Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclic ADP-ribose (cADPR) is an intracellular messenger that triggers the release of calcium ions from intracellular stores in a variety of cell types. The fluorometric cycling assay has become the preferred method for measuring cADPR due to its high level of sensitivity (in the sub-nanomolar range) and its use of commercially available reagents. Additionally, the assay is performed in multiwell plates, making it suitable for high throughput screening using a fluorescence plate reader. The findings reported in this paper present several problems that may be encountered during various stages of the assay, and provide solutions to these problems. Modifications to the assay address reduced recovery of sample and cADPR with removal of perchloric acid (PCA) using organic solvent, reduction in diaphorase activity with heat treatment, and effects on resorufin fluorescence by pH range. Using these modifications, we report an increase of approximately 15% in recovery of brain cADPR, and show that between-subject variability is greatly reduced. We hope that these observations will encourage more widespread application of this valuable assay.
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PMID:Modifications to increase the efficiency of the fluorometric cycling assay for cyclic ADP-ribose. 1701 83

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.
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PMID:Calcium signalling and pancreatic cell death: apoptosis or necrosis? 1743 16

Cyclic ADP-ribose (cADPR) is a Ca(2+)-mobilizing second messenger involved in the regulation of various physiological processes. The ability to detect changes in endogenous cADPR is a fundamental step in the identification of its role in signal transduction triggered by hormones and other stimuli. Because the intracellular concentration of cADPR can be very low, depending on the expression level of the ADP-ribosyl cyclase activity (forming cADPR and nicotinamide from NAD) in the cell type of interest, very sensitive and selective methods are required. The method presented here exploits the ability of the ADP-ribosyl cyclase to catalyze the reverse reaction (i.e., to synthesize NAD stoichiometrically starting from cADPR) in the presence of an excess of nicotinamide. The generation of NAD can be coupled to a cycling assay using the enzymes alcohol dehydrogenase and diaphorase. The former reduces NAD to NADH in the presence of ethanol and the latter oxidizes NADH to NAD in the presence of resazurin and flavin mononucleotide. The formation of the fluorescent reduced resazurin (resofurin) can be detected with a plate reader. Thus, this cycling assay for cADPR determination can be considered a high-throughput method, potentially screening cADPR concentration simultaneously in many samples.
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PMID:Cycling assay for determining intracellular cyclic adp-ribose levels. 2373 16

Oxygen and glucose deprivation (OGD) due to insufficient blood circulation can decrease cancer cell survival and proliferation in solid tumors. OGD increases the intracellular [AMP]/[ATP] ratio, thereby activating the AMPK. In this study, we have investigated the involvement of NQO1 in OGD-mediated AMPK activation and cancer cell death. We found that OGD activates AMPK in an NQO1-dependent manner, suppressing the mTOR/S6K/4E-BP1 pathway, which is known to control cell survival. Thus, the depletion of NQO1 prevents AMPK-induced cancer cell death in OGD. When we blocked OGD-induced Ca(2+)/CaMKII signaling, the NQO1-induced activation of AMPK was attenuated. In addition, when we blocked the RyR signaling, the accumulation of intracellular Ca(2+) and subsequent activation of CaMKII/AMPK signaling was decreased in NQO1-expressing cells under OGD. Finally, siRNA-mediated knockdown of CD38 abrogated the OGD-induced activation of Ca(2+)/CaMKII/AMPK signaling. Taken together, we conclude that NQO1 plays a key role in the AMPK-induced cancer cell death in OGD through the CD38/cADPR/RyR/Ca(2+)/CaMKII signaling pathway.
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PMID:NQO1-induced activation of AMPK contributes to cancer cell death by oxygen-glucose deprivation. 2558 69