Dataset Information

CDNA Isolation and Expression of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Cytochrome P450 Reductase Gene in the Chagas Disease Vector Triatoma infestans.

ABSTRACT: Pyrethroid resistance has been detected in Triatoma infestans (Hemiptera: Reduviidae), which was atributed to target site insensitivity and increased oxidative metabolism of the insecticide by cytochrome P450s. Nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase (CPR) plays an essential role in transferring electrons from NADPH to the P450-substrate complex. In this study, the full length CPR cDNA of T. infestans was isolated and gene expression was determined by quantitative polymerase chain reaction. The open reading frame is 2,046 bp long, encoding a protein of 682 amino acids. Amino acid sequence analysis indicates that the T. infestans CPR and the putative Rhodnius prolixus and Triatoma dimidiata CPRs present conserved ligand-binding domains. Congruent with a previous study of our laboratory, in which the expression of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6 genes) was induced by deltamethrin, the levels of T. infestans CPR mRNA were upregulated in the fat body of fifth instar nymphs after topical application of deltamethrin. Besides, as it was observed in the CYP4EM7 gene, it was detected overexpression of the CPR gene in the most resistant strain of T. infestans included in the study. These results suggest that CPR plays an essential role in P450-mediated resistance of T. infestans to insecticides.

SUBMITTER: Grosso CG

PROVIDER: S-EPMC5930885 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Publications

cDNA Isolation and Expression of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Cytochrome P450 Reductase Gene in the Chagas Disease Vector Triatoma infestans.

Grosso Carla G CG Stroppa María M MM Varela Gonzalo M GM García Beatriz A BA

The American journal of tropical medicine and hygiene 20180118 3

Pyrethroid resistance has been detected in Triatoma infestans (Hemiptera: Reduviidae), which was atributed to target site insensitivity and increased oxidative metabolism of the insecticide by cytochrome P450s. Nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase (CPR) plays an essential role in transferring electrons from NADPH to the P450-substrate complex. In this study, the full length CPR cDNA of T. infestans was isolated and gene expression was ...[more]

PMID: 29363459

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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

Dataset Information

CDNA Isolation and Expression of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Cytochrome P450 Reductase Gene in the Chagas Disease Vector Triatoma infestans.

Publications

cDNA Isolation and Expression of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Cytochrome P450 Reductase Gene in the Chagas Disease Vector <i>Triatoma infestans</i>.

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