Warm Cells, Hot Mitochondria: Achievements and Problems of Ultralocal Thermometry.

Temperature is a crucial regulator of the rate and direction of biochemical reactions and cell processes. The recent data indicating the presence of local thermal gradients associated with the sites of high-rate thermogenesis, on the one hand, demonstrate the possibility for the existence of "thermal signaling" in a cell and, on the other, are criticized on the basis of thermodynamic calculations and models. Here, we review the main thermometric techniques and sensors developed for the determination of temperature inside living cells and diverse intracellular compartments. A comparative analysis is conducted of the results obtained using these methods for the cytosol, nucleus, endo-/sarcoplasmic reticulum, and mitochondria, as well as their biological consistency. Special attention is given to the limitations, possible sources of errors and ambiguities of the sensor's responses. The issue of biological temperature limits in cells and organelles is considered. It is concluded that the elaboration of experimental protocols for ultralocal temperature measurements that take into account both the characteristics of biological systems, as well as the properties and limitations of each type of sensor is of critical importance for the generation of reliable results and further progress in this field.

Mitochondrial F-ATP Synthase Co-Migrating Proteins and Ca2+-Dependent Formation of Large Channels.

Monomers, dimers, and individual FOF1-ATP synthase subunits are, presumably, involved in the formation of the mitochondrial permeability transition pore (PTP), whose molecular structure, however, is still unknown. We hypothesized that, during the Ca2+-dependent assembly of a PTP complex, the F-ATP synthase (subunits) recruits mitochondrial proteins that do not interact or weakly interact with the F-ATP synthase under normal conditions. Therefore, we examined whether the PTP opening in mitochondria before the separation of supercomplexes via BN-PAGE will increase the channel stability and channel-forming capacity of isolated F-ATP synthase dimers and monomers in planar lipid membranes. Additionally, we studied the specific activity and the protein composition of F-ATP synthase dimers and monomers from rat liver and heart mitochondria before and after PTP opening. Against our expectations, preliminary PTP opening dramatically suppressed the high-conductance channel activity of F-ATP synthase dimers and monomers and decreased their specific "in-gel" activity. The decline in the channel-forming activity correlated with the reduced levels of as few as two proteins in the bands: methylmalonate-semialdehyde dehydrogenase and prohibitin 2. These results indicate that proteins co-migrating with the F-ATP synthase may be important players in PTP formation and stabilization.

Regulation of Mitochondrial Permeability Transition Pore Opening by Monovalent Cations in Liver Mitochondria.

The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K+ transport into mitochondria protects cells from ischemia/reperfusion. However, the role of K+ transport in PTP regulation is unclear. Here, we studied the role of K+ and other monovalent cations in the regulation of the PTP opening in an in vitro model. The registration of the PTP opening, membrane potential, Ca2+-retention capacity, matrix pH, and K+ transport was performed using standard spectral and electrode techniques. We found that the presence of all cations tested in the medium (K+, Na+, choline+, and Li+) strongly stimulated the PTP opening compared with sucrose. Several possible reasons for this were examined: the effect of ionic strength, the influx of cations through selective and non-selective channels and exchangers, the suppression of Ca2+/H+ exchange, and the influx of anions. The data obtained indicate that the mechanism of PTP stimulation by cations includes the suppression of K+/H+ exchange and acidification of the matrix, which facilitates the influx of phosphate. Thus, the K+/H+ exchanger and the phosphate carrier together with selective K+ channels compose a PTP regulatory triad, which might operate in vivo.

The Effect of Astaxanthin on Mitochondrial Dynamics in Rat Heart Mitochondria under ISO-Induced Injury.

Mitochondria are dynamic organelles that produce ATP in the cell and are sensitive to oxidative damage that impairs mitochondrial function in pathological conditions. Mitochondria are involved not only in a healthy heart but also in the development of heart disease. Therefore, attempts should be made to enhance the body's defense response against oxidative stress with the help of various antioxidants in order to decrease mitochondrial damage and reduce mitochondrial dysfunction. Mitochondrial fission and fusion play an important role in the quality control and maintenance of mitochondria. The ketocarotenoid astaxanthin (AX) is an antioxidant able to maintain mitochondrial integrity and prevent oxidative stress. In the present study, we investigated the effect of the protective effect of AX on the functioning of rat heart mitochondria (RHM). Changes in the content of proteins responsible for mitochondrial dynamics, prohibitin 2 (PHB2) as a protein that performs the function of quality control of mitochondrial proteins and participates in the stabilization of mitophagy, and changes in the content of cardiolipin (CL) in rat heart mitochondria after isoproterenol (ISO)-induced damage were examined. AX improved the respiratory control index (RCI), enhanced mitochondrial fusion, and inhibited mitochondrial fission in RHM after ISO injury. Rat heart mitochondria (RHM) were more susceptible to Ca2+-induced mitochondrial permeability pore (mPTP) opening after ISO injection, while AX abolished the effect of ISO. AX is able to perform a protective function in mitochondria, improving their efficiency. Therefore, AX can be considered an important ingredient in the diet for the prevention of cardiovascular disease. Therefore, AX can be examined as an important component of the diet for the prevention of heart disease.

Pioglitazone Is a Mild Carrier-Dependent Uncoupler of Oxidative Phosphorylation and a Modulator of Mitochondrial Permeability Transition.

Pioglitazone (PIO) is an insulin-sensitizing antidiabetic drug, which normalizes glucose and lipid metabolism but may provoke heart and liver failure and chronic kidney diseases. Both therapeutic and adverse effects of PIO can be accomplished through mitochondrial targets. Here, we explored the capability of PIO to modulate the mitochondrial membrane potential (ΔΨm) and the permeability transition pore (mPTP) opening in different models in vitro. ΔΨm was measured using tetraphenylphosphonium and the fluorescent dye rhodamine 123. The coupling of oxidative phosphorylation was estimated polarographically. The transport of ions and solutes across membranes was registered by potentiometric and spectral techniques. We found that PIO decreased ΔΨm in isolated mitochondria and intact thymocytes and the efficiency of ADP phosphorylation, particularly after the addition of Ca2+. The presence of the cytosolic fraction mitigated mitochondrial depolarization but made it sustained. Carboxyatractyloside diminished the PIO-dependent depolarization. PIO activated proton transport in deenergized mitochondria but not in artificial phospholipid vesicles. PIO had no effect on K+ and Ca2+ inward transport but drastically decreased the mitochondrial Ca2+-retention capacity and protective effects of adenine nucleotides against mPTP opening. Thus, PIO is a mild, partly ATP/ADP-translocase-dependent, uncoupler and a modulator of ATP production and mPTP sensitivity to Ca2+ and adenine nucleotides. These properties contribute to both therapeutic and adverse effects of PIO.

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site.

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD+ on: (1) the Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD+ increased the CRC of liver, heart, and brain mitochondria 1.5-2.5 times, insignificantly affecting the rate of Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca2+-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.

Dynamics of the permeability transition pore size in isolated mitochondria and mitoplasts.

The size of the permeability transition pore (PTP) is accepted to be ≤1.5 kDa. However, different authors reported values from 650 to 4000 Da. The present study is focused on the variability of the average PTP size in and between mitochondrial samples, its reasons and relations with PTP dynamics. Measurement of PTP size by the standard method revealed its 500 Da-range variability between mitochondrial samples. Sequential measurements in the same sample showed that the PTP size tends to grow with time and Ca2+ concentration. Selective damage to the mitochondrial outer membrane (MOM) reduced the apparent PTP size by ~200-300 Da. Hypotonic and hypertonic osmotic shock and partial removal of the MOM with the preservation of the mitochondrial inner membrane intactness decreased the apparent PTP size by ~50%. We developed an approach to continuous monitoring of the PTP size that revealed the existence of stable PTP states with different pore sizes (~700, 900-1000, ~1350, 1700-1800, and 2100-2200 Da) and transitions between them. The transitions were accelerated by elevating the Ca2+ concentration, temperature, and osmotic pressure, which demonstrates an increased capability of PTP to accommodate to large molecules (plasticity). Cyclosporin A inhibited the transitions between states. The analysis of PTP size dynamics in osmotically shocked mitochondria and mitoplasts confirmed the importance of the MOM for the stabilization of PTP structure. Thus, this approach provides a new tool for PTP studies and the opportunity to reconcile data on the PTP size and mitochondrial megachannel conductance.

Isoproterenol-Induced Permeability Transition Pore-Related Dysfunction of Heart Mitochondria Is Attenuated by Astaxanthin.

Mitochondria are key organelles of the cell because their main function is the capture of energy-rich substrates from the cytoplasm and oxidative cleavage with the generation of carbon dioxide and water, processes that are coupled with the synthesis of ATP. Mitochondria are subject to oxidative stress through the formation of the mitochondrial permeability transition pore (mPTP). Various antioxidants are used to reduce damage caused by oxidative stress and to improve the protection of the antioxidant system. Astaxanthin (AST) is considered to be a dietary antioxidant, which is able to reduce oxidative stress and enhance the antioxidant defense system. In the present investigation, the effect of AST on the functional state of rat heart mitochondria impaired by isoproterenol (ISO) under mPTP functioning was examined. It was found that AST raised mitochondrial respiration, the Ca2+ retention capacity (CRC), and the rate of TPP+ influx in rat heart mitochondria (RHM) isolated from ISO-injected rats. However, the level of reactive oxygen species (ROS) production increased. In addition, the concentrations of cardiolipin (CL), Mn-SOD2, and the proteins regulating mPTP rose after the injection of ISO in RHM pretreated with AST. Based on the data obtained, we suggest that AST has a protective effect in rat heart mitochondria.

Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells.

We have shown that hydroxycobalamin (vitamin В12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + В12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.

Astaxanthin Inhibits Mitochondrial Permeability Transition Pore Opening in Rat Heart Mitochondria.

The mitochondrion is the main organelle of oxidative stress in cells. Increased permeability of the inner mitochondrial membrane is a key phenomenon in cell death. Changes in membrane permeability result from the opening of the mitochondrial permeability transition pore (mPTP), a large-conductance channel that forms after the overload of mitochondria with Ca2+ or in response to oxidative stress. The ketocarotenoid astaxanthin (AST) is a potent antioxidant that is capable of maintaining the integrity of mitochondria by preventing oxidative stress. In the present work, the effect of AST on the functioning of mPTP was studied. It was found that AST was able to inhibit the opening of mPTP, slowing down the swelling of mitochondria by both direct addition to mitochondria and administration. AST treatment changed the level of mPTP regulatory proteins in isolated rat heart mitochondria. Consequently, AST can protect mitochondria from changes in the induced permeability of the inner membrane. AST inhibited serine/threonine protein kinase B (Akt)/cAMP-responsive element-binding protein (CREB) signaling pathways in mitochondria, which led to the prevention of mPTP opening. Since AST improves the resistance of rat heart mitochondria to Ca2+-dependent stress, it can be assumed that after further studies, this antioxidant will be considered an effective tool for improving the functioning of the heart muscle in general under normal and medical conditions.

Fusaricidin-Type Compounds Create Pores in Mitochondrial and Plasma Membranes of Mammalian Cells.

Fusaricidins and related LI-F compounds are effective bactericides and fungicides. Recently, we have found that they are highly toxic to mammalian cells. Here, we studied the effect of fusaricidin-type compounds (FTCs) on the membranes of mammalian cells. Ethanol extracts from Paenibacillus polymyxa strains, RS10 and I/Sim, were fractionated and analyzed by HPLC and mass spectrometry. The effects of FTCs on mitochondrial functions and integrity were studied by standard methods: measurements of swelling, membrane potential (ΔΨm), respiration rate, cytochrome c release, and pore sizes. Superoxide flashes were registered by 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA). Plasma membrane permeability was assessed by propidium iodide (PI) staining and ATP release. FTCs caused the permeabilization of the inner mitochondria membrane (IMM) to ions and low-molecular-weight (~750 Da) solutes. The permeabilization did not depend on the permeability transition pore (mPTP) but was strongly dependent on ΔΨm. Fusaricidins A plus B, LI-F05a, and LI-F05b-LI-F07b permeabilized IMM with comparable efficiency. They created pores and affected mitochondrial functions and integrity similarly to mPTP opening. They permeabilized the sperm cell plasma membrane to ATP and PI. Thus, the formation of pores in polarized membranes underlays the toxicity of FTCs to mammals. Besides, FTCs appeared to be superior reference compounds for mPTP studies.

Regulation of permeability transition pore opening in mitochondria by external NAD(H).

BACKGROUND: The opening of the permeability transition pore (PTP) in mitochondria plays a critical role in the pathogenesis of numerous diseases. Mitochondrial matrix pyridine nucleotides are potent regulators of the PTP, but the role of extramitochondrial nucleotides is unclear. METHODS: The PTP opening was explored in isolated mitochondria and mitochondria in permeabilized differentiated and undifferentiated cells in the presence of added NAD(P)(H) in combination with Mg2+, adenine nucleotides (AN), and the inhibitors of AN translocase (ANT), voltage-dependent anion channel (VDAC), and cyclophilin D. RESULTS: Added NAD(H) and AN, but not NADP(H), inhibited the PTP opening with comparable potency. PTP suppression required neither NAD(H) oxidation nor reduction. The protective effects of NAD(H) and cyclosporin A were synergistic, and the effects of NAD(H) and millimolar AN were additive. The conformation-specific ANT inhibitors were unable to cancel the protective effect of NADH even under total ANT inhibition. Besides, NAD(H) activated the efflux of mitochondrial AN via ANT. VDAC ligand (Mg2+) and blockers (G3139 and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) potentiated and attenuated the protective effect of NAD(H), respectively. However, in embryonic and cancer (undifferentiated) cells, in contrast to isolated differentiated hepatocytes and cardiocytes, the suppression of PTP opening by NADH was negligible though all cells tested possessed a full set of VDAC isoforms. CONCLUSIONS: The study revealed a novel mechanism of PTP regulation by external (cytosolic) NAD(H) through the allosteric site in the OM or the intermembrane space. GENERAL SIGNIFICANCE: The mechanism might contribute to the resistance of differentiated cells under different pathological conditions including ischemia/reperfusion.

Pyridine nucleotides regulate the superoxide anion flash upon permeabilization of mitochondrial membranes: An MCLA-based study.

The permeabilization of mitochondrial membranes via permeability transition pore opening or by the pore-forming peptide alamethicin causes a flash of superoxide anion (SA) and hydrogen peroxide production and the inhibition of matrix aconitase. It was shown using the SA probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) that the substrates of NAD-dependent dehydrogenases, inhibitors of the respiratory chain, and NAD(P)H at millimolar concentrations suppressed or delayed SA flashes. In the presence of added NADH and NADPH, SA flashes were observed only after considerable oxidation of pyridine nucleotides. The production of SA was maximal at NADPH and NADH redox potentials from -315 to -295 mV and from -325 to -270 mV, respectively, depending on NAD(P)H concentration. SA generation supported by NADPH was severalfold greater than that supported by NADH. In intact mitochondria, NADPH- and NADH-dependent SA generation was negligible. Respiratory substrates at physiological or lower concentrations were incapable of suppressing the NADPH-supported SA flash. These data indicate that, in conditions close to pathophysiological, matrix NADPH oxidoreductase(s), presumably, an adrenodoxin reductase in complex with adrenodoxin, can essentially contribute to SA flashes associated with transient or irreversible permeability transition pore opening or membrane permeabilization by another mechanism.

Effect of Melatonin on Rat Heart Mitochondria in Acute Heart Failure in Aged Rats.

Excessive generation of reactive oxygen species (ROS) in mitochondria and the opening of the nonselective mitochondrial permeability transition pore are important factors that promote cardiac pathologies and dysfunction. The hormone melatonin (MEL) is known to improve the functional state of mitochondria via an antioxidant effect. Here, the effect of MEL administration on heart mitochondria from aged rats with acute cardiac failure caused by isoprenaline hydrochloride (ISO) was studied. A histological analysis revealed that chronic intake of MEL diminished the age-dependent changes in the structure of muscle fibers of the left ventricle, muscle fiber swelling, and injury zones characteristic of acute cardiac failure caused by ISO. In acute heart failure, the respiratory control index (RCI) and the Ca2+ retention capacity in isolated rat heart mitochondria (RHM) were reduced by 30% and 40%, respectively, and mitochondrial swelling increased by 34%. MEL administration abolished the effect of ISO. MEL partially prevented ISO-induced changes at the subunit level of respiratory complexes III and V and drastically decreased the expression of complex I subunit NDUFB8 both in control RHM and in RHM treated with ISO, which led to the inhibition of ROS production. MEL prevents the mitochondrial dysfunction associated with heart failure caused by ISO. It was shown that the level of 2',3'-cyclicnucleotide-3'-phosphodiasterase (CNPase), which is capable of protecting cells in aging, increased in acute heart failure. MEL also retained the CNPase content in RHM both in control experiments and after ISO-induced heart damage. We concluded that an increase in the CNPase level promotes cardioprotection.

Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation.

Triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Qd-binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues.

Glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.

Chronic alcohol intoxication is associated with increased oxidative stress. However, the mechanisms by which ethanol triggers an increase in the production of reactive oxygen species (ROS) and the role of mitochondria in the development of oxidative stress has been insufficiently studied. The biochemical and proteomic data obtained in the present work suggest that one of the main causes of an increase in ROS generation is enhanced oxidation of glutamate in response to long-term alcohol exposure. In the course of glutamate oxidation, liver mitochondria from alcoholic rats generated more superoxide anion and H2O2 than in the presence of other substrates and more than control organelles. In mitochondria from alcoholic rats, rates of H2O2 production and NAD reduction in the presence of glutamate were almost twice higher than in the control. The proteomic study revealed a higher content of glutamate dehydrogenase in liver mitochondria of rats subjected to chronic alcohol exposure. Simultaneously, the content of mitochondrial catalase decreased compared to control. Each of these factors stimulates the production of ROS in addition to ROS generated by the respiratory chain complex I. The results are consistent with the conclusion that glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.

External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?

It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin. Paraquat was predominantly activated by internal mitochondria oxidoreductases. An increase in the ionic strength stimulated and suppressed the redox cycling of negatively and positively charged acceptors, as was expected for the Cyb5R3-mediated reduction. Antibodies against Cyb5R3 but not VDAC substantially inhibited the NADH-related oxidoreductase activities. The specific VDAC blockers G3139 and erastin, separately or in combination, in concentrations sufficient for the inhibition of substrate transport, exhibited minimal effects on the redox cycler-dependent NADH oxidation, ROS generation, and reduction of exogenous cytochrome c. In contrast, Cyb5R3 inhibitors (6-propyl-2-thiouracil, p-chloromercuriobenzoate, quercetin, mersalyl, and ebselen) showed similar patterns of inhibition of ROS generation and cytochrome c reduction. The analysis of the spectra of the endogenous cytochromes b5 and c in the presence of nitrofurantoin and the inhibitors of VDAC and Cyb5R3 demonstrated that the redox cycler can transfer electrons from Cyb5R3 to endogenous cytochrome c. This caused the oxidation of outer membrane-bound cytochrome b5, which is in redox balance with Cyb5R3. The data obtained argue against VDAC1 and in favor of Cyb5R3 involvement in the activation of redox cyclers in the outer mitochondrial membrane.

Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate, permeabilized cells, and tissue slices.

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.

Cereulide produced by Bacillus cereus increases the fitness of the producer organism in low-potassium environments.

Cereulide, produced by certain Bacillus cereus strains, is a lipophilic cyclic peptide of 1152 Da that binds K(+) ions with high specificity and affinity. It is toxic to humans, but its role for the producer organism is not known. We report here that cereulide operates for B. cereus to scavenge potassium when the environment is growth limiting for this ion. Cereulide-producing B. cereus showed higher maximal growth rates (µ(max)) than cereulide non-producing B. cereus in K(+)-deficient medium (K(+) concentration ~1 mM). The cereulide-producing strains grew faster in K(+)-deficient than in K(+)-rich medium with or without added cereulide. Cereulide non-producing B. cereus neither increased µ(max) in K(+)-deficient medium compared with K(+)-rich medium, nor benefited from added cereulide. Cereulide-producing strains outcompeted GFP-labelled Bacillus thuringiensis in potassium-deficient (K(+) concentration ~1 mM) but not in potassium-rich (K(+) concentration ~30 mM) medium. Exposure to 2 µM cereulide in potassium-free medium lacking an energy source caused, within seconds, a major efflux of cellular K(+) from B. cereus not producing cereulide as well as from Bacillus subtilis. Cereulide depleted the cereulide non-producing B. cereus and B. subtilis cells of a major part of their K(+) stores, but did not affect cereulide-producing B. cereus strains. Externally added 6-10 µM cereulide triggered the generation of biofilms and pellicles by B. cereus. The results indicate that both endogenous and externally accessible cereulide supports the fitness of cereulide-producing B. cereus in environments where the potassium concentration is low.

Sulfur-containing compounds quench 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one chemiluminescence: Discrimination between true antioxidants and quenchers using xanthine oxidase.

The probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) is widely used for studying the superoxide anion production and the efficiency of antioxidants in biological systems. Here we report that a number of sulfur-containing compounds applied in biochemical and cytological studies are able to suppress MCLA-derived chemiluminescence (MDCL) independent of their capability to scavenge superoxide anion. The most effective MDCL quenchers appeared to be the substances with thiocarbamoyl and thiocarbonyl groups coupled to cyclic molecules and several thiol- and disulfide-containing compounds. The analysis of MDCL kinetics in a xanthine oxidase system allows one to rapidly discriminate between true antioxidants and the quenchers of chemiluminescence.