Dataset Information

Cytochrome C Oxidase Subunit 1, Internal Transcribed Spacer 1, Nicotinamide Adenine Dinucleotide Hydrogen Dehydrogenase Subunits 2 and 5 of Clonorchis sinensis Ancient DNA Retrieved from Joseon Dynasty Mummy Specimens.

ABSTRACT: We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for molecular diagnosis between C. sinensis and the other trematode parasite species. The current result could improve our knowledge about genetic history of C. sinensis.

SUBMITTER: Hong JH

PROVIDER: S-EPMC6535405 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Cytochrome C Oxidase Subunit 1, Internal Transcribed Spacer 1, Nicotinamide Adenine Dinucleotide Hydrogen Dehydrogenase Subunits 2 and 5 of Clonorchis sinensis Ancient DNA Retrieved from Joseon Dynasty Mummy Specimens.

Hong Jong Ha JH Oh Chang Seok CS Chai Jong Yil JY Seo Min M Shin Dong Hoon DH

Journal of Korean medical science 20190527 20

We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for mol ...[more]

PMID: 31124326

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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

Cytochrome C Oxidase Subunit 1, Internal Transcribed Spacer 1, Nicotinamide Adenine Dinucleotide Hydrogen Dehydrogenase Subunits 2 and 5 of Clonorchis sinensis Ancient DNA Retrieved from Joseon Dynasty Mummy Specimens.

Publications

Cytochrome C Oxidase Subunit 1, Internal Transcribed Spacer 1, Nicotinamide Adenine Dinucleotide Hydrogen Dehydrogenase Subunits 2 and 5 of <i>Clonorchis sinensis</i> Ancient DNA Retrieved from Joseon Dynasty Mummy Specimens.

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