Biomedical subjects

A E Vercesi

Publications and source records attributed to A E Vercesi.

At least 19 recordsLinked to original sources

PubMed2004

Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans.

The respiration, membrane potential (Deltapsi), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 microM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Deltapsi respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 microM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 microM) inhibited respiration by 30% and 2 micro M antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Deltapsi induced by 5 mM ATP and 0.5% BSA, and Deltapsi decrease induced by 10 microM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

Candida albicans↗

PubMed2002

Opposite effects of Mn(III) and Fe(III) forms of meso-tetrakis(4-N-methyl pyridiniumyl) porphyrins on isolated rat liver mitochondria.

The relevance of porphyrins as therapeutic drugs targeted to mitochondria has been widely recognized. In this work, we studied the action of meso-tetrakis porphyrins (TMPyP) on respiring rat liver mitochondria. Mn(III)TMPyP exerted a protective effect against lipid peroxidation induced by Fe(II) or the azo initiator 4,4'-azobis(4-cyanopentanoic acid) (ABCPA), which partition in the hydrophobic phospholipid moiety, and 2,2'-azobis(2-amidinepropane)dihydrochloride (ABAP), which partitions in the aqueous phase. In contrast, Fe(III)TMPyP itself induced an intense lipid peroxidation, accompanied by mitochondrial permeability transition. Both mesoporphyrins studied promoted a release of mitochondrial state-4 respiration, in the concentration range of 1.0-20 microM. Based on the relative effects of Mn(III)TMPyP against ABAP and ABCPA-induced lipid peroxidation, we believe that meso-tetrakis porphyrins must concentrate preferably at membrane-water interfaces.

Animals↗

PubMed2001

Suramin inhibits respiration and induces membrane permeability transition in isolated rat liver mitochondria.

Suramin, a polysulfonated naphthylamine, caused a dose dependent inhibition of carbonyl cyanide p-(tri-fluoromethoxy)phenylhydrazone-stimulated respiration supported either by succinate or a cocktail of alphaketoglutarate, malate and isocitrate in isolated rat liver mitochondria. The half-maximum effect was obtained at 40 and 140 microM suramin for NADH- or FADH(2)-linked substrates, respectively. The respiration supported by N,N,N'N'-tetramethyl-p-phenylenediamine oxidation was unaffected by suramin (<or=500 microM). The latter respiratory substrate was used in the studies at examining the effects of suramin on the permeability properties of the inner mitochondrial membrane. These experiments provided evidence that suramin causes Ca(2+)-dependent mitochondrial swelling sensitive to the mitochondrial permeability transition (MPT) inhibitors cyclosporin A, ADP and Mg(2+). In addition, suramin decreased the content of reduced mitochondrial membrane protein thiols suggesting that thiol oxidation is the mechanism underlying suramin-induced MPT.

Adenosine Diphosphate↗

PubMed2001

Respiratory chain network in mitochondria of Candida parapsilosis: ADP/O appraisal of the multiple electron pathways.

In this study we demonstrated that mitochondria of Candida parapsilosis contain a constitutive ubiquinol alternative oxidase (AOX) in addition to a classical respiratory chain (CRC) and a parallel respiratory chain (PAR) both terminating by two different cytochrome c oxidases. The C. parapsilosis AOX is characterized by a fungi-type regulation by GMP (as a stimulator) and linoleic acid (as an inhibitor). Inhibitor screening of the respiratory network by the ADP/O ratio and state 3 respiration determinations showed that (i) oxygen can be reduced by the three terminal oxidases through four paths implying one bypass between CRC and PAR and (ii) the sum of CRC, AOX and PAR capacities is higher than the overall respiration (no additivity) and that their engagement could be progressive according to the redox state of ubiquinone, i.e. first cytochrome pathway, then AOX and finally PAR.

Adenosine Diphosphate↗

PubMed2001

Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli.

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.

Adenosine Diphosphate↗

PubMed2001

Ca(2+) transport into an intracellular acidic compartment of Candida parapsilosis.

In this report, we study Ca2+ transport in permeabilized Candida parapsilosis spheroplasts prepared by a new technique using lyticase. An intracellular non-mitochondrial Ca2+ uptake pathway, insensitive to orthovanadate and sensitive to the V-H(+)-ATPase inhibitor bafilomycin A(1), nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone was characterized. Acidification of the compartment in which Ca2+ accumulated was followed using the fluorescent dye acridine orange. Acidification was stimulated by the Ca2+ chelator EGTA and inhibited by Ca2+. These results, when added to the observation that Ca2+ induces alkalization of a cellular compartment, provide evidence for the presence of a Ca2+/nH(+) antiporter in the acid compartment membrane. Interestingly, like in acidocalcisomes of trypanosomatids, the antioxidant 3,5-dibutyl-4-hydroxytoluene inhibits the V-H(+)-ATPase. In addition, the antifungal agent ketoconazole promoted a fast alkalization of the acidic compartment. Ketoconazole effects were dose-dependent and occurred in a concentration range close to that attained in the plasma of patients treated with this drug.

Acridine Orange↗

PubMed2001

Mitochondrial permeability transition and oxidative stress.

Mitochondrial permeability transition (MPT) is a non-selective inner membrane permeabilization that may precede necrotic and apoptotic cell death. Although this process has a specific inhibitor, cyclosporin A, little is known about the nature of the proteinaceous pore that results in MPT. Here, we review data indicating that MPT is not a consequence of the opening of a pre-formed pore, but the consequence of oxidative damage to pre-existing membrane proteins.

Animals↗

PubMed2001

Ca2+ induces a cyclosporin A-insensitive permeability transition pore in isolated potato tuber mitochondria mediated by reactive oxygen species.

Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca2+ uniport, undergo inner membrane permeabilization when treated with Ca2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis-trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.

Animals↗

PubMed2001

The discovery of an uncoupling mitochondrial protein in plants.

This paper describes peculiar properties of plant mitochondria and summarizes the experiments that led to the discovery of an uncoupling protein in these mitochondria. Recent advances in the study of the biochemical and physiological properties as well as on genes encoding plant uncoupling proteins are described in articles by Borecky et al., Jezek et al., and Jarmuszkiewicz et al. in this issue.

Adipose Tissue, Brown↗

PubMed2001

Alternative oxidase and uncoupling protein: thermogenesis versus cell energy balance.

The physiological role of an alternative oxidase and an uncoupling protein in plant and protists is discussed in terms of thermogenesis and energy metabolism balance in the cell. It is concluded that thermogenesis is restricted not only by a lower-limit size but also by a kinetically-limited stimulation of the mitochondrial respiratory chain.

Animals↗

PubMed2001

Oxidative stress in Ca(2+)-induced membrane permeability transition in brain mitochondria.

Mitochondrial permeability transition (PT) is a non-selective inner membrane permeabilization, typically promoted by the accumulation of excessive quantities of Ca(2+) ions in the mitochondrial matrix. This phenomenon may contribute to neuronal cell death under some circumstances, such as following brain trauma and hypoglycemia. In this report, we show that Ca(2+)-induced brain mitochondrial PT was stimulated by Na(+) (10 mM) and totally prevented by the combination of ADP and cyclosporin A. Removal of Ca(2+) from the mitochondrial suspension by EGTA or inhibition of Ca(2+) uptake by ruthenium red partially reverted the dissipation of the membrane potential associated with PT. Ca(2+)-induced brain mitochondrial PT was significantly inhibited by the antioxidant catalase, indicating the participation of reactive oxygen species in this process. An increased detection of reactive oxygen species, measured through dichlorodihydrofluorescein oxidation, was observed after mitochondrial Ca(2+) uptake. Ca(2+)-induced dichlorodihydrofluorescein oxidation was enhanced by Na(+) and prevented by ADP and cyclosporin A, indicating that PT enhances mitochondrial oxidative stress. This could be at least in part a consequence of the extensive depletion in NAD(P)H that accompanied this Ca(2+)-induced mitochondrial PT. NADPH is known to maintain the antioxidant function of the glutathione reductase/peroxidase and thioredoxin reductase/peroxidase systems. In addition, the occurrence of mitochondrial PT was associated with membrane lipid peroxidation. We conclude that PT further increases Ca(2+)-induced oxidative stress in brain mitochondria leading to secondary damage such as lipid peroxidation.

Animals↗

PubMed2000

Characterization of the intracellular Ca(2+) pools involved in the calcium homeostasis in Herpetomonas sp. promastigotes.

Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present pro- and opisthomastigotes forms in their life cycles. In this study, we investigated the Ca(2+) transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca(2+), and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca(2+) transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca(2+) release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca(2+) uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca(2+) transport system, attributed to the endoplasmic reticulum, was also detected. Nigericin (1 microM), FCCP (1 microM), or bafilomycin A(1) (5 microM) had no effect on the vanadate-sensitive Ca(2+) transport. These data suggest the absence of a Ca(2+) transport mediated by a Ca(2+)/H(+) antiport. No evidence of a third Ca(2+) compartment with the characteristics of the acidocalcisomes described by A. E. Vercesi et al. (1994, Biochem. J. 304, 227-233) was observed. Thapsigargin and IP(3) were not able to affect the vanadate-sensitive Ca(2+) transport. Ruthenium red was able to inhibit the Ca(2+) uniport of mitochondria, inducing a slow mitochondrial Ca(2+) efflux, compatible with the presence of a Ca(2+)/H(+) antiport. Moreover, this efflux was not stimulated by the addition of NaCl, which suggests the absence of a Ca(2+)/Na(+) antiport in mitochondria.

Adenosine Diphosphate↗

PubMed2000

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore.

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca(2+) efflux without significant release of state-4 respiration, disruption of membrane potential (Deltapsi) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca(2+) efflux associated with a release of state-4 respiration, a disruption of Deltapsi and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.

Acetoacetates↗

PubMed2000

Catalases and thioredoxin peroxidase protect Saccharomyces cerevisiae against Ca(2+)-induced mitochondrial membrane permeabilization and cell death.

The involvement of reactive oxygen species in Ca(2+)-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the thioredoxin peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2, 4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca(2+) and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca(2+) and the dithiol binding reagent phenylarsine oxide. In contrast, S. cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(beta-aminoethyl ether) N,N, N',N'-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S. cerevisiae mitochondria against Ca(2+)-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition.

Adenosine Diphosphate↗

PubMed2000

Presence of a Na(+)/H(+) exchanger in acidocalcisomes of Leishmania donovani and their alkalization by anti-leishmanial drugs.

Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain most of the cellular calcium. The data presented here demonstrate that Leishmania donovani acidocalcisomes possess a Na(+)/H(+) exchanger. 3,5-Dibutyl-4-hydroxytoluene, in the concentration range of 0-20 microM, inhibited the Na(+)/H(+) exchanger, and strongly stimulated the activity of the vacuolar H(+)-ATPase responsible for vacuolar acidification. As occurs with Na(+), the cationic anti-leishmanial drugs pentamidine, WR-6026, and chloroquine promoted a fast and extensive alkalization of the L. donovani acidocalcisomes.

Acridine Orange↗

PubMed2000

Proton re-uptake partitioning between uncoupling protein and ATP synthase during benzohydroxamic acid-resistant state 3 respiration in tomato fruit mitochondria.

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl malonate. This constancy makes the determination of the contribution of the linoleic acid-induced energy-dissipating pathway by the ADP/O method possible. No decrease in membrane potential is observed in state 3 respiration with increasing concentration of n-butyl malonate, indicating that the rate of ATP synthesis is steeply dependent on membrane potential. Linoleic acid decreases the yield of oxidative phosphorylation in a concentration-dependent manner by a pure protonophoric process like that in the presence of FCCP. ADP/O measurements allow calculation of the part of respiration leading to ATP synthesis and the part of respiration sustained by the dissipative H(+) re-uptake induced by linoleic acid. Respiration sustained by this energy-dissipating process remains constant at a given LA concentration until more than 50% inhibition of state 3 respiration by n-butyl malonate is achieved. The energy dissipative contribution to oxygen consumption is proposed to be equal to the protonophoric activity of plant uncoupling protein divided by the intrinsic H(+)/O of the cytochrome pathway. It increases with linoleic acid concentration, taking place at the expense of ADP phosphorylation without an increase in the respiration.

Hydroxamic Acids↗

PubMed2000

First evidence and characterization of an uncoupling protein in fungi kingdom: CpUCP of Candida parapsilosis.

An uncoupling protein (UCP) was identified in mitochondria from Candida parapsilosis (CpUCP), a non-fermentative parasitic yeast. CpUCP was immunodetected using polyclonal antibodies raised against plant UCP. Activity of CpUCP, investigated in mitochondria depleted of free fatty acids, was stimulated by linoleic acid (LA) and inhibited by GTP. Activity of CpUCP enhanced state 4 respiration by decreasing DeltaPsi and lowered the ADP/O ratio. Thus, it was able to divert energy from oxidative phosphorylation. The voltage dependence of electron flux indicated that LA had a pure protonophoretic effect. The discovery of CpUCP proves that UCP-like proteins occur in the four eukaryotic kingdoms: animals, plants, fungi and protists.

Candida↗

PubMed2000

Calcium mobilization by arachidonic acid in trypanosomatids.

A recent report (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A, and arachidonic acid, Biochem J 1998:336:659-66) provided evidence that calcium entry in Trypanosoma brucei bloodstream trypomastigotes is regulated via a signaling pathway involving phospholipase A2-mediated generation of arachidonic acid and stimulation of a plasma membrane-located calcium channel. Here we show that Ca2+ influx in T. brucei procyclic trypomastigotes, Leishmania donovani promastigotes and T. cruzi amastigotes was also stimulated in a dose-dependent manner (50-400 nM) by the amphiphilic peptide melittin. This effect was blocked by the phospholipase A, inhibitor 3-(4-octadecyl)-benzoylacrylic acid. The unsaturated fatty acid arachidonic acid, in the range of 10-75 microM, induced Ca2+ entry by a mechanism sensitive to LaCl3. However, both melittin and arachidonic acid induced an increase in [Ca2+]i in T. brucei procyclic trypomastigotes incubated in Ca2+-free medium implying Ca2+ mobilization from intracellular stores. This hypothesis was supported by experiments showing that arachidonic acid promoted Ca2+ release from the acidocalcisomes of these cells. The results showing changes in mitochondrial membrane potential, release of acridine orange and Ca2+ from the acidocalcisomes and Ca2+ transport across the plasma membrane suggest that in addition to the possible stimulation of a Ca2+ channel-mediated process, arachidonic acid, in the range of concentrations used here, have other nonspecific effects on the trypanosomatids membranes.

Acridine Orange↗