Probing the redox metabolism in the strictly anaerobic,extremely thermophilic,hydrogen-producing <Emphasis Type="Italic">Caldicellulosiruptor</Emphasis><Emphasis Type="Italic">saccharolyticus</Emphasis> using amperometry

Changes in the redox metabolism in the anaerobic, extremely thermophilic, hydrogen-forming bacterium Caldicellulosiruptor saccharolyticus were probed for the first time in vivo using mediated amperometry with ferricyanide as a thermotolerant external mediator. Clear differences in the intracellular electron flow were observed when cells were supplied with different carbon sources. A higher electrochemical response was detected when cells were supplied with xylose than with sucrose or glucose. Moreover, using the mediated electrochemical method, it was possible to detect differences in the electron flow between cells harvested in the exponential and stationary growth phases. The electron flow of C. saccharolyticus was dependent on the NADH- and reduced ferredoxin generation flux and the competitive behavior of cytosolic and membrane-associated oxidoreductases. Sodium oxamate was used to inhibit the NADH-dependent lactate dehydrogenase, upon which more NADH was directed to membrane-associated enzymes for ferricyanide reduction, leading to a higher electrochemical signal. The method is noninvasive and the results presented here demonstrate that this method can be used to accurately detect changes in the intracellular electron flow and to probe redox enzyme properties of a strictly anaerobic thermophile in vivo.     

Electrochemical detection of wild type Saccharomyces cerevisiae responses to estrogens

The presence of an estrogen binding protein (EBP) and an endogenous ligand in three yeast species was first reported in 1982/1983. The ligand was shown to be 17beta-estradiol and the binding affinities of EBP were demonstrated to be similar to those of rat estrogen receptors. This report describes detection of the behaviour of a putative estrogen binding protein in Saccharomyces cerevisiae using a double mediator electrochemical detection system. The response to estrogen is shown to be quantitative with signals detectable from 10(-8) to 10(-14)M. An incubation period of 5h is established and a method to block electrochemical signals produced by the catabolism of exogenous substrates is demonstrated to be effective. The system provides a method that permits the use of wild type S. cerevisiae to quantify estrogens.     

Effect of ionophores on carrier-mediated electron translocation in ferricyanide-containing liposomes

Ferricyanide-containing liposomes were used as a system to compare the electron- and proton-translocating properties of six redox reagents commonly used as electron donors for biochemical systems. The effects of different ionophore combinations on the ferricyanide-reduction rate were generally consistent with the expected proton- and electron-translocating properties of the mediators. The transmembrane pH gradient produced by hydrogen carriers was demonstrated. Nigericin or valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone are capable of collapsing this gradient and of stimulating ferricyanide reduction mediated by this type of carrier. No pH gradient is produced with the electron carrier 1,1'-dibutylferrocene. In the presence of tetraphenylboron anion, which is needed for this carrier to act as an efficient mediator, addition of valinomycin alone is sufficient to obtain full stimulation of ferricyanide reduction. NNN'N'-Tetramethyl-p-phenylenediamine does not behave as a simple electron carrier. During NNN'N'-tetramethyl-p-phenylenediamine-mediated ferricyanide reduction protons are translocated across the membrane and accumulated in the vesicles. This is not due to the presence of demethylated impurities in the NNN'N'-tetramethyl-p-phenylenediamine sample, but may be the result of an accumulation of oxidation products other than the Wurster's Blue radical. These results suggest a reconsideration of studies on protonmotive forces across membranes where NNN'N'-tetramethyl-p-phenylenediamine is used as a mediator.     

Modification of redox properties of cells by ortho-benzoquinones

Ortho-benzoquinones have been studied for their effect on the processes of methemoglobin formation in the rabbit erythrocytes and reduction of potassium ferricyanide by the HeLa cells. It is established that the incubation of cells in the presence of lipophilic quinone OBQ-1 results in the formation of the intracellular methemoglobin in erythrocytes and the intensification of the ferricyanide reduction by HeLa cells. OBQ-2 differing in the presence of the polar sulphogroup does not react with the intracellular oxyhemoglobin and exerts no effect on the ferricyanide reduction. It is supposed that OBQ-1 may change the ratio of the oxidized and reduced metabolites in a cell, thus inducing in it a state of the oxidative stress. A conclusion is drawn hat ortho-benzoquinones are able to modify the redox properties of cells, the efficiency of modification depending on the chemical structure of the compounds.     

Topography of the active site of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> plasmalemmal dicarboxylate transporter studied using lipophilic derivatives of its substrates

2-Alkylmalonates and O-acyl-L-malates have been found to competitively inhibit the dicarboxylate transporter of Saccharomyces cerevisiae cells, and the substrate derivatives chosen did not penetrate across the plasmalemma under the experiment conditions. Probing of the active site of this transporter has revealed a large lipophilic area stretching between the 0.72 to 2.5 nm from the substrate-binding site. Itaconate inhibited the transport fivefold more effectively than L-malate. This suggests the existence of a hydrophobic region immediately near the dicarboxylate-binding site (to 0.72 nm). The yeast plasmalemmal transporter was different from the rat liver mitochondrial dicarboxylate transporter. An area with variable lipophilicity adjoining the substrate-binding site has been revealed in the latter by a similar method. This area is mainly hydrophobic at distances up to 1.76 nm from the binding site and is separated by a hydrophilic region from 0.38 to 0.88 nm. Fumarate but not maleate competitively inhibited succinate transport into the S. cerevisiae cells. It is suggested that the plasmalemmal transporter binds the substrate in the trans-conformation. The prospects of the proposed approach for scanning lipophilic profiles of channels of different transporters are discussed.     

Aqueous Electrochemical Energy Storage with a Mediator‐Ion Solid Electrolyte

This study presents a battery concept with a “mediator‐ion” solid electrolyte for the development of next‐generation electrochemical energy storage technologies. The active anode and cathode materials in a single cell can be in the solid, liquid, or gaseous form, which are separated by a sodium‐ion solid‐electrolyte separator. The uniqueness of this mediator‐ion strategy is that the redox reactions at the anode and the cathode are sustained by a shuttling of a mediator sodium ion between the anolyte and the catholyte through the solid‐state electrolyte. Use of the solid‐electrolyte separator circumvents the chemical‐crossover problem between the anode and the cathode, overcomes the dendrite‐problem when employing metal‐anodes, and offers the possibility of using different liquid electrolytes at the anode and the cathode in a single cell. The battery concept is demonstrated with two low‐cost metal anodes (zinc and iron), two liquid cathodes (bromine and potassium ferricyanide), and one gaseous cathode (air/O₂) with a sodium‐ion solid electrolyte. This novel battery strategy with a mediator‐ion solid electrolyte is applicable to a wide range of electrochemical energy storage systems with a variety of cathodes, anodes, and mediator‐ion solid electrolytes.     

EPR characterization of a high-spin system in carbon monoxide dehydrogenase from Methanothrix soehngenii.

Carbon monoxide dehydrogenase and methyl-coenzyme M reductase were purified from 61Ni-enriched and natural-abundance nickel-grown cells of the methanogenic archae Methanothrix soehngenii. The nickel-EPR signal from cofactor F-430 in methyl-CoM reductase was of substoichiometric intensity and exhibited near-axial symmetry with g = 2.153, 2.221 and resolved porphinoid nitrogen superhyperfine splittings of approximately 1 mT. In the spectrum from 61Ni-enriched enzyme a well-resolved parallel I = 3/2 nickel hyperfine splitting was observed, A parallel = 4.4 mT. From a computer simulation of this spectrum the final enrichment in 61Ni was estimated to be 69%, while the original enrichment of the nickel metal was 87%. Carbon monoxide dehydrogenase isolated from the same batch exhibited four different EPR spectra. However, in none of these signals could any splitting or broadening from 61Ni be detected. Also, the characteristic g = 2.08 EPR signal found in some other carbon monoxide dehydrogenases and ascribed to a Ni-Fe-C complex, was never observed by us under any conditions of detection (4 to 100 K) and incubation in the presence of ferricyanide, dithionite, CO, coenzyme A, or acetyl-coenzyme A. Novel, high-spin EPR was found in the oxidized enzyme with effective g-values at g = 14.5, 9.6, 5.5, 4.6, 4.2, 3.8. The lines at g = 14.5 and 5.5 were tentatively ascribed to an S = 9/2 system (approximately 0.3 spins/alpha beta) with rhombicity E/D = 0.047 and D less than 0. The other signals were assigned to an S = 5/2 system (0.1 spins/alpha beta) with E/D = 0.27. Both sets of signals disappear upon reduction with Em,7.5 = - 280 mV. With a very similar reduction potential, Em,7.5 = - 261 mV, an S = 1/2 signal (0.1 spins/alpha beta) appears with the unusual g-tensor 2.005, 1.894, 1.733. Upon further lowering of the potential the putative double cubane signal also appears. At a potential E approximately - 320 mV the double cubane is only reduced by a few percent and this allows the detection of individual cubane EPR not subjected to dipolar interaction; a single spectral component is observed with g-tensor 2.048, 1.943, 1.894.     

A microelectrochemical technique to measure trans‐plasma membrane electron transport in plant tissue and cells in vivo

A novel electrochemical technique was developed to enable high‐resolution measurements of trans‐plasma membrane reductase activity in vivo in growing plant tissue and single cells. Carbon fibre microelectrodes (CFMEs) with a tip diameter of 5 µm were used for electrochemical mapping of the reduction of the external impermeant electron acceptor ferricyanide along the root tip surface of 4‐d‐old maize seedlings. Ferricyanide reduction was detected in all locations along the first 12 mm of the growing root apex. However, a distinct peak in activity was detected at the proximal end of the elongation zone (1·5–4·5 mm from the apex), where reductase activity was three times greater than in more apical or distal regions. The inhibition of the ferricyanide reduction at all locations along the growing apex, by the vitamin K antagonists warfarin and dicumarol, supports previous data showing that electron transfer by the constitutive trans‐plasma membrane reductase is achieved via a quinone shuttle. We demonstrate that in addition to their utility in whole‐tissue/‐organ studies, CFMEs are sensitive enough to monitor trans‐plasma membrane electron transport in single cells.     

Electrochemical detection of DNA hybridization using a change in flexibility

A novel electrochemical method of detecting DNA hybridization is presented based on the change in flexibility between the single and double stranded DNA. A recognition surface based on gold nanoparticles (GNPs) is firstly modified via mixing self-assembled monolayer of thiolated probe DNA and 1,6-hexanedithiol. The hybridization and electrochemical detection are performed on the surface of probe-modified GNPs and electrode, respectively. Here in our method the charge transfer resistance (R(ct)) signal is enhanced by blocking the surface of electrode with DNA covered GNPs. The GNPs will be able to adsorb on the gold electrode when covered with flexible single stranded DNA (ssDNA). On the contrary, it will be repelled from the electrode, when covered with stiff double stranded DNA (dsDNA). Therefore, different R(ct) signals are observed before and after hybridization. The hybridization events are monitored by electrochemical impedance spectroscopy (EIS) measurement based on the R(ct) signals without any external labels. This method provides an alternative route for expanding the range of detection methods available for DNA hybridization.     

Evidence for Ca++-calmodulin control of transplasmalemma electron transport in carrot cells

Cultured carrot cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system, which may be associated with an iron reduction and uptake system in plant roots. Here we provide evidence for the inhibition of transplasma membrane ferricyanide reduction by four different Ca2+-calmodulin type antagonists, calmidazolium, trifluoperazine, pimozide and fluphenazine. These compounds inhibit in low concentrations (approximately 5-10 microM) in a time-dependent manner. Higher concentrations (50-100 microM) are required to inhibit transmembrane ferricyanide reduction in 10 min rather than in 30 min. The permeable calcium chelator, TMB-8, also inhibits transmembrane ferricyanide reduction in carrot cells. Since the redox system is controlled by hormones, the effects of anticalmodulin agents on hormone response may be mediated through the redox system.     

Postreplication repair and PCNA modification in Schizosaccharomyces pombe

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.     

Selective thiol inhibition of ferricyanide reduction in photosystem II of spinach chloroplasts

Selective inhibition of ferricyanide reduction in photosystem II by lipophilic thiols indicates a unique pathway of electron transport, which is not involved in reduction of class III acceptors or transfer of electrons to photosystem I. Both aromatic and aliphatic thiols induce the inhibition, but thiol binding reagents such as p-hydroxymercuribenzoate or N-ethylmaleimide do not inhibit. The inhibition can be observed using either dibromothymoquinone or bathophenanthroline to direct electrons away from photosystem I. No pretreatment of chloroplasts with thiols in the light was necessary to inhibit ferricyanide reduction by photosystem II or the O₂ evolution associated with ferricyanide reduction.     

Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.     

Amphiphilic amine-N-oxides with aliphatic alkyl chain act as efficient superoxide dismutase mimics, antioxidants and lipid peroxidation blockers in yeast

Amphiphilic 3-(alkanoylamino)propyldimethylamine-N-oxides with different length of the alkyl chain, i.e. different hydrophilic-lipophilic balance, act in micromolar concentrations as SOD mimics by lifting the inhibition of aerobic growth caused by SOD deletions in Saccharomyces cerevisiae. They also enhance the survival of sod mutants of S. cerevisiae exposed to the hydrophilic superoxide-generating prooxidant paraquat and the amphiphilic hydroperoxide-producing tert-butylhydroperoxide (TBHP), and largely prevent TBHP-induced peroxidation of isolated yeast plasma membrane lipids. Unlike the SOD-mimicking effect, the magnitude of these effects depends on the alkyl chain length of the amine-N-oxides, which incorporate into S. cerevisiae membranes, causing fluidity changes in both the hydrophilic surface part of the membrane and the membrane lipid matrix. Unlike wild-type strains, the membranes of sod mutants were found to contain polyunsaturated fatty acids; the sensitivity of the mutants to lipophilic pro-oxidants was found to increase with increasing content of these acids. sod mutants are useful in assessing pro- and antioxidant properties of different compounds.     

Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment

Eukaryotic cells respond to DNA damage by activating checkpoint signalling pathways. Checkpoint signals are transduced by a protein kinase cascade that also requires non-kinase mediator proteins. One such mediator is the Saccharomyces cerevisiae Dpb11 protein, which binds to and activates the apical checkpoint kinase, Mec1. Here, we show that a ternary complex of Dpb11, Mec1 and another key mediator protein Rad9 is required for efficient Rad9 phosphorylation by Mec1 in vitro, and for checkpoint activation in vivo. Phosphorylation of Rad9 by cyclin-dependent kinase (CDK) on two key residues generates a binding site for tandem BRCT repeats of Dpb11, and is thereby required for Rad9 recruitment into the ternary complex. Checkpoint signalling via Dpb11, therefore, does not efficiently occur during G1 phase when CDK is inactive. Thus, Dpb11 coordinates checkpoint signal transduction both temporally and spatially, ensuring the initiator kinase is specifically activated in proximity of one of its critical substrates.     

Use of an electrochemical technique to study plasmamembrane redox reactions in cultured cells of Daucus carota L.

By exploiting the electron transfer reactions of ferricyanide-ferrocyanide at a gold electrode, an electrochemical technique was devised and successfully employed for the measurement of extracellular ferricyanide reduction or ferrocyanide oxidation by carrot (Daucus carota L.) cells grown in suspension culture. This technique eliminated problems of cell damage and insensitivity encountered when these activities are measured spectrophotometrically in magnetically stirred cell suspensions. Cells harvested from the mid-exponential phase of culture growth catalysed a rapid reduction of ferricyanide which was accompanied by H⁺ extrusion and was stimulated by ethanol. These cells also oxidised ferrocyanide, which was not associated with H⁺ extrusion. These observations can be explained on the basis of a plasmamembrane located, H⁺-translocating redox system. The electrochemical technique is a useful method of studying this system.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Growth kinetics of Chlorella vulgaris and its use as a cathodic half cell

The kinetics of growth of the algal species Chlorella vulgaris has been investigated using CO(2) as the growth substrate. The growth rate was found to increase as the dissolved CO(2) increased to 150 mg/L, but fell dramatically at higher concentrations. Increasing the radiant flux also increased growth rate. With a radiant flux of 32.3 mW falling directly on the 500 mL culture media, the growth rate reached up to 3.6 mg of cells/L-h. Both pH variation (5.5-7.0) and mass transfer rate of CO(2) (K(L)a between 6h(-1) and 17 h(-1)) had little effect on growth rate. Growing on glucose, the yeast Saccharomyces cerevisiae produced a stable 160 mV potential difference when acting as a microbial fuel cell anode with ferricyanide reduction at the cathode. The algal culture was observed to be a workable electron acceptor in a cathodic half cell. Using an optimum methylene blue mediator concentration, a net potential difference of 70 mV could be achieved between the growing C. vulgaris culture acting as a cathode and a 0.02 M potassium ferrocyanide anodic half cell. Surge current and power levels of 1.0 microA/mg of cell dry weight and 2.7 mW/m(2) of cathode surface area were measured between these two half cells.