Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O₂⁻). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O₂⁻ generation, besides complex III, stimulated NO degradation. Larger amounts of O₂⁻ were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O₂⁻ production were stimulated by free Ca²⁺ concentration. Together, these results indicate that Ca²⁺-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O₂⁻ production that favors NO degradation in potato tuber mitochondria.     

Metformin induces suppression of NAD(P)H oxidase activity in podocytes

Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms.The experiments were performed in normal (NG, 5.6 mM) and high (HG, 30 mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-³H]-glucose.ROS production increased by about 27% (187 ± 8 vs. 238 ± 9 arbitrary units AU, P < 0.01) in HG. Metformin (2 mM, 2 h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58 ± 0.07 to. 0.99 ± 0.06, and from 0.53 ± 0.03 to 0.64 ± 0.03; P < 0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C.We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.     

Vitamin B12 protects against superoxide-induced cell injury in human aortic endothelial cells

Superoxide (O₂^•−) is implicated in inflammatory states including arteriosclerosis and ischemia-reperfusion injury. Cobalamin (Cbl) supplementation is beneficial for treating many inflammatory diseases and also provides protection in oxidative-stress-associated pathologies. Reduced Cbl reacts with O₂^•− at rates approaching that of superoxide dismutase (SOD), suggesting a plausible mechanism for its anti-inflammatory properties. Elevated homocysteine (Hcy) is an independent risk factor for cardiovascular disease and endothelial dysfunction. Hcy increases O₂^•− levels in human aortic endothelial cells (HAEC). Here, we explore the protective effects of Cbl in HAEC exposed to various O₂^•− sources, including increased Hcy levels. Hcy increased O₂^•− levels (1.6-fold) in HAEC, concomitant with a 20% reduction in cell viability and a 1.5-fold increase in apoptotic death. Pretreatment of HAEC with physiologically relevant concentrations of cyanocobalamin (CNCbl) (10-50 nM) prevented Hcy-induced increases in O₂^•− and cell death. CNCbl inhibited both Hcy and rotenone-induced mitochondrial O₂^•− production. Similarly, HAEC challenged with paraquat showed a 1.5-fold increase in O₂^•− levels and a 30% decrease in cell viability, both of which were prevented with CNCbl pretreatment. CNCbl also attenuated elevated O₂^•− levels after exposure of cells to a Cu/Zn-SOD inhibitor. Our data suggest that Cbl acts as an efficient intracellular O₂^•− scavenger.     

Peroxynitrite-Induced Apoptosis in Epithelial (T84) and Macrophage (RAW 264.7) Cell Lines: Effect of Legume-Derived Polyphenols (Phytolens)

Peroxynitrite (ONOO⁻) has been proposed as a mediator of gut inflammation and as an inducer of cell death by apoptosis. Phytolens (PHY), a water-soluble extract of polyphenolic antioxidants from nonsoy legumes (Biotics Research Corp, patent pending), was evaluated as a cytoprotective agent in human colonic (T84) and murine macrophage (RAW 264.7) cell lines. In the antioxidant testing, PHY showed a significant free radical scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and superoxide (O₂^•) radicals with an IC₅₀of 4.44 and 5.87 μg/ml against DPPH and O₂^•, respectively. Apoptosis (DNA fragmentation) was measured by an ELISA technique. Cells were exposed to oxidative stress by treating them with peroxynitrite (100–300 μM) for 4 h in the presence and absence of PHY. Peroxynitrite elicited a dose-dependent increase in DNA fragmentation in both cell lines compared to the control group receiving decomposed ONOO⁻. PHY (10, 30, or 50 μg/ml) significantly attenuated the degree of apoptosis in T84 cells induced by ONOO⁻(P< 0.05). PHY (10–100 μg/ml) did not directly affect T84 cell viability or induce apoptosis after 4 h or overnight exposure. RAW 264.7 cells exposed to PHY alone (>30 μg/ml) for 4 h displayed decreased cell viability (P< 0.05) and increased apoptosis (P< 0.05). Phytolens may have beneficial effects on inflammation by attenuating peroxynitrite-induced apoptosis. The sparing of epithelial cells while compromising the viability of macrophages suggests that PHY may be beneficial in autoimmune disorders.     

Effect of zooplankton availability on the rates of photosynthesis, and tissue and skeletal growth in the scleractinian coral Stylophora pistillata

This work investigated the effect of light and feeding on tissue composition as well as on rates of photosynthesis and calcification in the zooxanthellae (zoox) scleractinian coral, Stylophora pistillata. Microcolonies were maintained at three different light levels (80, 200, 300 μmol m⁻² s⁻¹) and subjected to two feeding regimes (starved and fed) over 9 weeks. Corals were fed both natural plankton and Artemia salina nauplii four times a weeks and samplings were made after 2, 5, and 9 weeks. Results confirmed that feeding enhances coral growth rate and increases both the dark and light calcification rates. These rates were 50-75% higher in fed corals (FC; 60±20 and 200±40 nmol Ca²⁺ cm⁻² h⁻¹ for dark and light calcification, respectively) compared to control corals (CC; 30±9 and 124±23 nmol Ca²⁺ cm⁻² h⁻¹). The dark calcification rates, however, were four times lower than the rates of light calcification (independent of trophic status). After 5 weeks, chlorophyll a (chl-a) concentrations were four to seven times higher in fed corals (7-21 μg cm⁻²) than in control corals (2-5 μg cm⁻²). The amount of protein was also significantly higher in fed corals (2.11-2.50 mg cm⁻²) than in control corals (1.08-1.52 mg cm⁻²). Rates of photosynthesis in fed corals were 2-10 times higher (1.24±0.75 μmol O₂ h⁻¹ cm⁻²) than those measured in control corals (0.20±0.08 μmol O₂ h⁻¹ cm⁻²).     

Superoxide production and oxygen consumption in endothelium-intact and -denuded artery stimulated by angiotensin II

Arteries stimulated by angiotensin II (AII) to contract do not display the expected augmentation of O₂ consumption seen with other cardiovascular contractile agonists. We tested the hypothesis that superoxide (O₂⁻) or other reactive oxidant species generated by AII played a role in the paradoxical O₂ consumption response in porcine carotid artery, with or without an intact endothelium. Endothelium-denuded arteries were incubated with either 1 μM diphenylene iodonium (DPI), an inhibitor of NAD(P)H oxidase, 300 u/ml superoxide dismutase (SOD), a scavenger of O₂⁻, or 20 U/ml catalase, an enzyme which promotes conversion of O₂⁻ (scavenged in the form of H₂O₂) to O₂. DPI treatment resulted in the expected increase in O₂ consumption upon contractile activation with AII challenge (1.05± 0.23 μmol/g/min; n = 6, p < .01), as did treatment with SOD (0.67± 0.20 μmol/g/min; n = 4, p < .05). Catalase incubation resulted in a burst of O₂ generation upon AII challenge (1.30 ± 0.21 μmol/g/min; n = 10, p < .001). In endothelium-intact arteries, O₂ consumption was again not augmented with AII challenge; instead, a burst of O₂ production was observed (0.66 ± 0.22 μmol/g/min; n = 9, p < .05), which was not affected further by addition of catalase. Thus, the absence of apparent augmentation of O₂ consumption during contractile activation of endothelium-denuded arteries was attributed to simultaneous NAD(P)H oxidase-dependent production of O₂⁻, and attendant H₂O₂ and O₂ generation which either and masked the detection of O₂ consumed or suppressed mitochondrial uptake of O₂, or both. An intact endothelium was required to manifest the burst of O₂ generation with AII stimulation under normal conditions. (Mol Cell Biochem xxx: 235–239, 2005)     

The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25 μg/ml) decreased nitric oxide (^•NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10 μg/ml) stimulated a Ca²⁺ influx linked to apocynin-sensitive superoxide radical anion (O₂^•−) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4 h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24 h. Therefore, in addition to modulating ^•NO bioavailability by stimulating O₂^•− production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, μg/ml) before (not after) SAA treatment ameliorated the Ca²⁺ influx and O₂^•− production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating ^•NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.     

Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry

Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern–Volmer constant (K_Cl^-) for chloride in water solution was 115.0 ± 2.8 M⁻¹, whereas the intracellular K_Cl^- was 17.8 ± 0.8 M⁻¹, for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.     

Modeling of biopterin-dependent pathways of eNOS for nitric oxide and superoxide production

Endothelial dysfunction is associated with increase in oxidative stress and low NO bioavailability. The endothelial NO synthase (eNOS) uncoupling is considered an important factor in endothelial cell oxidative stress. Under increased oxidative stress, the eNOS cofactor tetrahydrobiopterin (BH₄) is oxidized to dihydrobiopterin, which competes with BH₄ for binding to eNOS, resulting in eNOS uncoupling and reduction in NO production. The importance of the ratio of BH₄ to oxidized biopterins versus absolute levels of total biopterin in determining the extent of eNOS uncoupling remains to be determined. We have developed a computational model to simulate the kinetics of the biochemical pathways of eNOS for both NO and O₂^•− production to understand the roles of BH₄ availability and total biopterin (TBP) concentration in eNOS uncoupling. The downstream reactions of NO, O₂^•−, ONOO⁻, O₂, CO₂, and BH₄ were also modeled. The model predicted that a lower [BH₄]/[TBP] ratio decreased NO production but increased O₂^•− production from eNOS. The NO and O₂^•− production rates were independent above 1.5 μM [TBP]. The results indicate that eNOS uncoupling is a result of a decrease in [BH₄]/[TBP] ratio, and a supplementation of BH₄ might be effective only when the [BH₄]/[TBP] ratio increases. The results from this study will help us understand the mechanism of endothelial dysfunction.     

Influence of daylength on metabolic rate and daily water loss in the male prosimian primate Microcebus murinus

In its natural habitat, Microcebus murinus, a small malagasy prosimian primate, is exposed to seasonal shortage of water and resources. During the winter dry season, animals enter a pronounced fattening period with concurrent decrease in behavioural/physiological activities, whereas the breeding season is restricted to the rainy summer months. To determine the role of daylength on metabolic rate and water loss in this nocturnal primate, we measured body mass, oxygen consumption at 25°C (RMR), circadian water loss through urine output (UO) and evaporation (EWL) in eight males exposed to either short days (8L:16D SD) or long days (14L:10D LD), under controlled captive conditions. Exposure to SD led to a ponderal increase (maximal body mass: 125±4 g, N=8), and to significant changes in RMR and water loss, both reaching lowest values after 3 months under SD (0.84±0.04 ml O₂ h⁻¹ g⁻¹ and 38±0.3 mg H₂O g⁻¹ day⁻¹, respectively). Following exposure to LD, body mass decreased to 77±3 g (N=8), whereas both RMR and water loss, mainly through EWL, significantly increased (P<0.001), the highest value occurring after 2 months (1.51±0.08 ml O₂ h^.−1 g⁻¹ and 87±7 mgH₂O g⁻¹ day⁻¹, respectively). Moreover, independent of daylength, circadian changes in EWL were characterized by significantly reduced values during the diurnal rest. The results demonstrate that daylength variations affect the physiology of this tropical primate, allowing anticipatory adaptation to seasonal environmental constraints.     

AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes

In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate ( [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} ). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions (>200 μM O₂) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . At physiologically relevant [O₂] (<40 μM O₂), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} with no change in sensitivity to [O₂] as assessed by estimates of cellular P₅₀ and P₉₀ values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} in normoxia. Treatment of hepatocytes with ≤200 μM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.     

Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase

Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 μmol mg protein⁻¹ min⁻¹). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (μ_aerobic 0.09±0.03 h⁻¹). Co-expression of Lactococcus lactis β-phosphoglucomutase accelerated maltose utilization via this route (μ_aerobic 0.21±0.01 h⁻¹, μ_anaerobic 0.10±0.00 h⁻¹). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.     

High-performance liquid chromatography ultraviolet assay for human erythrocytic catalase activity by measuring glutathione as o-phthalaldehyde derivative

The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H₂O₂) absorbance decrease at 240 nm. Here we report an alternative high-performance liquid chromatography (HPLC) assay for human erythrocytic CAT (heCAT) activity measurement based on glutathione (GSH) analysis as a highly stable, H₂O₂-insensitive o-phthalaldehyde (OPA) derivative. The method was developed and validated using an isolated heCAT in phosphate-buffered saline at pH 7.4 and was applied to measure CAT activity in lysed human erythrocytes. heCAT activity was measured at initial concentrations of 5 nM for heCAT, 5 mM for H₂O₂, and 10 mM for GSH, and the incubation time was 10 min. Nitrite (NO₂⁻) was found to be an uncompetitive inhibitor of heCAT activity (IC₅₀ = 9 μM) and of CAT activity in hemolysate (IC₅₀ ∼ 750 μM). Nitrate (NO₃⁻) at concentrations up to 100 μM did not inhibit heCAT activity. Azide (N₃⁻) was found to be a very strong inhibitor of the heCAT (IC₅₀ = 0.2 nM) but a relatively weak CAT inhibitor (IC₅₀ ∼ 10 μM) in human hemolysates. The novel CAT activity assay works under redox conditions that more closely resemble those prevailing in cells and allows high-throughput analysis despite the required HPLC step.     

Flavin-mediated reduction of ferric leghemoglobin from soybean nodules

The reduction of ferric leghemoglobin (Lb³⁺) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb³⁺ to oxyferrous (Lb²⁺ · O₂) or deoxyferrous (Lb²⁺) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O₂ the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb³⁺ reduced) than in the presence of O₂; this phenomenon was probably because O₂ competes with Lb³⁺ for reductant, thus generating activated O₂ species. The flavin-mediated reduction of Lb³⁺ did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb³⁺ directly. The NAD(P)H + flavin system also reduced the complexes Lb³⁺ · nicotinate and Lb³⁺ · acetate to Lb²⁺ · O₂, Lb²⁺ or Lb²⁺ · nicotinate, depending on the concentrations of ligands and of O₂. In the presence of 200 M nitrite most Lb remained as Lb³⁺ in aerobic conditions but the nitrosyl complex (Lb²⁺ · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb³⁺ at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb³⁺ in vivo.Abbreviations and Terminology FLbR ferric leghemoglobin reductase - Hb²⁺ /Hb³⁺ hemoglobin containing Fe²⁺ /Fe²⁺ - Lb²⁺ /Lb³⁺ leghemoglobin containing Fe²⁺ /Fe³⁺ - Lb³⁺ · nicotinate/acetate Lb in which nicotinate or acetate are complexed to Lb³⁺ - Lb²⁺ · O₂/CO/NO/nicotinate Lb in which O₂, CO, NO or nicotinate are complexed to Lb²⁺ - Rfl riboflavin - SOD superoxide dismutase (EC 1.15.1.1) Published as Paper No. 9237, Journal Series, Nebraska Agricultural Research DivisionWe thank M.B. Crusellas for his skillful drawings. M. Becana thanks the Spanish Ministry of Education and Science/Fulbright Commission for financial support.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

Au-nanoclusters incorporated 3-amino-5-mercapto-1,2,4-triazole film modified electrode for the simultaneous determination of ascorbic acid, dopamine, uric acid and nitrite

A novel biosensor has been constructed by the electrodeposition of Au-nanoclusters (nano-Au) on poly(3-amino-5-mercapto-1,2,4-triazole) (p-TA) film modified glassy carbon electrode (GCE) and employed for the simultaneous determination of dopamine (DA), ascorbic acid (AA), uric acid (UA) and nitrite (NO₂⁻). NH₂ and SH groups exposed to the p-TA layer are helpful for the electrodeposition of nano-Au. The combination of nano-Au and p-TA endow the biosensor with large surface area, good biological compatibility, electricity and stability, high selectivity and sensitivity and flexible and controllable electrodeposition process. In the fourfold co-existence system, the linear calibration plots for AA, DA, UA and NO₂⁻ were obtained over the range of 2.1–50.1 μM, 0.6–340.0 μM, 1.6–110.0 μM and 15.9–277.0 μM with detection limits of 1.1 × 10⁻⁶ M, 5.0 × 10⁻⁸ M, 8.0 × 10⁻⁸ M and 8.9 × 10⁻⁷ M, respectively. In addition, the modified biosensor was applied to the determination of AA, DA, UA and NO₂⁻ in urine and serum samples by using standard adding method with satisfactory results.     

Active trans-plasma membrane water cycling in yeast is revealed by NMR

Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal ¹H₂O NMR relaxation time constant (T₁) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τ_i), where τ_i⁻¹ is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O₂/5%CO₂ (O₂) or 95%N₂/5%CO₂ (N₂). ATP was high during O₂, and τ_i⁻¹ was 3.1 s⁻¹ at 25°C. After changing to N₂, ATP decreased and τ_i⁻¹ was 1.8 s⁻¹. The principal active yeast ion transport protein is the plasma membrane H⁺-ATPase. Studies using the H⁺-ATPase inhibitor ebselen or a yeast genetic strain with reduced H⁺-ATPase found reduced τ_i⁻¹, notwithstanding high ATP. Steady-state water exchange correlates with H⁺-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity.     

Complexation of curium(III) by adenosine 5′-triphosphate (ATP): A time-resolved laser-induced fluorescence spectroscopy (TRLFS) study

The complex formation of curium(III) with adenosine 5′-triphosphate (ATP) was determined by time-resolved laser-induced fluorescence spectroscopy (TRLFS). The interaction between soluble species of curium(III) with ATP was studied at trace Cm(III) concentrations (3 × 10⁻⁷ M). The concentrations of ATP were varied between 6.0 × 10⁻⁷ and 1.5 × 10⁻⁴ M in the pH range of 1.5-7.0 using 0.154 M NaCl as background electrolyte.Three Cm-ATP species, M_pH_qL_r, could be identified from the fluorescence emission spectra: (i) CmH₂ATP⁺ with a peak maximum at 598.6 nm, (ii) CmHATP with a peak maximum at 600.3 nm, and (iii) CmATP⁻ with a peak maximum at 601.0 nm. The formation constants of these complexes were calculated from TRLFS measurements to be log β₁₂₁ = 16.86 ± 0.09, log β₁₁₁ = 13.23 ± 0.10, and log β₁₀₁ = 8.19 ± 0.16. The hydrated Cm-ATP species showed fluorescence lifetimes between 88 and 96 μs; whereas the CmATP⁻ complex has a significantly longer fluorescence lifetime of 187 ± 7 μs.     

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl⁻ current (I_Cl.PKA) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I_Cl.PKA and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I_Cl.PKA in a concentration- and voltage-dependent fashion (IC₅₀ at +100 mV = 0.3 ± 1.5 μM and at −100 mV = 5.1 ± 1.3 μM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I_Cl.PKA (30 μM) was tested on other cardiac ion currents. Inward current at −120 mV, comprised predominantly of the inward-rectifier background K⁺ current, I_K1, was reduced by ∼43% (n = 5). Under selective recording conditions, the Na⁺ current (I_Na) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at −40 mV of ∼82%; n = 8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca²⁺ channel current (I_Ca,L); fractional block at +10 mV of ∼49% and of ∼28% at −10 mV; n = 11, with a ∼−3 mV shift in the voltage-dependence of I_Ca,L activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I_Cl.PKA channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.