Overestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts

Several limitations have recently been described for lucigenin, a probe frequently used to assess the activity of vascular NAD(P)H oxidase, a major superoxide source. The preferential reducing substrate of such oxidase remains unclear. We assessed whether lucigenin artifacts could affect detection of NAD(P)H oxidase activity. Initial chemiluminescence assays were performed with vascular rings or homogenates at 5, 50, or 250 microM concentrations. Results showed preferential signals with NADPH (vs. NADH) with 5 and 50 microM lucigenin, which were blocked by diphenylene iodonium (DPI), superoxide dismutase (SOD), or its cell-permeable mimetic MnTBAP. With 250 microM lucigenin, the relative signal with NADH became larger than with NADPH, and was poorly inhibited by all three antagonists above. All SOD/DPI-resistant signals were effectively blocked by the electron acceptor nitrobluetetrazolium. Spin trapping with DMPO showed an approximate doubling of DMPO-OH radical adduct signal upon addition of 5 microM lucigenin to homogenates incubated with either NADPH or NADH. With 50 or 250 microM lucigenin, much larger increases were observed with NADH, as opposed to NADPH. Furthermore, oxygen consumption measurements showed analogous results. In summary, our data suggest that: (i) Lucigenin redox-cycling is detectable in vascular tissue even at 5 microM concentrations, while at 250 microM redox-cycling becomes predominant and is markedly increased when NADH is the assayed substrate; and (ii) With 250 microM lucigenin, preferentially with NADH, signals are further overestimated by direct, oxidase-dependent, superoxide-independent two-electron transfer. Therefore, previous reports of preferential NADH affinity of the vascular oxidase may have been due to these artifacts.     

The Superoxide Synthases of Plasma Membrane Preparations from Cultured Rose Cells

Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O2- when incubated with either NADH or NADPH, as measured by an O2--specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The Km for NADH was 159 [mu]M; that for NADPH was 19 [mu]M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O2- seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells.     

A New Type-II NADH Dehydrogenase from the Archaeon Acidianus ambivalens: Characterization and in vitro Reconstitution of the Respiratory Chain

A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens. This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron–sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids. Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein^-1/min^-1) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain. The apparent K_m for NADH was found to be 6 M, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site. Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment. A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa₃ type quinol oxidase. This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase.     

Partial purification and some properties of biopterin synthase and dihydropterin oxidase from Drosophila melanogaster

An enzyme which has been named biopterin synthase has been discovered in Drosophila melanogaster. This enzyme, which has been purified 200-fold from extracts of Drosophila, catalyzes the conversion of sepiapterin to dihydrobiopterin, or oxidized sepiapterin to biopterin. The K _m values for the two substrates are 63 µm for sepiapterin and 10 µm for oxidized sepiapterin. NADPH is required in this enzymatic reaction. An analysis of enzyme activity during development in Drosophila indicates a correlation between enzyme activity and biopterin content at various development stages. Another enzyme, called dihydropterin oxidase, was also discovered and partially purified. This enzyme catalyzes the oxidation of dihydropterin compounds to the corresponding pterin compounds. For example, sepiapterin (a dihydropterin) is oxidized to oxidized sepiapterin in the presence of this enzyme. The only dihydropterin that has been tested that is not a substrate for this enzyme is dihydroneopterin triphosphate, the compound thought to be a precursor for all naturally occurring pterins and dihydropterins. Since the action of dihydropterin oxidase is reduced significantly when the concentration of oxygen is very low, it is likely that this enzyme uses molecular oxygen as the oxidizing agent during the oxidation of dihydropterins. Neither NAD⁺ or NADP⁺ is required. In the presence of the two enzymes dihydropterin oxidase and biopterin synthase, sepiapterin is converted to biopterin. However, in the presence of biopterin synthase alone, sepiapterin is converted to dihydrobiopterin.This work was supported by research grants from the National Institutes of Health (AMO3442) and the National Science Foundation (PCM75-19513 AO2).     

Induction of phytoalexin formation in crown gall and hairy root culture of Salvia miltiorrhiza by methyl viologen

Methyl viologen (MV) (20–150 M), a generator of superoxide anion (O₂ ^–), but not hydrogen peroxide (H₂O₂) (10 M–2 mM) triggered the formation of cryptotanshinone (a phytoalexin) in cultures of both crown galls and hairy roots of Salvia miltiorrhiza. MV also inhibited the biomass formation and decreased the contents of phenolic acids in both cultures whereas H₂O₂ did not. In addition, MV and yeast elicitor induced cryptotanshinone formation synergistically only in crown gall cultures. Treatment of the cultures with 3.3 M diphenylene iodonium, an inhibitor of NAD(P)H oxidase, did not exhibit any detrimental effect on the yeast elicitor-induced cryptotanshinone formation in hairy root cultures whereas 1 M diphenylene iodonium was inhibitory on yeast elicitor-induced cryptotanshinone formation in crown gall cultures.     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Purification and characterization of ferredoxin-NADP<Superscript>+</Superscript> reductase encoded by <Emphasis Type="Italic">Bacillus subtilis yumC</Emphasis>

From Bacillus subtilis cell extracts, ferredoxin-NADP⁺ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with K_m values of 0.57 M vs >200 M. K_cat values of YumC with NADPH were 22.7 s^–1 and 35.4 s^–1 in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.     

The stereospecificity,purification, and characterization of an NADH-ferricyanide reductase from spinach leaf plasma membrane

Summary The stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane, peroxisomal membrane, plasma membrane and tonoplast are all specific for the -hydrogen of NADH whereas the reductases in the ER, the Golgi and the outer mitochondrial membrane are -specific. This shows unequivocally that the NADH-ferricyanide activity in the plasma membrane is not caused by ER contamination. In all the membranes one or several polypeptides with an apparent size of 45–50 kDa cross-react with antibodies raised against a microsomal NADH-ferricyanide reductase. An NADH-ferricyanide reductase was purified from spinach leaf plasma membranes. The enzyme was released from the membrane by CHAPS solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size exclusion chromatography on FPLC. A major band of 45 kDa was detected by SDS-PAGE and it cross-reacted with the anti-NADH-ferricyanide reductase antibodies. The native size of the enzyme is 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Isoelectric focusing revealed three isoenzymes between pH 5.3 and 5.6. The enzyme shows typical FAD fluorescence spectra with excitation peaks at 371 and 468 nm and an emission peak at 525 nm. It is specific for the -hydrogen of NADH and prefers NADH over NADPH as electron donor. It is highly specific for ferricyanide as electron acceptor and it is therefore unlikely to be the enzyme responsible for iron reduction on the outer surface of the plasma membrane.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate - DQ duroquinone - FPLC fast protein liquid chromatography; Ferricyanide hexacyanoferrate(III) - NEM N-ethylmaleimide - PCMB p-chloromercurobenzoate - SHAM salicylhydroxamic acid - SMP submitochondrial particles     

2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg^-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg^-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl₂. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 M chloramphenicol, 10 M 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits (49 kDa), (39 kDa) and (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxy-glutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of and but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric -structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the -subunits.Non-standard abbreviations DTT dithiothreitol - PAGE polyaccrylamide gel electrophoresis - VIS visible     

Modeling of Alternative Pathways of Electron Transport to Photosystem I in Isolated Thylakoids

Kinetics of dark decay of absorbance changes at 830 nm (₈₃₀) was examined in thylakoids isolated from leaves of pea seedlings at various concentrations of exogenous NADPH or NADH. Absorbance changes were induced by far-red light to avoid electron donation from photosystem II. In the presence of either biological reductant, the kinetics of ₈₃₀ decay reflecting dark reduction of 700⁺, the primary electron donor of photosystem I, was fitted by a single exponential term. The rate of 700⁺ reduction increased with the rise in the concentration of both NADPH and NADH. The values of K _M and V _max for 700⁺ reduction estimated from concentration dependences were 105 ± 21 M and 0.32/s for NADPH or 21 ± 8 M and 0.12/s for NADH. The rate of P700⁺ reduction by either NADPH or NADH significantly increased in the presence of rotenone, a specific inhibitor of chloroplast reductase. The value of V _max was changed only in the presence of rotenone, whereas K _m was practically unaffected. Unlike the chloroplasts of intact leaves, the only enzyme mediating the input of reducing equivalents from NADPH or NADH to the electron transport chain was concluded to be present in thylakoids.     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells

Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O2*- production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O2*-. To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O2*- with DEPMPO. We confirmed that the main source of O2*- from VSMC membranes is an NAD(P)H oxidase and that the O2*- formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O2*- when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- +/- 0.4-fold higher than the NADH signal). The two substrates had similar EC(50)'s ( approximately 10-50 microM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 +/- 8% and 83 +/- 1% increase above control, respectively), with less of an effect on NADH-dependent O2*- (17 +/- 3% and 36 +/- 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O2*-. In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O2*- production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme.     

Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii

Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl₂, and inhibited by CaCl₂, MgCl₂, NiCl₂, and ZnCl₂.     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification

Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O₂ with H₂ at a maximal specific rate of 0.4 U (mol/min) per mg protein with an apparent K _m for O₂ of 30 M. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F₄₂₀-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F₄₂₀H₂ with 1 O₂ to 2 F₄₂₀ and 2 H₂O. The apparent K _m for F₄₂₀ was 30 M and that for O₂ was 2 M with a V _max of 240 U/mg at 37°C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H₂O₂ as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H–X–E–X–D–X₆₂–H–X₁₈–D–X₆₀–H. Sequence comparisons revealed that the F₄₂₀H₂ oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30–40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F₄₂₀H₂ oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F₄₂₀H₂ oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.Dedicated to Hans Schlegel on the occasion of his 80th birthday.     

A variety of epistatic interactions can occur between partially homologous transgene loci brought together by sexual crossing

Summary Epistatic interactions between unlinked transgene loci in tobacco plants were studied following sexual crosses between different transgenic lines. Three potential modifier transgene loci, which were structurally similar but integrated at different chromosomal locations, were tested for their ability to influence the expression of a partially homologous target transgene locus. After introduction of an individual modifier locus, the target locus could be either unaffected, completely inactivated and methylated or differentially sensitive, showing more complete inactivation and methylation when homozygous than when hemizygous. The implications of these results for inbreeding depression in plants are discussed.     

Oligogalacturonide signal transduction,induction of defense-related responses and protection of grapevine against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H₂O₂, followed by differential expression of nine defense-related genes and stimulation of chitinase and -1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and -1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55–65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.Abbreviations AOS Active oxygen species - Chit Chitinase - DPI Diphenylene iodonium - -Glu -1,3-Glucanase - GST Glutathione-S-transferase - MAP Mitogen-activated protein - OGA Oligogalacturonides - PAL Phenylalanine ammonia-lyase - PR Pathogenesis-related - PGIP Polygalacturonase inhibiting protein - PIN Serine-proteinase inhibitor - STS Stilbene synthase