The protozoan toxin climacostol and its derivatives: Cytotoxicity studies on 10 species of free-living ciliates

Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to resorcinolic lipids, a group of compounds that show antimicrobial, antiparasitic, and anticancer activities. We investigated the cytotoxic activity of the chemically synthesized toxin and its alkyl and alkynyl derivatives on C. virens and nine other common species of free-living freshwater ciliates. Our results show that the cytotoxic potency of climacostol can be modulated by the substitution of the double bond present in the aliphatic chain of the toxin with a single or a triple one that was previously obtained during the synthesis of the unsaturated and saturated derivatives of the parent molecule. We demonstrated that the cytotoxicity level of the molecules considered in this study appears to be inversely correlated to the unsaturation level of their aliphatic chains, and that the potency of their action is also related to the target organism.     

Rate constants in two dimensions of electron transfer between pyruvate oxidase, a membrane enzyme, and ubiquinone (coenzyme Q8), its water-insoluble electron carrier

The functionality of the membrane-bound, ubiquinone-dependent pyruvate oxidase from the respiratory chain of Escherichia coli was reconstituted with a supported lipidic structure. The artificial structure was especially designed to allow the electrochemical control of the quinone pool through the lateral mobility of the ubiquinone (Q(8)) molecules. The kinetic coupling of the enzyme bound to the lipid structure with the quinone pool was ensured by the regeneration of the oxidized form of ubiquinone at the electrochemical interface. Such an experimental approach enabled us to carry out an unprecedented determination of the kinetic parameters controlling the reaction between the enzyme bound and the electron carrier under conditions taking rigorously into account the fact that the freedom of motion is restricted to two dimensions. The kinetic constants we found show that the activated enzyme can be efficiently regulated by the oxidation level of the quinone pool in natural membranes.     

The NADPH:quinone oxidoreductase P1-zeta-crystallin in Arabidopsis catalyzes the alpha,beta-hydrogenation of 2-alkenals: detoxication of the lipid peroxide-derived reactive aldehydes

P1-zeta-crystallin (P1-ZCr) is an oxidative stress-induced NADPH:quinone oxidoreductase in Arabidopsis thaliana, but its physiological electron acceptors have not been identified. We found that recombinant P1-ZCr catalyzed the reduction of 2-alkenals of carbon chain C(3)-C(9) with NADPH. Among these 2-alkenals, the highest specificity was observed for 4-hydroxy-(2E)-nonenal (HNE), one of the major toxic products generated from lipid peroxides. (3Z)-Hexenal and aldehydes without alpha,beta-unsaturated bonds did not serve as electron acceptors. In the 2-alkenal molecules, P1-ZCr catalyzed the hydrogenation of alpha,beta-unsaturated bonds, but not the reduction of the aldehyde moiety, to produce saturated aldehydes, as determined by gas chromatography/mass spectrometry. We propose the enzyme name NADPH:2-alkenal alpha,beta-hydrogenase (ALH). A major portion of the NADPH-dependent HNE-reducing activity in A. thaliana leaves was inhibited by the specific antiserum against P1-ZCr, indicating that the endogenous P1-ZCr protein has ALH activity. Because expression of the P1-ZCr gene in A. thaliana is induced by oxidative stress treatments, we conclude that P1-ZCr functions as a defense against oxidative stress by scavenging the highly toxic, lipid peroxide-derived alpha,beta-unsaturated aldehydes.     

Inhibition of PSII in atrazine-tolerant tobacco cells by barbatic acid, a lichen-derived depside

In atrazine-tolerant tobacco cells with Ser to Thr mutation at the 264th amino acid of PsbA polypeptide in photosystem II (PSII), electron trannsport around the secondary quinone acceptor (Q(B)) site was inhibited to a greater extent by barbatic acid than in wild-type cells. Further characterization suggests similar mode of action of barbatic acid and phenyl-type herbicides.     

Single chain antibodies specific for fatty acids derived from a semi-synthetic phage display library

The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic. We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor alpha release in mononuclear cells; bound to hydrophobic bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure--function analysis of lipoproteins in animal cells.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3‐nitropropionic acid (3‐NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum‐isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC), and that 3‐NPA increased DA, reactive oxygen species (ROS), DA‐quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3‐NPA‐induced increase in DA, ROS, lipid peroxidation, and DA‐quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α‐tocopherol. Results show that the increase in DA induced by 3‐NPA was inhibited by both 25 μM vinpocetine and 50 μM α‐tocopherol. Vinpocetine, as α‐tocopherol, also inhibited 3‐NPA‐induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA‐RS formation), and DA‐quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3‐NPA, 3‐NPA increases DA‐oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α‐tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA‐rich structures.     

Anti-Salmonella activity of (2E)-alkenals

AIMS: This study investigated the effect of a series of naturally occurring aliphatic (2E)-alkenals against Salmonella choleraesuis subsp. choleraesuis ATCC 35640 and evaluated their antibacterial action. METHOD AND RESULTS: A homologous series of aliphatic (2E)-alkenals from C5-C13 were tested for their antibacterial activity against Salm. Choleraesuis. The antibacterial action of (2E)-alkenals against Salm. choleraesuis increases with increasing carbon chain length. (2E)-Dodecenal (C12) was the most effective against this food-borne bacterium with minimum bactericidal concentration (MBC) of 6.25 microg ml-1 (34 micromol l-1), followed by (2E)-undecenal (C11) with an MBC of 12.5 microg ml-1 (77 micromol l-1). The activity was found to correlate with the hydrophobic alkyl chain length from the hydrophilic aldehyde group. The time-kill curve study showed that (2E)-dodecenal was bactericidal against Salm. choleraesuis at any growth stage. CONCLUSIONS: The antibacterial activity of (2E)-alkenals against Salm. choleraesuis was found to correlate with the hydrophobic alkyl chain length. The conjugated double bond is not essential in eliciting the activity but is associated with increasing it. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of their easy availability and wide distribution in many edible plants, (2E)-alkenals can be used as anti-Salmonella agents.     

Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria

Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wieckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151-163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death.     

Antibacterial activity of cerein 8A, a bacteriocin-like peptide produced by Bacillus cereus.

The mode of action of cerein 8A, a bacteriocin produced by the soil bacterium Bacillus cereus 8A, was investigated. The effect of cerein 8A was tested against Listeria monocytogenes and a bactericidal effect at 400 arbitrary units (AU)/ml was observed. In addition, cerein 8A was bactericidal against Bacillus cereus at 200 AU/ml, and inhibited the growth of Escherichia coli and Salmonella Enteritidis. Stronger inhibition of these gram-negative bacteria was achieved when the chelating agent EDTA was added together with bacteriocin. The effect of cerein 8A on B. cereus and L. monocytogenes was also investigated by Fourier transform infrared spectroscopy (FTIR). Treated cells had an important frequency increase at 2920 cm-1 and a decrease at 1400 cm-1, corresponding to assignments of fatty acids. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material. These results suggest that the mode of action of cerein 8A is to interfere with cell membranes and the cell wall.     

Characterization of the Na-dependent respiratory chain NADH:quinone oxidoreductase of the marine bacterium, Vibrio alginolyticus, in relation to the primary Na pump

The Na⁺-dependent respiratory chain NADH: quinone oxidoreductase of the marine bacterium, Vibrio alginolyticus, was extracted from membrane by a detergent, Liponox DCH, and was purified by chromatography on QAE-Sephadex and Bio-Gel HTP. The activity of NADH oxidation was separated into two fractions. The one fraction could react with several artificial electron acceptors including Q-1, but could not reduce ubiquinone and menaquinone such as Q-5 and menaquinone-4, which was called NADH dehydrogenase. The other fraction could reduce Q-5 and menaquinone-4 in addition to the NADH dehydrogenase activity, which was called quinone reductase. The purified NADH dehydrogenase consumed NADH in excess of the amount of Q-1 and the reduced Q-1 (quinol) was not produced at all due to an oxidation-reduction cycle of semiquinone radicals. The quinone reductase, however, consumed NADH with the quantitative formation of quinol on account of a dismutation reaction of semiquinone radicals. Identical to the membrane-bound NADH: quinone oxidoreductase, the quinone reductase specifically required Na⁺ for the activity and was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. The electron transfer in the quinone reductase was formulated in a form of quinone cycle and the dismutation reaction of semiquinone radicals was assigned to be coupled to the Na⁺ pump in the respiratory chain of this organism.     

Loss of Cytochrome c Oxidase Activity and Acquisition of Resistance to Quinone Analogs in a Laccase-Positive Variant of Azospirillum lipoferum

Laccase, a p-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa(3) type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc(1) complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.     

Electrochemical measurement of lateral diffusion coefficients of ubiquinones and plastoquinones of various isoprenoid chain lengths incorporated in model bilayers.

The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%). The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ10 or PQ9 was 2.0 +/- 0.4 x 10(-8) cm2 s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ2 or PQ2 to UQ10, the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.     

Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.     

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.     

Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.     

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.     

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

The second respiratory chain of Candida parapsilosis: a comprehensive study

The yeast C. parapsilosis CBS7157 is strictly dependent on oxidative metabolism for growth since it lacks a fermentative pathway. It is nevertheless able to grow on high glucose concentrations and also on a glycerol medium supplemented with antimycin A or drugs acting at the level of mitochondrial protein synthesis. Besides its normal respiratory chain C. parapsilosis develops a second electron transfer chain antimycin A-insensitive which allows the oxidation of cytoplasmic NAD(P)H resulting from glycolytic and hexose monophosphate pathways functioning through a route different from the NADH-coenzyme Q oxidoreductase described in S. cerevisiae or from the alternative pathways described in numerous plants and microorganisms. The second respiratory chain of C. parapsilosis involves 2 dehydrogenases specific for NADH and NADPH respectively, which are amytal and mersalyl sensitive and located on the outer face of the inner membrane. Since this antimycin A-insensitive pathway is fully inhibited by myxothiazol, it was hypothesized that electrons are transferred to a quinone pool that is different from the classical coenzyme Q-cytochrome b cycle. Two inhibitory sites were evidenced with myxothiazol, one related to the classical pathway, the other to the second pathway and thus, the second quinone pool could bind to a Q-binding protein at a specific site. Elimination of this second pool leads to a fully antimycin A-sensitive NADH oxidation, whereas its reincorporation in mitochondria allows recovery of an antimycin A-insensitive, myxothiazol sensitive NADH oxidation. The third step in this second respiratory chain involves a specific pool of cytochrome c which can deliver electrons either to a third phosphorylation site or to an alternative oxidase, cytochrome 590. This cytochrome is inhibited by high cyanide concentrations and salicylhydroxamates.