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In timber infested by brown-rot fungi, a rapid loss of strength is attributed to production of hydroxyl radicals (HO.). The hydroxyl radicals are produced by the Fenton reaction [Fe(II)/H2O2], but the pathways leading to Fe(II) and H2O2 have remained unclear. Cellobiose dehydrogenase, purified from cultures of Coniophora puteana, has been shown to couple oxidation of cellodextrins to conversion of Fe(III) to Fe(II). Two characteristics of brown rot are release of oxalic acid and lowering of the local pH, often to about pH 2. Modelling of Fe(II) speciation in the presence of oxalate has revealed that Fe(II)-oxalate complexes are important at pH 4-5, but at pH 2 almost all Fe(II) is in an uncomplexed state which reacts very slowly with dioxygen. Diffusion of Fe(II) away from the hyphae will promote conversion to Fe(II)-oxalate and autoxidation with H2O2 as product. Thus the critical Fe(II)/H2O2 combination will be generated at a distance, enabling hydroxyl radicals to be formed without damage to the hyphae.

To explore aromatase inhibition and to broaden the structural diversity of dual aromatase-sulfatase inhibitors (DASIs), we introduced the steroid sulfatase (STS) inhibitory pharmacophore to letrozole. Letrozole derivatives were prepared bearing bis-sulfamates or mono-sulfamates with or without adjacent substituents. The most potent of the achiral and racemic aromatase inhibitor was 40 (IC 50 = 3.0 nM). Its phenolic precursor 39 was separated by chiral HPLC, and the absolute configuration of each enantiomer was determined using vibrational and electronic circular dichroism in tandem with calculations of the predicted spectra. Of the two enantiomers, ( R)-phenol ( 39a) was the most potent aromatase inhibitor (IC 50 = 0.6 nM, comparable to letrozole), whereas the ( S)-sulfamate, ( 40b) inhibited STS most potently (IC 50 = 553 nM). These results suggest that a new structural class of DASI for potential treatment of hormone-dependent breast cancer has been identified, and this is the first report of STS inhibition by an enantiopure nonsteroidal compound.

How can professional foresters ensure they practise with due diligence when they are charged with making decisions in which the advice of non-forestry specialists is one of the factors they must take into account? The case law on due diligence in Canada supports the need for appropriate expertise when potential regulatory offences are at stake. In a forestry context, the appropriate expertise is sometimes found among non-forestry specialists. As a result, professional foresters are growing increasingly reliant on the advice of such specialists. But especially in those provinces — BC, Ontario, and Quebec — with legislated exclusive professional forestry practice, professional foresters are often placed in the awkward position of having to judge the advice of specialists even when these foresters do not possess the expertise to make that judgment. Can foresters overrule the advice of non-forestry specialists? The case law on due diligence suggests they do so at their peril. By way of two hypothetical case studies, this paper highlights this dilemma, but also suggests that sharing the "decision-making space" may be a partial way to ensure that forestry decisions meet the demands of due diligence case law. Key words: professional ethics, professional forester, reasonable care, absolute liability, strict liability, R. v. Sault Ste. Marie

Forty-six (1.5%) of nearly 3000 isolates of Mycosphaerella graminicola assayed in vitro were resistant to the QOI fungicide azoxystrobin, but on sub-culturing only ten remained resistant. Cross-resistance extended to other QOIs, but varied between different isolates. In planta the resistant isolates were not well controlled, especially at lower azoxystrobin dose rates. Propyl gallate, an inhibitor of alternative oxidase, potentiated the activity of azoxystrobin in vitro so that resistance was no longer observed. The growth of resistant strains in the presence of azoxystrobin led to alternative oxidase activation. This increased flexibility in respiration allows resistant strains to survive in the presence of a QOI fungicide. Under these conditions, selection for target-site mutations can occur. Using QOIs preventatively reduces the risk of resistance since the alternative oxidase cannot by itself generate all the energy needed for germination and early infection.

Limnetic-benthic sympatric species pairs of threespine stickleback (Gasterosteus spp.) are unique to lakes of southwestern British Columbia. One pair has gone extinct and the remaining three pairs are listed as endangered. The biodiversity conservation policies that could potentially protect these species are examined. The plight of the stickleback pairs serves as a test of the sufficiency of Canada's constellation of biodiversity conservation policies. This article finds that until very recently not even the combination of national and provincial policies offered unequivocal protection for these species. A new national Species at Risk Act now offers more promise. Nevertheless, a more fundamental issue remains. A misconception concerning the value of biodiversity may be impeding the formulation of adequate policies and plans for biodiversity conservation in general, both in Canada and worldwide.

This summary describes a functional role for the alternative respiratory pathway of the wheat ‘take-all’ fungus, Gaeumannomyces graminis var tritici (Ggt), which utilises the alternative oxidase as its terminal oxidase. Unlike other plant and fungal alternative oxidases, the alternative oxidase of Ggt is both constitutively expressed and active. We have demonstrated that for cellular respiration, and therefore for ATP synthesis, to occur both the cytochrome and alternative pathways must remain active. © 1999 Society of Chemical Industry

An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with d-cellobiose or d-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of 1H-NMR spectra in D2O, the product of d-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included d-cellobiose, l-arabinose, d-xylose and sucrose, but the specificity parameter (k cat/K m) was highest for d-glucose. Two electron acceptors were identified, namely 2,6-dichloroindophenol and p-benzoquinone, but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of d-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus.

EPR spectroscopy has been employed for the direct detection of a variety of free radicals formed from reaction of Fe(II)–oxalate and H2O2 in the presence of carbohydrates and related compounds: this system has been designed to model the proposed mode of action of brown rot fungi. The observed hyperfine splittings allow characterization of individual radicals formed at different positions in the carbohydrate rings. Relative signal intensities in steady-state spectra indicate the rapid generation of the hydroxyl radical, followed by relatively unselective attack of ˙OH on the substrates’ C–H bonds: the rapidity of oxidation by Fe(III) of oxygen-conjugated carbon-centred radicals (typically k 108 dm3 mol–1 s–1) is significantly reduced if there is an eclipsing β-oxygen substituent. The relevance of these findings to cellulose-cleaving reactions of certain fungi is discussed.

The value of biodiversity is usually confused with the value of biological resources, both actual and potential. A sharp distinction between biological resources and biodiversity offers a clearer insight into the value of biodiversity itself and therefore the need to preserve it. Biodiversity can be defined abstractly as the differences among biological entities. Using this definition, biodiversity can be seen more appropriately as: (a) a necessary precondition for the long term maintenance of biological resources, and therefore, (b) an essential environmental condition. Three values of biodiversity are identified and arranged in a hierarchy: (1) the self-augmenting phenomenon of biodiversity maintains (2) the conditions necessary for the adaptive evolution of species and higher taxa, which in turn is necessary for providing humans with (3) a range of biological resources in the long term. Two broad policy implications emerge: increments of biodiversity should not be traded off against biological resources as if they were the same, and the conservation of biodiversity should be a constraint on the public interest, not a goal in service of the public interest.

Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana. Strong cross-reaction was observed with antibodies to cellobiose:quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium. Kinetic measurements were made with cellobiose as electron donor. Ferricyanide and DCPIP both showed a pH optimum close to pH 4, but activity with ferricyanide declined more rapidly when the pH was raised. Dioxygen reduction to hydrogen peroxide was observed, but at a much lower rate than for other acceptors. These properties are similar to those of cellobiose dehydrogenase from P. chrysosporium, despite differences between brown and white rot modes of decay.

4-{[(4-Cyanophenyl)(4H-1,2,4-triazol-4-yl)amino]methyl}phenyl sulfamate and its ortho-halogenated (F, Cl, Br) derivatives are first-generation dual aromatase and sulfatase inhibitors (DASIs). Structure–activity relationship studies were performed on these compounds, and various modifications were made to their structures involving relocation of the halogen atom, introduction of more halogen atoms, replacement of the halogen with another group, replacement of the methylene linker with a difluoromethylene linker, replacement of the para-cyanophenyl ring with other ring structures, and replacement of the triazolyl group with an imidazolyl group. The most potent in vitro DASI discovered is an imidazole derivative with IC50 values against aromatase and steroid sulfatase in a JEG-3 cell preparation of 0.2 and 2.5 nm, respectively. The parent phenol of this compound inhibits aromatase with an IC50 value of 0.028 nm in the same assay.

To determine the effect of liver disease on the pharmacokinetics of rocuronium, the authors administered 0.6 mg/kg (twice the ED95) to 10 patients with liver disease and compared these results to values in 10 healthy surgical patients. Anesthesia was induced with thiopental and maintained with isoflurane (0.9%-1.1% end-tidal concentration) and nitrous oxide (60%). Venous blood samples were obtained for 6 h after rocuronium injection and plasma concentrations were measured using gas chromatography. Pharmacokinetic differences between groups were determined using a population-based pharmacokinetic analysis (NONMEM). Hepatic impairment did not alter the plasma clearance of rocuronium (217 +/- 21.8 mL/min, mean +/- SE, for both groups), but did increase the volume of the central compartment (5.96 +/- 1.01 L for controls, 7.87 +/- 1.33 L for patients with liver disease) and volume of distribution at steady state (16.4 L for controls, 23.4 L for patients with liver disease). In turn, elimination half-life was longer in patients with liver disease (111 min) compared to controls (75.4 min). The authors conclude that liver disease alters the pharmacokinetics of rocuronium by increasing its volume of distribution. The longer elimination half-life might result in a longer duration of action of rocuronium in patients with liver disease, particularly after prolonged administration.

Many forms of Fe(II) react with H 2 0 2 to generate hydroxyl radicals (Fenton reaction). There is evidence that hydroxyl radicals are important in brown-rot, while they can be formed by secondary reactions during lignin breakdown by white-rot fungi. Their involvement in cellulose breakdown creates a range of oxidized sugars. The two reactants of Fenton's reagent can be generated by Fe(II) autoxidation, or by superoxide in reaction with Fe(III). A rapid autoxidation is not possible for complexes with a high Fe(III)/Fe(II) redox potential. Turning to specific pathways for formation of Fenton's reagent, decomposition of Fe(III)-oxalate is probably solely a photochemical process. Lignin peroxidases can act indirectly as a source of superoxide, either by reactions that lead to a peroxyradical, or by 1-electron oxidation of an aliphatic compound creating a strong reductant. Cellobiose dehydrogenase can provide a direct enzymic source for Fenton's reagent (S.M. Kremer and P.M. Wood (1992) Eur. J. Biochem. 208, 807–814). In the experiments as published, hydroxyl radical production was limited by the slow interaction of cellobiose dehydrogenase with O 2 . This limitation can be removed by the presence of an iron complex with an autoxidizable Fe(lI) state. The successful use of Fenton's reagent by a living organism requires a spatial separation between initiating enzyme(s) and the site of production of hydroxyl radicals. The mobility of the extra electron on Fe(II) by intermolecular transfer may be important for achieving this separation.

[reaction: see text]. Irradiation of benzotriazole with a variety of maleimide derivatives leads to the stereo- and regioselective formation of aryl [2 + 2] photocycloaddition products. Further studies with 2-alkyl benzotriazole derivatives indicates that in the case of the parent benzotriazole this cycloaddition proceeds selectively via the 2H-tautomer.

Concurrent inhibition of aromatase and steroid sulfatase (STS) may provide a more effective treatment for hormone-dependent breast cancer than monotherapy against individual enzymes, and several dual aromatase-sulfatase inhibitors (DASIs) have been reported. Three aromatase inhibitors with sub-nanomolar potency, better than the benchmark agent letrozole, were designed. To further explore the DASI concept, a new series of letrozole-derived sulfamates and a vorozole-based sulfamate were designed and biologically evaluated in JEG-3 cells to reveal structure-activity relationships. Amongst achiral and racemic compounds, 2-bromo-4-(2-(4-cyanophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)phenyl sulfamate is the most potent DASI (aromatase: IC₅₀ =0.87 nM; STS: IC₅₀ =593 nM). The enantiomers of the phenolic precursor to this compound were separated by chiral HPLC and their absolute configuration determined by X-ray crystallography. Following conversion to their corresponding sulfamates, the S-(+)-enantiomer was found to inhibit aromatase and sulfatase most potently (aromatase: IC₅₀ =0.52 nM; STS: IC₅₀ =280 nM). The docking of each enantiomer and other ligands into the aromatase and sulfatase active sites was also investigated.

The design and synthesis of a series of bicyclic ring containing dual aromatase-sulfatase inhibitors (DASIs) based on the aromatase inhibitor (AI) 4-[(4-bromobenzyl)(4H-1,2,4-triazol-4-yl)amino]benzonitrile are reported. Biological evaluation with JEG-3 cells revealed structure-activity relationships. The X-ray crystal structure of sulfamate 23 was determined, and selected compounds were docked into the aromatase and steroid sulfatase (STS) crystal structures. In the sulfamate-containing series, compounds containing a naphthalene ring are both the most potent AI (39, IC(50 AROM)=0.25 nM) and the best STS inhibitor (31, IC(50 STS)=26 nM). The most promising DASI is 39 (IC(50 AROM)=0.25 nM, IC(50 STS)=205 nM), and this was evaluated orally in vivo at 10 mg kg(-1), showing potent inhibition of aromatase (93 %) and STS (93 %) after 3 h. Potent aromatase and STS inhibition can thus be achieved with a DASI containing a bicyclic ring system; development of such a DASI could provide an attractive new option for the treatment of hormone-dependent breast cancer.

Cellobiose oxidase activity from Phanerochaete chrysosporium was monitored continuously by two techniques that are independent of the presence of a solid substrate, measuring either ferricyanide by the electrode potential of anaerobic ferricyanide plus ferrocyanide, or O 2 with a specific electrode. Both methods demonstrated that microcrystalline cellulose (Avicel) is a substrate for oxidation. All experiments were at pH 4.0 and 30°C. Rates were evaluated for two-electron transfer. For Avicel → ferricyanide, a study of initial rates gave k cat = 0.40 ± 0.05 s −1 , much slower than k cat = 3.6 ± 0.3 s −1 with cellobiose as substrate. With Avicel plus 0.2 μM cellobiose oxidase, the rate fell to half its initial value in 3–8 min, indicating a heterogeneity of reducing ends. For Avicel → O 2 , k cat = 0.057 ± 0.004 s −1 . Half maximal rates corresponded to K m ( Avicel ) = 8 ± 2 gl −1 with ferricyanide, and 3.3 ± 0.5 gl −1 with O 2 as acceptor.

Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30 degrees C, with excess cellobiose as substrate and O2 or Fe(III) as acceptor. With O2 at 230 microM as sole electron acceptor, the O2 uptake rate corresponded to a one-electron turnover number of 0.13 +/- 0.01 s-1. Measurements at different O2 concentrations indicated Km(O2) greater than 120 microM. In separate experiments, the reduction of Fe(III) acetate was monitored at 340 nm in the absence of oxygen. The maximum velocity of Fe(III)-acetate reduction (Vmax) was 4.5 +/- 0.7 s-1, while Km[Fe(III) acetate] was 34 +/- 12 microM. With ferricyanide in place of Fe(III) acetate, the corresponding values were 6.9 +/- 0.7 s-1 and 23 +/- 5 microM. Redox titrations established the potential of the haem prosthetic group of the oxidase at pH 4.0 as +165 mV. The midpoint potential for Fe(III)/Fe(II) acetate at pH 4.0 is much higher, a value of +535 mV being obtained with 200 microM Fe. Cellobiose oxidase resembles yeast flavocytochrome b2 and differs from the neutrophil NADPH oxidase in having the potential of its haem group far above the potential for one-electron reduction of O2 to superoxide (Em,4 = -110 mV). A kinetic comparison led to the conclusion that the role of cellobiose oxidase is as an Fe(III) reductase. Fe(II) may have a biological importance as a component of Fenton's reagent [Fe(II)/H2O2]. The concentration of cellobiose oxidase in the growth medium at harvest (0.3 microM) can provide a far higher flux of Fe(II) than a non-enzymic proposal in the literature.

Phanerochaete chrysosporium releases two enzymes that oxidize cellobiose and higher cellodextrins: the flavohaemoprotein cellobiose oxidase and the flavoprotein cellobiose:quinone oxidoreductase (CBQase). Partial digestion of these enzymes with Staphylococcal V8 proteinase or cyanogen bromide yielded many identical bands on SDS-polyacrylamide gels. A polyclonal antibody to either purified protein gave cross-reaction. The purification procedure also yielded a haem protein that ran on dodecyl sulphate gels at Mr 31,000, as compared with 91,000 for cellobiose oxidase and 63,000 for CBQase. The 31 kDa haem protein cross-reacted with polyclonal antibody to cellobiose oxidase, but not with antibody to CBQase. Sulphite bleached the flavin of cellobiose oxidase, but gave no reaction with the 31 kDa haem protein, suggesting an absence of flavin. It is proposed that CBQase and the 31 kDa haem protein are formed from cellobiose oxidase by proteolytic cleavage.

The absolute configuration of a newly designed, letrozole-based chiral aromatase inhibitor that could not be defined by crystallographic techniques has been determined by means of vibrational and electronic circular dichroism and by optical rotation measurements combined with density functional theory calculations on possible conformers. The same absolute configurational assignment can be applied to the individual enantiomeric sulfamate esters, which are derived from the corresponding enantiomers of the chirally separated parent phenols, based on the similarity of the ECD spectrum of the sulfamate derivative to that of its phenolic precursor. The two enantiomeric sulfamate esters studied here are the first examples of nonsteroidal dual aromatase-sulfatase inhibitor whose activities have been evaluated on optically resolved enantiomers. Chirality 21:802-808, 2009. (C) 2009 Wiley-Liss, Inc.

Implicitly, the "greatest good for the greatest number" serves as the dominant land-use ethic of professional foresters, but is flawed for several reasons that are discussed in this paper. In effect, when foresters invoke this ethic when they recommend or make land-use decisions, they are "taking sides" politically in exactly those situations in which professionals are ethically required to be politically neutral. I suggest that this problem may have contributed to the profession's recent lack of public credibility, but it also represents an opportunity for the profession to enhance its credibility by designing and explicitly adopting a more appropriate land-use ethic.

Mitochondrial respiration conserves energy by linking NADH oxidation and electron-coupled proton translocation with ATP synthesis, through a core pathway involving three large protein complexes. Strobilurin fungicides block electron flow through one of these complexes (III), and disrupt energy supply. Despite an essential need for ATP throughout fungal disease development, strobilurins are largely preventative; indeed some diseases are not controlled at all, and several pathogens have quickly developed resistance. Target-site variation is not the only cause of these performance difficulties. Alternative oxidase (AOX) is a strobilurin-insensitive terminal oxidase that allows electrons from ubiquinol to bypass Complex III. Its synthesis is constitutive in some fungi but in many others is induced by inhibition of the main pathway. AOX provides a strobilurin-insensitive pathway for oxidation of NADH. Protons are pumped as electrons flow through Complex I, but energy conservation is less efficient than for the full respiratory chain. Salicylhydroxamic acid (SHAM) is a characteristic inhibitor of AOX, and several studies have explored the potentiation of strobilurin activity by SHAM. We present a kinetic-based model which relates changes in the extent of potentiation during different phases of disease development to a changing importance of energy efficiency. The model provides a framework for understanding the varying efficacy of strobilurin fungicides. In many cases, AOX can limit strobilurin effectiveness once an infection is established, but is unable to interfere significantly with strobilurin action during germination. A less stringent demand for energy efficiency during early disease development could lead to insensitivity towards this class of fungicides. This is discussed in relation to Botrytis cinerea, which is often poorly controlled by strobilurins. Mutations with a similar effect may explain evidence implicating AOX in resistance development in normally well-controlled plant pathogens, such as Venturia inaequalis.

This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici, While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a Delta psi across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide, Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase, Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici, These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis

The reaction of Fe(II) oxalate with hydrogen peroxide and dioxygen was studied for oxalate concentrations up to 20 mM and pH 2-5, under which conditions mono- and bis-oxalate complexes (Fe[II](ox) and Fe[II](ox)2[2-]) and uncomplexed Fe2+ must be considered. The reaction of Fe(II) oxalate with hydrogen peroxide (Fe2+ + H2O2 --> Fe3+ + .OH + OH-) was monitored in continuous flow by ESR with t-butanol as a radical trap. The reaction is much faster than for uncomplexed Fe2+ and a rate constant, k = 1 x 10(4) M(-1) s(-1) is deduced for Fe(II)(ox). The reaction of Fe(II) oxalate with dioxygen is strongly pH dependent in a manner which indicates that the reactive species is Fe(II)(ox)2(2-), for which an apparent second order rate constant, k = 3.6 M(-1) s(-1), is deduced. Taken together, these results provide a mechanism for hydroxyl radical production in aqueous systems containing Fe(II) complexed by oxalate. Further ESR studies with DMPO as spin trap reveal that reaction of Fe(II) oxalate with hydrogen peroxide can also lead to formation of the carboxylate radical anion (CO2-), an assignment confirmed by photolysis of Fe(II) oxalate in the presence of DMPO.

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H2O2, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(III) complexes, including Fe(III) acetate. Many Fe(II) complexes react with H2O2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H2O2 + Fe2+----HO. + HO- + Fe3+. The hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(II) plus H2O2 and with cellobiose oxidase plus cellobiose, O2 and Fe(III). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(III) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O2 electrode. Addition of H2O2 to Fe(II) acetate (pH 4.0) or Fe(II) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH+HO.----R. + H2O; R. + O2----RO2.----products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged. Oxygen uptake was monitored for mixtures of Avicel (5 g.1-1), cellobiose oxidase, O2 and Fe(III) (30 microM). An addition of catalase after 20-30 min indicated very little accumulation of H2O2, but caused a 70% inhibition of the O2 uptake rate. This was observed with either phosphate (pH 2.8) or 10 mM acetate (pH 4.0) as buffer, and is further evidence that oxidative damage had been taking place, until the Fenton reaction was suppressed by catalase. A separate binding study established that with 10 mM acetate as buffer, almost all (98%) of the Fe(III) would have been bound to the Avicel. In the presence of Fe(III), cellobiose oxidase could provide a biological method for disrupting the crystalline structure of cellulose.

Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in μmol·min−1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a Kmof 4.8 ± 1.0 (g cellulose)·1−1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 μmol·(g cellulase)−1·min−1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 μmol·(g cellulose)−1·min−1.

Summary A peroxidase has 1-electron oxidation as its characteristic activity, while that of cytochrome P-450 is hydroxylation. Both catalytic cycles involve similar high valency states of iron. However, a peroxidase can only accept electrons at the haem edge, while the substrate for cytochrome P-450 is bound in a precise orientation before the active state is created.

An earlier paper (Wood, P.M. (1983) FEBS Lett. 164, 223–226) proposed that the covalent links that characterize c-type haem originated in order to prevent the haem being lost into the external medium. This is discussed in relation to Gram-negative and Gram-positive bacteria, cyanobacterial thylakoids and eukaryotes. The practical application is for unravelling complex electron transfer chains: c-type cytochromes may be assumed to be absent from the cytoplasm. Sulphate reducing bacteria provide the only confirmed exception to this rule. Examples of non-covalent haem at risk of being lost are considered. Analogies are drawn with flavin and pyrroloquinoline quinone as prosthetic groups.

This article examines species-at-risk policy initiatives of the British Columbia provincial government and evaluates their sufficiency for meeting international and national standards, agreements, and expectations. We briefly outline the evolution of the relevant policies and an array of policy drivers that influence (or should influence) government decisions. From our analyses, we find that the BC government has not sufficiently met its national and international obligations due to its low performance in the legal listing of scientifically recognized species at risk of extinction, a misconception of the purpose of species-at-risk legislation, arbitrary constraints on the protection of these species, and a lack of legal commitment to their recovery. As a result of its poor performance, the provincial government is vulnerable to punitive actions from governments and non-governmental organizations external to British Columbia.

The hypothesis presented is that the different classes of c-type cytochrome originated as proteins located in the bacterial periplasmic space, or on the periplasmic side of the cytoplasmic membrane. In these locations, covalent bonds between haem and protein prevented the haem from being lost to the surrounding medium. Subsequent evolution has led to internal location of c-type cytochromes in eucaryotes and cyanobacteria. The covalent links have been retained because of their structural role; a b-type cytochrome could be created with similar molecular properties, but its formation would require a large evolutionary jump. If this hypothesis is correct, it should be useful in unravelling electron transport chains with unconventional donors or acceptors. Apparent exceptions deserve further investigation.

The use of O2 uptake as a valid assay for non-cyclic photosynthetic electron flow in membranes from Chlorobium limicola is discussed. It is recommended that methyl viologen, catalase and superoxide dismutase should be added to the experimental medium. The addition of methyl viologen more than doubled the rate of O2 uptake observed on illumination with 1 mM sulphide as donor. Superoxide dismutation was shown to be efficient under the experimental conditions by means of standard additions of potassium superoxide dissolved in dimethylsulphoxide. The highest rates of light stimulated O2 uptake were obtained with sulphide as electron donor, and approached 50 μmol O2 · h-1 · mg bacteriochlorophyll c -1 with 0.2 mM sulphide. The presence of 5 mM 2-mercaptoethanol or 3 mM sulphite as electron donor led to lower light stimulated rates of O2 uptake, while 5 mM thiosulphate had little effect. The rates were insensitive to uncoupler. The light stimulated O2 uptake with 0.2 mM sulphide as donor was 20–30% inhibited by 10 μM antimycin A and 50 μM cyanide.

Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole cells of Nitrosomonas europaea. Positive results were obtained with tri- and tetramethylhydroquinone. An earlier study showed that phenol was converted into hydroquinone by the monooxygenase. Cells were therefore incubated with trimethylphenol, to see if its hydroxylation to trimethylhydroquinone would lead to a self-sufficient conversion of trimethylphenol into trimethylquinone. No trimethylquinone could be detected. The maximal rates of propene epoxidation obtained with tri-and tetramethylhydroquinone were 1.8 and 4.6 μmol · h−1 · mg protein−1, respectively.

SUMMARY: Reduction of cells or extracts of Nitrosomonas europaea with dithionite leads to a peak in absorption spectra at 460 nm. Most of this chromophore is bound to hydroxylamine oxidase, but a small fraction exists as a separate protein, namely cytochrome P-460. An improved purification of both these proteins is described. The mobility of cytochrome P-460 on gel electrophoresis in the presence of SDS corresponded to a molecular weight of 18000, whereas a molecular weight of 52000 was determined by gel filtration. The band obtained by electrophoresis gave a yellow-green fluorescence in UV light. Electrophoresis in the presence of 8 m-urea, or after acid-treatment, resulted in red fluorescence from cytochrome P-460. We previously reported that extracts from N. europaea gave eight fluorescence bands in SDS-polyacrylamide gels; cytochrome P-460 was found to correspond to the previously unassigned band VI. Electrophoresis of purified hydroxylamine oxidase did not give a band in common with cytochrome P-460. Highly purified cytochrome P-460 gave no detectable flavin-type fluorescence. There was no antigenic similarity between hydroxylamine oxidase and cytochrome P-460.

Incubation of whole cells of the nitrifying bacterium Nitrosomonas europaea with ethylene led to the formation of ethylene oxide. Ethylene oxide production was prevented by inhibitors of ammonium ion oxidation, and showed properties implying that ethylene is a substrate for the ammonia oxidising enzyme, ammonia monooxygenase. Endogenous substrates, hydroxylamine, hydrazine and ammonium ions were compared as sources of reducing power in terms of rates and stoichiometries of ethylene oxidation. The highest rates of ethylene oxide formation (15 μmol h-1 mg protein-1) were obtained with hydrazine as donor. The data suggest that at high concentrations of ethylene the rate of oxidation is limited by the rate at which reducing power can be supplied to the monooxygenase, not by an intrinsic V max. Ethylene had an inhibitory effect on the rate of ammonium ion utilisation; an approximate K i of 80 μM was derived, but the results deviated from simple competitive behaviour. Measurement of relative rates of ethylene oxide formation and ammonium ion utilization led to a k cat/K m value for ethylene of 1.1 relative to NH 4 + , or 0.04 relative to the true natural substrate, NH3. The effects of higher concentrations of ethylene oxide on oxygen uptake rates were also investigated. The results imply that ethylene oxide is also a substrate for the monooxygenase, but with a much lower affinity than ethylene.

SUMMARY: The soluble cytochrome oxidase of Nitrosomonas europaea has been highly purified and shown to be a copper protein devoid of haem, not a cytochrome o as was previously assumed. The native molecular weight was 120000 and the subunit molecular weight 35000. Soluble cytochrome oxidase activity co-purified with nitrite reductase activity; both activities were almost certainly associated with the same protein. The possible physiological role of the nitrite reductase activity is discussed.

A membrane fraction was isolated from the purple photosynthetic bacterium Rhodopseudomonas viridis. It contained the membrane cytochromes c-552 and c-557. A limit on the concentration of the soluble cytochrome c2 was set at 1% of total c-type haem by a technique involving fluorescent gels. Cytochrome c-557 had extremely slow kinetics of re-reduction after oxidation by a flash of light: τ = 40 s. The rate of re-reduction was considerably accelerated by adding back purified cytochrome c2. Cytochromes c-557 and c2 were monitored individually by appropriate wavelength pairs. Their kinetics subsequent to a flash of light fitted a linear mechanism with cytochrome c2 as the donor to cytochrome c-557: X being an uncharacterised donor and P960 the photoreactive bacteriochlorophyll. Approximate rate constants were obtained: k1 = 2 · 105 M−1 · s−1 and k2 = 1 · 106 M−1 · s−1. The presence of 5 μM antimycin slowed k1 by 30% but had no effect on k2. Dark reduction of oxidised cytochrome c2, added to membranes plus succinate, showed kinetics similar to those observed after a flash of light, and antimycin had the same effect in both cases.

The oxidation of benzene to phenol by whole cells of Nitrosomonas europaea is catalysed by ammonia monooxygenase, and therefore requires a source of reducing power. Endogenous substrates, hydrazine, hydroxylamine and ammonium ions were compared as reductants. The highest rates of benzene oxidation were obtained with 4 mM benzene and hydrazine as reductant, and equalled 6 μmol· h-1·mg protein-1. The specificity of ammonia monooxygenase for benzene as a substrate was determined by measuring k cat/K m for benzene relative to k cat/K m for uncharged ammonia, a value of 0.4 being obtained. Phenol was found to be further hydroxylated to yield hydroquinone. This reaction, like benzene oxidation, was sensitive to the ammonia monooxygenase inhibitor allylthiourea. Catechol and resorcinol were not detected as products of phenol oxidation, implying that at least 88% of the hydroxylation is para-directed.

A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas. Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome f, with alpha-band maxima near 554 nm, very asymmetrical beta-bands, and gamma-band maxima at 421 nm. The cytochrome from Anacystis had alpha and gamma-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as +350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum. A very fast rate of electron transfer occurred between this cytochrome purified from Euglena and cytochrome c-552. The roles of the membrane-bound and soluble c-type cytochromes in algal photosynthesis are discussed, and it is recommended that the name cytochrome f should be reserved for the membrane-bound cytochrome (to emphasize its affinity with higher plant cytochrome f), while the soluble one should be named by its alpha-band (c-552, c-553, etc.) to make clear its distinctness from higher plant cytochrome f and homology with mitochondrial cytochrome c.

Membranes from Nitrosomonas europaea were found to contain two b-type cytochromes. One had an α-band centred at 562 nm and Em,7 = + 155 mV; the other had an α-band maximum close to 558 nm and Em,7 = + 40 mV. A b-type cytochrome ran at an apparent Mr of 32000 on lithium dodecyl sulphate/polyacrylamide gels at 4°C.

The products of incubation of Nitrosomonas europaea with methanol were studied by 3C NMR spectroscopy. The methanol was converted into formaldehyde, which was at least partially oxidized to formate. Methanol oxidation was prevented by inhibitors of the ammonia oxidizing enzyme, ammonia monooxygenase. The effects of hydrazine and NH,+ were as found in earlier studies with other substrates for ammonia monooxygenase. From experiments in which ammonia and methanol were simultaneously oxidized, the specificity parameter kcat/Km was 0.008 for methanol, relative to unity for uncharged ammonia. The rate of growth was not enhanced by methanol, and attempts to grow the cells with methanol as energy source were unsuccessful. Formaldehyde proved much more toxic than methanol when added to shake-flask cultures. Hydroxylamine, an intermediate in ammonia oxidation, reacts with formaldehyde to form formaldoxime. Formaldoxime inhibited hydroxylamine oxidation, providing a specific mechanism for formaldehyde toxicity.