Consumer group consists of different people who change in consumption habits. This situation lead... more Consumer group consists of different people who change in consumption habits. This situation leads to overproduction or underproduction. Analyzation of this group is a very crucial factor so as to make production properly. In addition to the owner of the business should determine the eating habits of customers as likes or dislikes. End of the day, They have to check portion control. Method of menu engineering is based on profit and popularity of the type of food located in the menu.
Journal of visualized experiments : JoVE, Jan 16, 2017
Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in ma... more Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable. Recently, we expanded the pallet of NO• indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO•) probes (geNOps) that directly respond to cellular NO• fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO• signals in response to two different chemical NO•-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (N...
In this issue of Cell Chemical Biology, Navas-Navarro et al. (2016) demonstrate that fusion of en... more In this issue of Cell Chemical Biology, Navas-Navarro et al. (2016) demonstrate that fusion of engineered derivatives of the long-known jellyfish proteins green fluorescent protein (GFP) and aequorin yield optimized genetically encoded fluorescent probes for detecting Ca(2+) signals within the endoplasmic reticulum (ER) of living animals.
Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specifi... more Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results: Resveratrol and piceatannol selectively ...
Mitochondrial Ca(2+) uptake regulates diverse endothelial cell functions and has also been relate... more 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...
Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitrogl... more Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but l...
Changes in intra- and extracellular potassium ion (K) concentrations control many important cellu... more Changes in intra- and extracellular potassium ion (K) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K changes is severely limited by the lack of practicable detection methods. We developed K-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K signals. Hitherto unknown Ca-triggered, organelle-specific K changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diver...
American Journal of Physiology-Heart and Circulatory Physiology
We previously described a novel "chemogenetic" animal model of heart failure that recap... more We previously described a novel "chemogenetic" animal model of heart failure that recapitulates a characteristic feature commonly found in human heart failure: chronic oxidative stress. This heart failure model uses a chemogenetic approach to activate a recombinant yeast D-amino acid oxidase in rat hearts in vivo in order to generate oxidative stress, which then rapidly leads to the development of a dilated cardiomyopathy. Here we apply this new model to drug testing by studying its response to treatment with the angiotensin-II (Ang-II) receptor blocker valsartan, administered either alone or with the neprilysin inhibitor sacubitril. Echocardiographic and F18-deoxyglucose positron emission tomographic imaging revealed that valsartan in the presence or absence of sacubitril reverses the anatomical and metabolic remodeling induced by chronic oxidative stress. Markers of oxidative stress, mitochondrial function, and apoptosis- as well as classical heart failure biomarkers- al...
Consumer group consists of different people who change in consumption habits. This situation lead... more Consumer group consists of different people who change in consumption habits. This situation leads to overproduction or underproduction. Analyzation of this group is a very crucial factor so as to make production properly. In addition to the owner of the business should determine the eating habits of customers as likes or dislikes. End of the day, They have to check portion control. Method of menu engineering is based on profit and popularity of the type of food located in the menu.
Journal of visualized experiments : JoVE, Jan 16, 2017
Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in ma... more Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable. Recently, we expanded the pallet of NO• indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO•) probes (geNOps) that directly respond to cellular NO• fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO• signals in response to two different chemical NO•-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (N...
In this issue of Cell Chemical Biology, Navas-Navarro et al. (2016) demonstrate that fusion of en... more In this issue of Cell Chemical Biology, Navas-Navarro et al. (2016) demonstrate that fusion of engineered derivatives of the long-known jellyfish proteins green fluorescent protein (GFP) and aequorin yield optimized genetically encoded fluorescent probes for detecting Ca(2+) signals within the endoplasmic reticulum (ER) of living animals.
Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specifi... more Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results: Resveratrol and piceatannol selectively ...
Mitochondrial Ca(2+) uptake regulates diverse endothelial cell functions and has also been relate... more 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...
Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitrogl... more Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but l...
Changes in intra- and extracellular potassium ion (K) concentrations control many important cellu... more Changes in intra- and extracellular potassium ion (K) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K changes is severely limited by the lack of practicable detection methods. We developed K-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K signals. Hitherto unknown Ca-triggered, organelle-specific K changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diver...
American Journal of Physiology-Heart and Circulatory Physiology
We previously described a novel "chemogenetic" animal model of heart failure that recap... more We previously described a novel "chemogenetic" animal model of heart failure that recapitulates a characteristic feature commonly found in human heart failure: chronic oxidative stress. This heart failure model uses a chemogenetic approach to activate a recombinant yeast D-amino acid oxidase in rat hearts in vivo in order to generate oxidative stress, which then rapidly leads to the development of a dilated cardiomyopathy. Here we apply this new model to drug testing by studying its response to treatment with the angiotensin-II (Ang-II) receptor blocker valsartan, administered either alone or with the neprilysin inhibitor sacubitril. Echocardiographic and F18-deoxyglucose positron emission tomographic imaging revealed that valsartan in the presence or absence of sacubitril reverses the anatomical and metabolic remodeling induced by chronic oxidative stress. Markers of oxidative stress, mitochondrial function, and apoptosis- as well as classical heart failure biomarkers- al...
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