application-genetically-encoded-fluorescent-nitric-oxide-no-probes

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Medical University of Graz

Cleistocalyx nervosum var. paniala, an edible fruit found in Northern Thailand, contains high amounts of phenolic compounds with in vitro antioxidant activity. The aqueous extract of the ripe fruit was evaluated for its safety and beneficial effects using genotoxicity and toxicity tests. The C. nervosum extract was not only non-mutagenic in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of metabolic activation, but exhibited also moderate antimutagenic effects against aflatoxin B1 and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline-induced mutagenesis. Electrospray ionization-mass spectrometric analysis revealed the major anthocyanins, which included cyanidin-3,5-diglucoside, cyanidin-3-glucoside and cyanidin-5-glucoside. The administration of C. nervosum at concentration of 5,000 mg/kg bw did not induce acute toxicity in rats. A liver micronucleus test was performed to detect clastogenicity and anticlastogenicity. The extract in the dose of 1,000 mg/kg did not cause micronucleus formation in the liver of rats. Furthermore, in rats administered 100-1,000 mg/kg of the extract, no anticlastogenic effect against diethylnitrosamine-induced hepatic micronucleus formation was observed. These studies provide data concerning the safety and antimutagenic potency of an aqueous extract of C. nervosum fruit.

Assessment of genotoxicity and antigenotoxicity of an aqueous extract of Cleistocalyx nervosum var. paniala in in vitro and in vivo models.

Nitric oxide () is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca(2+) sensor allowed us to visualize and Ca(2+) signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics.

Mitochondrial Ca(2+) uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO(•)) production. However, it is not entirely clear if the organelles support or counteract NO(•) biosynthesis by taking up Ca(2+). The objective of this study was to verify whether or not mitochondrial Ca(2+) uptake influences Ca(2+)-triggered NO(•) generation by endothelial NO(•) synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO(•) probes, the geNOps, and Ca(2+) sensors to monitor single cell NO(•) and Ca(2+) dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP3)-generating agonist. Mitochondrial Ca(2+) uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca(2+) uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca(2+) uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca(2+)-triggered NO(•) increase independently of global cytosolic Ca(2+) signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca(2+) sequestration and Ca(2+)-induced NO(•) signals. The positive correlation between mitochondrial Ca(2+) elevation and NO(•) production was independent of eNOS phosphorylation at serine(1177). Our findings emphasize that manipulating mitochondrial Ca(2+) uptake may represent a novel strategy to control eNOS-mediated NO(•) production.

Article	Year
Assessment of genotoxicity and antigenotoxicity of an aqueous extract of Cleistocalyx nervosum var. paniala in in vitro and in vivo models. Interdisciplinary toxicology\| PubMed ID: 23554564	2012
Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics. Nature communications\| PubMed ID: 26842907	2016
Intact mitochondrial Ca(2+) uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS). Free radical biology & medicine\| PubMed ID: 27923677	2017

Suphachai Charoensin

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