A new simple fluorimetric method to assay cytosolic ATP content: application to durum wheat seedlings to assess modulation of mitochondrial potassium channel and uncoupling protein activity under hyperosmotic stress

Biologia ◽  
2013 ◽  
Vol 68 (3) ◽  
Author(s):  
Mario Soccio ◽  
Maura Laus ◽  
Daniela Trono ◽  
Donato Pastore
Keyword(s):  
Durum Wheat ◽  
Uncoupling Protein ◽  
Hyperosmotic Stress ◽  
K Channel ◽  
Acid Extraction ◽  
Atp Content ◽  
Protein Activity ◽  
Simple Method ◽  
Wheat Seedlings ◽  
Stress Dependent

AbstractThe assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2.68 ± 0.04, 1.69 ± 0.12 (−40%) and 1.35 ± 0.16 (−50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K+ channel (PmitoKATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoKATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.

The Transcript Levels of two Plant Mitochondrial Uncoupling Protein (pUCP)-Related Genes are not Affected by Hyperosmotic Stress in Durum Wheat Seedlings Showing an Increased Level of pUCP Activity

2006 ◽  
Vol 26 (3) ◽  
pp. 251-261 ◽  
Author(s):  
Daniela Trono ◽  
Mario Soccio ◽  
Anna M. Mastrangelo ◽  
Vanessa De Simone ◽  
Natale Di Fonzo ◽  
...  
Keyword(s):  
Durum Wheat ◽  
Uncoupling Protein ◽  
Maximal Activity ◽  
Hyperosmotic Stress ◽  
Mitochondrial Uncoupling ◽  
Wheat Seedlings ◽  
Potential Measurement ◽  
Transcript Levels ◽  
Mitochondrial Uncoupling Protein ◽  
Plant Uncoupling Protein

Etiolated early seedlings of durum wheat submitted to moderate and severe salt (NaCl) and osmotic (mannitol) stress showed no relevant increase of both transcript levels of two plant uncoupling protein (pUCP)-related genes and maximal pUCP activity in purified mitochondria (which estimates protein level); contrarily, pUCP functioning due to endogenous free fatty acids strongly increased. These results show that pUCP activation under hyperosmotic stress may be due to modulation of pUCP reaction rather than to an increased protein synthesis. Finally, a properly developed method, based on a single membrane potential measurement, to evaluate both pUCP maximal activity and functioning, is reported.

Mitochondrial proline oxidation is affected by hyperosmotic stress in durum wheat seedlings

2010 ◽  
Vol 157 (1) ◽  
pp. 1-11 ◽  
Author(s):  
M. Soccio ◽  
M.N. Laus ◽  
G.P. Spera ◽  
D. Trono ◽  
M. Pompa ◽  
...  
Keyword(s):  
Durum Wheat ◽  
Hyperosmotic Stress ◽  
Wheat Seedlings

scholarly journals Acute Oxygen Sensing in Heme Oxygenase-2 Null Mice

2006 ◽  
Vol 128 (4) ◽  
pp. 405-411 ◽  
Author(s):  
Patricia Ortega-Sáenz ◽  
Alberto Pascual ◽  
Raquel Gómez-Díaz ◽  
José López-Barneo
Keyword(s):  
Gene Expression ◽  
K Channel ◽  
Α Subunit ◽  
Organ Growth ◽  
K Channels ◽  
Channel Gene ◽  
Sensitive Organ ◽  
Stress Dependent ◽  
Acute Oxygen Sensing ◽  
O2 Sensing

Hemeoxygenase-2 (HO-2) is an antioxidant enzyme that can modulate recombinant maxi-K+ channels and has been proposed to be the acute O2 sensor in the carotid body (CB). We have tested the physiological contribution of this enzyme to O2 sensing using HO-2 null mice. HO-2 deficiency leads to a CB phenotype characterized by organ growth and alteration in the expression of stress-dependent genes, including the maxi-K+ channel α-subunit. However, sensitivity to hypoxia of CB is remarkably similar in HO-2 null animals and their control littermates. Moreover, the response to hypoxia in mouse and rat CB cells was maintained after blockade of maxi-K+ channels with iberiotoxin. Hypoxia responsiveness of the adrenal medulla (AM) (another acutely responding O2-sensitive organ) was also unaltered by HO-2 deficiency. Our data suggest that redox disregulation resulting from HO-2 deficiency affects maxi-K+ channel gene expression but it does not alter the intrinsic O2 sensitivity of CB or AM cells. Therefore, HO-2 is not a universally used acute O2 sensor.

Hyperosmotic stress-dependent NFκB activation is regulated by reactive oxygen species and IGF-1 in cultured cardiomyocytes

FEBS Letters ◽  
2006 ◽  
Vol 580 (18) ◽  
pp. 4495-4500 ◽  
Author(s):  
Verónica Eisner ◽  
Alfredo Criollo ◽  
Clara Quiroga ◽  
Claudio Olea-Azar ◽  
Juan Francisco Santibañez ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Hyperosmotic Stress ◽  
Oxygen Species ◽  
Stress Dependent ◽  
Cultured Cardiomyocytes

A simple method for acid-extraction of cadmium from tissue

1975 ◽  
Vol 13 (1) ◽  
pp. 97-100 ◽  
Author(s):  
Tore L. M. Syversen ◽  
Gaby B. Syversen
Keyword(s):  
Acid Extraction ◽  
Simple Method

Determination of Benzo (a) pyrene in Foods

1978 ◽  
Vol 61 (1) ◽  
pp. 129-135
Author(s):  
Yukio Saito ◽  
Hiroshi Sekita ◽  
Mitsuharu Takeda ◽  
Mitsuru Uchiyama
Keyword(s):  
Sulfuric Acid ◽  
Silica Gel ◽  
Column Chromatography ◽  
Analytical Method ◽  
Selective Extraction ◽  
Acid Extraction ◽  
Simple Method ◽  
Extraction Step ◽  
Concentrated Sulfuric Acid

Abstract An analytical method was developed for determining benzo (a) pyrene in foods, suitable for routine use. The method consists of 4 cleanup steps: (1) alkali cleavage of sample, (2) preliminary silica gel column chromatography, (3) selective extraction with concentrated sulfuric acid, and (4) further silica gel column chromatography. Recoveries of benzo- (a) pyrene added to 50 g (or 10 g) food at levels of 0.4 ppb (or 2 ppb) ranged from 70% for short-necked clam and mackerel to 85% for chicken meat. The sulfuric acid extraction step affords a simple method for isolating benzo (a)- pyrene from various kinds of interfering substances which could not be separated by existing methods.

scholarly journals The Molecular and Functional Characterization of the Durum Wheat Lipoxygenase TdLOX2 Suggests Its Role in Hyperosmotic Stress Response

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1233
Author(s):  
Valeria Menga ◽  
Daniela Trono
Keyword(s):  
Durum Wheat ◽  
Propyl Gallate ◽  
Superoxide Anion ◽  
Functional Characterization ◽  
High Sensitivity ◽  
Nacl Stress ◽  
Hyperosmotic Stress ◽  
Strong Increase ◽  
Superoxide Anion Generation ◽  
Oxidative Damages

In plants, lipoxygenases (LOXs) are involved in various processes, such as growth, development, and response to stress cues. In the present study, the expression pattern of six durum wheat LOX-encoding genes (TdLpx-B1.1, TdLpx-B1.2, TdLpx-A2, TdLpx-B2, TdLpx-A3 and TdLpx-B3) under hyperosmotic stress was investigated. With osmotic (0.42 M mannitol) and salt (0.21 M NaCl) stress imposed at the early stages of seedling growth, a strong induction of the TdLpx-A2 gene expression in the shoots paralleled an equally strong increase in the LOX activity. Enhanced levels of malondialdehyde (MDA) and increased rates of superoxide anion generation were also observed as a result of the stress imposition. Sequence analysis of the TdLOX2 encoded by the TdLpx-A2 gene revealed that it belonged to the type-1 9-LOX group. When overexpressed in E. coli, TdLOX2 exhibited normal enzyme activity, high sensitivity to specific LOX inhibitors, with 76% and 99% inhibition by salicylhydroxamic and propyl gallate, respectively, and a preference for linoleic acid as substrate, which was converted exclusively to its corresponding 13-hydroperoxide. This unexpected positional specificity could be related to the unusual TV/K motif that in TdLOX2 replaces the canonical TV/R motif of 9-LOXs. Treatment of seedlings with propyl gallate strongly suppressed the increase in LOX activity induced by the hyperosmotic stress; the MDA accumulation was also reduced but less markedly, whereas the rate of superoxide anion generation was even more increased. Overall, our findings suggest that the up-regulation of the TdLpx-A2 gene is a component of the durum wheat response to hyperosmotic stress and that TdLOX2 may act by counteracting the excessive generation of harmful reactive oxygen species responsible for the oxidative damages that occur in plants under stress.

In Phosphorylating Acanthamoeba castellanii Mitochondria the Sensitivity of Uncoupling Protein Activity to GTP Depends on the Redox State of Quinone

2005 ◽  
Vol 37 (2) ◽  
pp. 97-107 ◽  
Author(s):  
Wieslawa Jarmuszkiewicz ◽  
Aleksandra Swida ◽  
Malgorzata Czarna ◽  
Nina Antos ◽  
Claudine M. Sluse-Goffart ◽  
...  
Keyword(s):  
Redox State ◽  
Uncoupling Protein ◽  
Acanthamoeba Castellanii ◽  
Protein Activity

Salt-induced accumulation of glycine betaine is inhibited by high light in durum wheat

2011 ◽  
Vol 38 (2) ◽  
pp. 139 ◽  
Author(s):  
Petronia Carillo ◽  
Danila Parisi ◽  
Pasqualina Woodrow ◽  
Giovanni Pontecorvo ◽  
Giuseppina Massaro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Durum Wheat ◽  
Glycine Betaine ◽  
High Light ◽  
Light Conditions ◽  
Salt Treatment ◽  
Wheat Seedlings ◽  
Triticum Durum Desf ◽  
Tyrosine Content

In this study, we determined the effects of both salinity and high light on the metabolism of durum wheat (Triticum durum Desf. cv. Ofanto) seedlings, with a special emphasis on the potential role of glycine betaine in their protection. Unexpectedly, it appears that high light treatment inhibits the synthesis of glycine betaine, even in the presence of salt stress. Additional solutes such as sugars and especially amino acids could partially compensate for the decrease in its synthesis upon exposure to high light levels. In particular, tyrosine content was strongly increased by high light, this effect being enhanced by salt treatment. Interestingly, a large range of well-known detoxifying molecules were also not induced by salt treatment in high light conditions. Taken together, our results question the role of glycine betaine in salinity tolerance under light conditions close to those encountered by durum wheat seedlings in their natural environment and suggest the importance of other mechanisms, such as the accumulation of minor amino acids.

Sign in / Sign up

Export Citation Format

Share Document