20240722_183501

Component	Description / assay
ATP synthesis (=the capacity of the respiratory chain to generate ATP within the mitochondria.)	[각 MC chain 별 ATP Production] complex I-linked ADP phosphorylation with glutamate/malate as substrate Complex II/III-linked ADP phosphorylation using succinate as substrate complex IV-linked ADP phosphorylation with ascorbate/TMPD as substrate. Mortiboys: performed on digitonin treated fibroblasts as described elsewhere (PMID 19918262) (heart) there is complete turnover of the myocardial ATP pool in every 10 s (56), the number of ATP molecules produced for each oxygen atom consumed by the mitochondria (termed the P/O ratio) can vary [(Salin, 2015 #1165)]. One factor underlying this variation is the number of H+ pumped into the inter-membrane space per unit of oxygen consumed by the ETC (the H/O ratio is substrate-specific. For example, mitochondria oxidizing succinate exhibit an H/O ratio of 6, whereas this ratio is 10 when the substrate is pyruvate/malate [22]. Despite well-established methods to determine which metabolic substrate is being oxidized (box 2), and direct evidence of substrate type [22,42], most studies of energy metabolism do not take into the efficiency of ATP production. A second potential mechanism alters the proton pumps. This slippage results in either fewer protons being pumped into the space per electron transferred by the ETC, or fewer ATP molecules produced through ATP synthase [22,43–45]. However, while such slip reactions can occur in vitro, their importance under physiological conditions remains uncertain [22]. Assay: below luciferase assay + glutamate/malate 도
ATP Basal = [total cellular ATP] :	MPP+ (MPTP's active metabolite) causes a rapid and profound depletion of cellular ATP in cultured cell models, including hepatocytes (Di Monte et al. 1986), in brain synaptosomal preparations (Scotcher et al. 1990), and in mouse brain tissues (Chan et al. 1991). In mice, however, MPTP causes only a mild (~20% reduction) in striatal and midbrain ATP levels (Chan et al. 1991). Human plasma ATP concentration is reported in many studies as roughly 1000 nmol/L., in brain ? In house; ATP detected in plasma [ATP assay] There are several ATP tests, such as colorimetric, fluorescence, and bioluminescence analyses. As bioluminescent ATP test has the dvantages such as high sensitivity, simple and homogeneous protocol, and rapid operation, the bioluminescent ATP assay is widely used.
Luciferin + Ultra-Glo™ Luciferase + ATP / O2 / Mg²⁺ → Oxyluciferin + Light (inline chemistry schematic; preserved as body_r02 evidence)
The bioluminescent ATP assay uses the luminescence reaction of the firefly luciferase enzyme to measure ATP amount inside cells. When the reagent is added, living cells are lysed and ATP is released. Eg: (Barksdale, 2010 #1502) ATPlite kit (Perkin Elmer) (MORtiboys) Nishimura Taka: ATP is affected by too many factors (hormone, diurnal variability, easily degraded) and ATP/ADP might be more meaningful?
MC I subunit	20 kDa ND6 protein (Grünewald, 2016 #1071), midbrain postmortem (both formalin-fixed & frozen samples) : anti-NDUFB8 (NADHdehydrogenase 1 β subcomplex 8; ab110242) and anti-NDUFA13 (NADH dehydrogenase 1 alpha subcomplex 13; ab110240); CII, anti-SDHA (succinate dehydrogenase complexsubunit A; ab14715); CIII, anti-UQCRC2 (ubiquinol–cytochrome c reductase core protein II; ab14745); and CIV, anti-COXI (cytochrome c oxidase subunit I; ab14705). These subunits are situated in functional domains correlating with the overall function of the complexes. Cf) To rule out that the samples' PMIs negatively impacted on the abundance of the targeted protein, we performed Spearman correlation analyses with PMI delay and mitochondrial protein levels as variables. This did not show a significant decline in protein levels as a result of higher PMI intervals. below table is from Keeney 2006 (fig 2) Product: (including Anti-NDUFB8 antibody) for antibody cocktail 39KDa 30KDa 20KDa 15KDa 8KDa CTL 2101 / 401 2034 / 432 3071 / 221 2622 / 170 2942 / 333 PD 2326 / 259 2290 / 269 3410 / 255 2796 / 196 1959 / 626 P-value 0.15 0.13 0.005 0.05 0.0004 Figure 2. Western blot of CTL and PD mitochondria for complex I subunits. The same mitochondrial preparations from frontal cortex samples were used for all figures. The blot images are shown for CTL and PD mitochondrial samples, and localization of the complex I subunit bands was performed as described previously (Triepels et al., 2001; Murray et al., 2003). The table at the bottom gives the actual mean band densities with the SDs underneath; p-values indicated highly significant differences for the 20 and 8 kDa subunits. See Materials and Methods for details. Resources for below: Wirth 2016
NDUFA3	NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3,
NDUFA13	(NADH dehydrogenase 1 alpha subcomplex 13;)
NDUFB8	(NADH dehydrogenase 1 β subcomplex 8;) = NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, complex I), 19 kDa, Accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), that is believed not to be involved in catalysis
NDUFS1	The protein encoded by this gene belongs to the complex I 75 kDa subunit family. This protein has NADH dehydrogenase activity and oxidoreductase activity. It transfers electrons from NADH to the respiratory chain. The immediate electron acceptor for the enzyme is believed to be ubiquinone. This protein is the largest subunit of complex I and it is a component of the iron-sulfur (IP) fragment of the enzyme. It may form part of the active site crevice where NADH is oxidized. Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) that is believed to belong to the minimal assembly required for catalysis https://www.mybiosource.com/ndufs1-human-elisa-kits/nadh-dehydrogenase-ubiquinone-fe-s-protein-1-75kda-nadh-coenzyme-q-reductase/9316968, ELISA kit)
NDUFS4
NDUFS7	25 kDa, also known as NADH-ubiquinone oxidoreductase 20 kDa subunit, Complex I-20kD (CI-20kD), or PSST subunit, one of the ubiquinone binding sites
NDUFS8	= TYKY, one of the ubiquinone binding sites
SDHA (succinate dehydrogenase complex subunit A)
UQCRC2 (ubiquinol-cytochrome c reductase core protein II)	AO (Antimycin A/Oligomycin A) treatment induces depolarization in mitochondria by inhibiting complex III and V respectively (Misa from 2018 Ordureau).
COXI (cytochrome c oxidase subunit I)
(Complex V)	AO (Antimycin A/Oligomycin A) treatment induces depolarization in mitochondria by inhibiting complex III and V respectively (Misa from 2018 Ordureau).
biogenesis (transcription factors)	PGC-1a, NRF1/2 or Tfam, TFB2M (mtDNA transcription factors), PGC-1α induces mitochondrial biogenesis by stimulating the genetic expression of two transcription factors called nuclear respiratory factor 1 (NRF1) and nuclear respiratory factor 2 (NRF2). NRF1 and NRF2 ensure the coordinated expression of nuclear and mitochondrial genomes.
mitophagy	(cf: 다음은 mito가 아니라 parkin의 function임!) EGFP-Parkin variants to relocalize from the cytosol to perinuclear regions, using high content imaging (HCI). This objective assay relies on the ratio of EGFP-Parkin signal intensities between cytosol and nucleus correlates with activation and translocation of Parkin to damaged mitochondria. DRP1
mtDNA copy number / mass	mtDNA COPY number using two different mtDNA markers (COX and CYTB) To confirm whether mitochondrial number is actually reduced , real-time quantitative PCR Measured in human WBC (Davis, 2020 #1090) [commercial kit] https://www.detroitrandd.com/store/human-mitochondrial-dna This DNA analysis kit is for the determination of human mitochondrial DNA copy number in vivo and in vitro, by the comparison of mitochondrial (mt) and nuclear (n) DNA content using real-time qPCR. (Grünewald, 2016 #935): A triplex real-time PCR assay for simultaneous detection of 3 targets within the mitochondrial genome (ND1 [NADH dehydrogenase1], ND4, and D-loop) was performed.15 The quantification of mtDNA was conducted as previously published.16 For mtDNA copy number quantification, the ND1 concentration relative to nuclear DNA. To detect mt DNA major arc deletions, the ND1:ND4 was determined. The third target amplified in the triplex real-time PCR was a small area of the noncoding region that is preserved in 99% of deleted mtDNA species reported. As an integral part of mtDNA structure and replication, a linear DNA molecule, that is, 7S DNA, is incorporated in this particular noncoding region of the mtDNA molecule, resulting in a triple-stranded structure denominated as "D-loop." 17,19 The D-loop:ND1 ratio is therefore representative of the mtDNA replication status in a given cell, with a higher ratio representing more actively transcribing or replicating mtDNA molecules.20 상식: DNA Copy number 에는 qPCR Otake: PhRET (actually, former CVM-DDU that you may not know) has developed mtDNA assay. It just quantitates MT-ND1 and MT-ND4 copy number by real-time qPCR. Genomic region B2M is selected as internal control. (Qi, 2023 #2429): = in PD PBMC (Tresse, 2023 #2491) ↓ ND4, ND5 (also, in mice looked at D-Loop, 8-OHdG (pseudocoloured green), Coal (mitochondria, pseudocoloured red) in mice midbrains (Sanders, 2014 #1411) Methods ARP staining: detects abasic sites (aldehyde-reactive probe), ARP reacts with a ring-opened sugar...

Uncertain Spans

location	transcription	uncertainty
ATP synthesis row	the H/O ratio is substrate-specific. For example, mitochondria oxidizing succinate exhibit an H/O ratio of 6, whereas this ratio is 10 when the substrate is pyruvate/malate [22].	Specific numeric ratios (6 / 10) match the Salin 2015 #1165 highlighted text but are small.
MC I subunit row table	CTL/PD numeric pairs (2101 / 401, etc.)	Each cell shows a pair “mean / SD”; the slash separator is faint and could be a comma.
Triplex PCR	ND1, ND4, D-loop	The D-loop annotation could be “D-loop” or “D loop”; preserved verbatim.
mtDNA copy number row	Davis, 2020 #1090	Trailing close-paren after the citation is faint.
mitophagy row Korean fragment	(cf: 다음은 mito가 아니라 parkin의 function임!)	The exclamation mark could be a parenthesis-mark; preserved verbatim.