Genetic implications of phylogeographical patterns in the conservation of the boreal wetland butterfly Colias palaeno (Pieridae)

The boreo‐montane wetland butterfly species Colias palaeno has a European distribution from the Alps to northern Fennoscandia. Within its European range, the species’ populations have shrunk dramatically in recent historical times. Therefore, detailed baseline knowledge of the genetic makeup of the species is pivotal in planning potential conservation strategies. We collected 523 individuals from 21 populations across the entire European range and analyzed nuclear (20 allozyme loci) and mitochondrial (600 bp of the cytochrome c oxidase subunit I gene) genetic markers. The markers revealed contrasting levels of genetic diversity and divergence: higher in allozymes and lower in mitochondrial sequences. Five main groups were identified by allozymes: Alps, two Czech groups, Baltic countries, Fennoscandia, and Poland. The haplotype mitochondrial network indicates a recent range expansion. The most parsimonious interpretation for our results is the existence of a continuous Würm glacial distribution in Central Europe, with secondary disjunction during the Last Glacial Maximum into a south‐western and a north‐eastern fragment and subsequent moderate differentiation. Both groups present signs of postglacial intermixing in the Czech Republic. However, even a complete extinction in this region would not considerably affect the species’ genetic basis, as long as the source populations in the Alps and in northern Europe, comprising the most relevant evolutionary units for conservation, are surviving.     

Revisiting Kadenbach: Electron flux rate through cytochrome c‐oxidase determines the ATP‐inhibitory effect and subsequent production of ROS

Mitochondrial respiration is the predominant source of ATP. Excessive rates of electron transport cause a higher production of harmful reactive oxygen species (ROS). There are two regulatory mechanisms known. The first, according to Mitchel, is dependent on the mitochondrial membrane potential that drives ATP synthase for ATP production, and the second, the Kadenbach mechanism, is focussed on the binding of ATP to Cytochrome c Oxidase (CytOx) at high ATP/ADP ratios, which results in an allosteric conformational change to CytOx, causing inhibition. In times of stress, ATP‐dependent inhibition is switched off and the activity of CytOx is exclusively determined by the membrane potential, leading to an increase in ROS production. The second mechanism for respiratory control depends on the quantity of electron transfer to the Heme aa3 of CytOx. When ATP is bound to CytOx the enzyme is inhibited, and ROS formation is decreased, although the mitochondrial membrane potential is increased.     

The oxidation of apocynin catalyzed by myeloperoxidase: proposal for NADPH oxidase inhibition

Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidases. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO/H2O2 or activated neutrophils and has as products dimer and trimer derivatives. Since apocynin impedes the migration of the cytosolic component p47phox to the membrane and this effect could be related to its conjugation with essential thiol groups, we studied the reactivity of apocynin and its MPO-catalyzed oxidation products with glutathione (GSH). We found that apocynin and its oxidation products do not react with GSH. However, this thiol compound was efficiently oxidized by the apocynin radical during the MPO-catalyzed oxidation. We suggest that the reactivity of apocynin radical with thiol compounds could be involved in the inhibitory effect of this methoxy-catechol on NADPH oxidase complex.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a₃ as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK_a values during the pumping process are derived for comparison to experiments.     

Cardiolipin is associated with the terminal oxidase of an extremely halophilic archaeon

Membranes having an a high content of cardiolipin were isolated from an extremely halophilic archaeon Halorubrum sp. Absorbance difference spectra of detergent-solubilized plasma membranes reduced by dithionite suggested the presence of b-type cytochromes. Non-denaturing gel electrophoresis revealed only one fraction having TMPD-oxidase activity in which cardiolipin was the major lipid component. The electroeluted fraction showed a cytochrome c oxidase activity characterized by the reduced minus oxidized difference spectra as a terminal heme-copper oxidase. The cytochrome c oxidase activity of the archaeal cardiolipin-rich membranes was inhibited by the cardiolipin-specific fluorescent marker 10-N-nonyl acridine orange (NAO) in a dose-dependent manner. The results indicate that an archaeal analogue of cardiolipin is tightly associated to archaeal terminal oxidases and is required for its optimal functioning.     

Calculation of immobilized enzyme reaction progress curves from nested ordered-sequential rate expressions

A method for estimating immobilized enzyme reaction progress curves, using simultaneous non-linear regression analysis of 2–3 substrate concentrations with time, is presented. These facile procedures involve using nested Gauss–Newton curve fitting algorithms on a Microsoft EXCEL spreadsheet. We refer to our technique as "nested" because the analysis consists of two or three mutually parameter-dependent sets of computations associated with bi- or termolecular enzyme-catalyzed reactions, respectively. We have applied the method to immobilized glucose oxidase-catalyzed reactions ([ -glucose] and [O₂] with time) and found that the kinetic parameters from initial velocity data were similar to those determined by the nested curve fitting method discussed herein.     

Programme of senescence in petals and carpels of Pisum sativum L. flowers and its control by ethylene

The role of ethylene in the control of senescence of both petals and unpollinated carpels of pea was investigated. An increase in ethylene production accompanied senescence, and the inhibitors of ethylene action were effective in delaying senescence symptoms in different flower verticils. Pollination did not seem to trigger the senescence syndrome in the corolla as deduced from the observation that petals from pollinated and unpollinated flowers and from flowers whose carpels had been removed senesced at the same time. A cDNA clone encoding a putative ethylene-response sensor (psERS) was isolated from pea flowers, and the pattern of expression of its mRNA was studied during development and senescence of different flower tissues. The levels of psERS mRNA paralleled ethylene production (and also levels of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) mRNA) in both petals and styles. Silver thiosulfate treatments were efficient at preventing ACO and psERS mRNA induction in petals. However, the same inhibitor showed no ability to modify expression patterns in pea carpels around the anthesis stage, suggesting different controls for ethylene synthesis and sensitivity in different flower organs. Received: 18 June 1998 / Accepted: 22 December 1998