The schistosome excretory system: a key to regulation of metabolism, drug excretion and host interaction

There is a gulf between the enormous information content of the various genome projects and the understanding of the life of the parasite in the host. In vitro studies with adult Schistosoma mansoni using several substrates suggest that the excretory system contains both P-glycoproteins and multiresistance proteins. If both these families of protein were active in vivo, they could regulate parasite metabolism and be responsible for the excretion of drugs. During skin penetration, membrane-impermeant molecules of a wide range of molecular weights can be taken into the cercaria and schistosomulum through the nephridiopore, through the surface membrane or through both. We speculate that this uptake process might stimulate novel signalling pathways involved in growth and development.

Inhibition of cytochrome P450-mediated metabolism enhances ex vivo susceptibility of Fasciola hepatica to triclabendazole

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP P450) system was inhibited using piperonyl butoxide (PB). The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP P450 system was inhibited by a 2 h pre-incubation in PB (100 mu M). Flukes were then incubated for a further 22 h in NCTC medium containing either PB; PB + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); PB + NADPH + TCBZ (15 mu g/ml); or PB + NADPH + TCBZ.SO (15 mu g/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible than the resistant isolate. However, co-incubation with PB and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant Oberon isolate than with each drug on its own. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action, and only with TCBZ.SO. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Patterns of variation at a mitochondrial sequence-tagged-site locus provides new insights into the postglacial history of European Pinus sylvestris populations

Due to their maternal mode of inheritance, mitochondrial markers can be regarded as almost 'ideal' tools in evolutionary studies of conifer populations. In the present study, polymorphism was analysed at one mitochondrial intron (nad 1, exon B/C) in 23 native European Pinus sylvestris populations. In a preliminary screening for variation using a polymerase chain reaction-restriction fragment length polymorphism approach, two length variants were identified. By fully sequencing the 2.5 kb region, the observed length polymorphism was found to result from the insertion of a 31 bp sequence, with no other mutations observed within the intron. A set of primers was designed flanking the observed mutation, which identified a novel sequence-tagged-site mitochondrial marker for P. sylvestris. Analysis of 747 trees from the 23 populations using these primers revealed the occurrence of two distinct haplotypes in Europe. Within the Iberian Peninsula, the two haplotypes exhibited extensive population differentiation (Phi(ST) = 0.59; P less than or equal to 0.001) and a marked geographical structuring. In the populations of central and northern Europe, one haplotype largely predominated, with the second being found in only one individual of one population.

Lack of Oxygen Deactivates Mitochondrial Complex I IMPLICATIONS FOR ISCHEMIC INJURY?

For S-nitrosothiols and peroxynitrite to interfere with the activity of mitochondrial complex I, prior transition of the enzyme from its active (A) to its deactive, dormant (D) state is necessary. We now demonstrate accumulation of the D-form of complex I in human epithelial kidney cells after prolonged hypoxia. Upon reoxygenation after hypoxia there was an initial delay in the return of the respiration rate to normal. This was due to the accumulation of the D-form and its slow, substrate-dependent reconversion to the A-form. Reconversion to the A-form could be prevented by prolonged incubation with endogenously generated NO. We propose that the hypoxic transition from the A-form to the D-form of complex I may be protective, because it would act to reduce the electron burst and the formation of free radicals during reoxygenation. However, this may become an early pathophysiological event when NO-dependent formation of S-nitrosothiols or peroxynitrite structurally modifies complex I in its D-form and impedes its return to the active state. These observations provide a mechanism to account for the severe cell injury that follows hypoxia and reoxygenation when accompanied by NO generation.

MEASUREMENT OF SUPEROXIDE FORMATION BY MITOCHONDRIAL COMPLEX I OF YARROWIA LIPOLYTICA

Complex I (NADH: ubiquinone oxidoreductase) is generally regarded as one of the major sources of mitochondrial reactive oxygen species (ROS). Mitochondrial membranes from the obligate aerobic yeast Yarrowia lipolytica, as well as the purified and reconstituted enzyme, can be used to measure complex I-dependent generation of superoxide (O-2(center dot-)). The use of isolated complex I excludes interference with other respiratory chain complexes and matrix enzymes during superoxide dismutase-sensitive reduction of acetylated cytochrome c. Alternately. hydrogen peroxide formation can be measured by the Amplex Red/horseradish peroxidase assay. Both methods allow the determination of complex I-generated ROS, depending on substrates (NADH, artificial ubiquinones), membrane potential, and active/deactive transition. ROS production by Yorrowia complex I in the

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I

Mitochondrial complex I (NADH: ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide: oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

S-nitrosation of mitochondrial complex I depends on its structural conformation

Nitric oxide is known to cause persistent inhibition of mitochondrial respiration as a result of S-nitrosation of NADH: ubiquinone oxidoreductase (complex I) (Clementi, E., Brown, G. C., Feelisch, M., and Moncada, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7631-7636). Little is known about whether such nitrosation occurs in physiological conditions and, if so, what are the possible cellular mechanisms. We have now found that the conformational state (active/deactive transition (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185)) of mitochondrial complex I is an important factor for the interaction of the enzyme with nitrosothiols and peroxynitrite. Only the deactivated, idle form of complex I was susceptible to inhibition by nitrosothiols and peroxynitrite. In contrast, the active form of the enzyme was insensitive to such treatment. Neither form of complex I was inhibited by nitric oxide itself. Our data suggest that the process of active/deactive transition plays an important role in the regulation of complex I activity and cellular respiration by nitric oxide. The implications of this finding for hypoxic or pathophysiological conditions in vivo are discussed.

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H(->+)/2e(-) indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H(->+)/2e(-). This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme. (c) 2006 Elsevier B.V. All rights reserved.

HDQ (1-hydroxy-2-dodecyl-4(1H)quinolone), a high affinity inhibitor for mitochondrial alternative NADH dehydrogenase

Alternative NADH dehydrogenases (NADH:ubiquinone oxidoreductases) are single subunit respiratory chain enzymes found in plant and fungal mitochondria and in many bacteria. It is unclear how these peripheral membrane proteins interact with their hydrophobic substrate ubiquinone. Known inhibitors of alternative NADH dehydrogenases bind with rather low affinities. We have identified 1-hydroxy-2-dodecyl-4(1H)quinolone as a high affinity inhibitor of alternative NADH dehydrogenase from Yarrowia lipolytica. Using this compound, we have analyzed the bisubstrate and inhibition kinetics for NADH and decylubiquinone. We found that the kinetics of alternative NADH dehydrogenase follow a ping-pong mechanism. This suggests that NADH and the ubiquinone headgroup interact with the same binding pocket in an alternating fashion.