia cells and human natural killer cells has been reported in some articles with similar function to that of the classic 3.8kb P-glycoprotein.[34]


Identification and characterization of primate P-glycoprotein.

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

Tissue expression of P-glycoprotein in primates. Total RNA was extracted from 50mg of liver, brain, kidney and intestine using stratagen RNA extraction protocol and amplified by superscript II one step RT-PCR protocol from Invitrogen
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Tissue expression of P-glycoprotein in primates. Total RNA was extracted from 50mg of liver, brain, kidney and intestine using stratagen RNA extraction protocol and amplified by superscript II one step RT-PCR protocol from Invitrogen
Mentions: To demonstrate the assumption, specimens from freshly frozen liver, brain, kidney, and intestine of M. nemestrinas (n = 3) were screened for the expression of P-glycoprotein. Tissues were provided by the Washington Regional Primate Research Center at the University of Washington. tRNA was extracted from 50 mg of each tissues by stratagen RNA extraction according to the manufacturers protocol and amplified by superscript II one step RT-PCR protocol from Invitrogen. 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. All the forward and reverse primers were acquired from Invitrogen (Life technologies, Grand Island, NY). These findings suggest that in addition to the full length P-glycoprotein, there could be a possibility of existence of a smaller size transporter at least in the brain and kidney of the nemestrinas. Substantiating these findings, the existence of another form of P-glycoprotein with shorter length (mini p-glycoprotein) in murine leukemia cells and human natural killer cells has been reported in some articles with similar function to that of the classic 3.8kb P-glycoprotein.[34]

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.