João P. Monteiro

Mitochondrial membrane lipid remodeling in pathophysiology: a new target for diet and therapeutic interventions.

Mitochondria are arbiters in the fragile balance between cell life and death. These organelles present an intricate membrane system, with a peculiar lipid composition and displaying transverse as well as lateral asymmetry. Some lipids are synthesized inside mitochondria, while others have to be imported or acquired in the form of precursors. Here, we review different processes, including external interventions (e.g., diet) and a range of biological events (apoptosis, disease and aging), which may result in alterations of mitochondrial membrane lipid content. Cardiolipin, the mitochondria lipid trademark, whose biosynthetic pathway is highly regulated, will deserve special attention in this review. The modulation of mitochondrial membrane lipid composition, especially by diet, as a therapeutic strategy for the treatment of some pathologies will be also addressed.

Structure-Bioactivity Relationships of Methylxanthines: Trying to Make Sense of All the Promises and the Drawbacks

Methylxanthines are a group of phytochemicals derived from the purine base xanthine and obtained from plant secondary metabolism. They are unobtrusively included in daily diet in common products as coffee, tea, energetic drinks, or chocolate. Caffeine is by far the most studied methylxanthine either in animal or epidemiologic studies. Theophylline and theobromine are other relevant methylxanthines also commonly available in the aforementioned sources. There are many disseminated myths about methylxanthines but there is increased scientific knowledge to discuss all the controversy and promise shown by these intriguing phytochemicals. In fact, many beneficial physiologic outcomes have been suggested for methylxanthines in areas as important and diverse as neurodegenerative and respiratory diseases, diabetes or cancer. However, there have always been toxicity concerns with methylxanthine (over)consumption and pharmacologic applications. Herein, we explore the structure-bioactivity relationships to bring light those enumerated effects. The potential shown by methylxanthines in such a wide range of conditions should substantiate many other scientific endeavors that may highlight their adequacy as adjuvant therapy agents and may contribute to the advent of functional foods. Newly designed targeted molecules based on methylxanthine structure may originate more specific and effective outcomes.

Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan

Microorganisms are very powerful tools for the supply of information about the toxic effects of lipophilic compounds, since an impairment of cell growth usually occurs as a result of perturbations related, in most cases, with the partition of toxicants in membranes. The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify alpha- and beta-endosulfan interactions with the membrane possibly related with the insecticide toxicity. Two approaches have been pursued: (a) bacterial growth is followed and the effects of endosulfan isomers determined; (b) biophysical studies with the fluorescent fluidity probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to assess the effects of alpha- and beta-endosulfan on the organization of the membrane lipid bilayer. The effects on growth were quantitatively evaluated by determination of growth parameters, namely the lag phase, the specific growth rate and the cell density reached by cultures in the stationary phase. Growth inhibition by alpha and beta-endosulfan dependent on the concentration is diminished or removed by the addition of 2.5 mM Ca2+ to bacterial cultures. Fluorescence DPH polarization consistently showed opposite effects of Ca2+ and alpha- and beta-endosulfan on the physical state of bacterial polar lipid dispersions.

Nimesulide interaction with membrane model systems : are membrane physical effects involved in nimesulide mitochondrial toxicity?

Nimesulide (NIM), a widely used nonsteroidal anti-inflammatory drug (NSAID), is known to interfere with mitochondrial physiology and to cause idiosyncratic hepatotoxicity. In this study, we characterized the effects of NIM on the physical properties of membrane models containing the main phospholipid classes of the inner mitochondrial membrane: phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL). NIM binding/incorporation was observed with the mitochondrial membrane mimicking model composed of dioleoyl PC (DOPC), dioleoyl PE (DOPE) and tetraoleoyl CL (TOCL) at a 1:1:1M ratio, as well as an increase of membrane permeability, monitored by calcein release, and an increase of lipid disorder, evaluated by fluorescence anisotropy of DPH-PA. Consistently, DSC thermograms of dipalmitoyl PC (DPPC) and a mixture of dipalmitoyl PE (DPPE) and TOCL (7:3 M ratio) showed a NIM-induced decrease of the cooperativity of the phase transition and a shift of the DPPC endotherm to lower temperatures. On the other hand, (31)P NMR studies with the ternary lipid model indicated a stabilizing effect of NIM on the lipid bilayer structure. Quenching of the fluorescent probes DPH and DPH-PA revealed a peripheral insertion of NIM in the hydrophobic portion of the bilayer. Our data indicate that NIM may influence mitochondria physiological processes by interfering with membrane structure and dynamics. The relevance of these findings will be discussed in terms of the reported NIM effects on mitochondria transmembrane potential, protonophoresis, and induction of the permeability transition pore.

Interaction of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) with lipid membrane systems: a biophysical approach with relevance to mitochondrial uncoupling

FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), a classical uncoupler of mitochondrial oxidative phosphorylation, is used in this study as a model to clarify how interactions of uncouplers with membrane lipid bilayers may influence membrane biophysics and their protonophoric activity itself. In order to disclose putative effects that may be important when considering using uncouplers for pharmacological purposes, an extensive characterization of FCCP membrane lipid interactions using accurate biophysical approaches and simple model lipid systems was carried out. Differential scanning calorimetry studies showed that FCCP molecules disturb lipid bilayers and favor lateral phase separation in mixed lipid systems. 31P NMR assays indicated that FCCP alters the curvature elastic properties of membrane models containing non-bilayer lipids, favoring lamellar/HII transition, probably by alleviation of hydrocarbon-packing constraints in the inverted hexagonal phase. Taking advantage of FCCP quenching effects on the fluorescent probes DPH (1,6-diphenyl-1,3,5-hexatriene) and DPH-PA (3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid), it is demonstrated that FCCP distributes across the bilayer thickness in both a single and a ternary lipid system mimicking the inner mitochondrial membrane. This behavior is consistent with the ability of the compound to migrate through the thickness of the inner mitochondrial membrane, an event required for its protonophoric activity. Finally, the study of the membrane fluidity in different lipid systems, as reported by the rotational correlation time (θ) of DPH or DPH-PA, showed that the extension at which FCCP disturbs membrane properties associated with the dynamics and the order of lipid molecules depends on the lipid composition of the model lipid system assayed.

Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene : Relevance for toxicological assessment

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.

Molecular mechanisms of the metabolite 4-hydroxytamoxifen of the anticancer drug tamoxifen: use of a model microorganism

A strain of the thermophilic eubacterium Bacillus stearothermophilus was used as a model system to identify membrane mediated cytotoxic effects of 4-hydroxytamoxifen, following previous studies with tamoxifen. With this experimental approach we attempted to further clarify tamoxifen and 4-hydroxytamoxifen membrane interactions often evoked as responsible for their multiple cellular effects. Bacterial growth and the oxygen consumption rate provided quantitative data of the cytotoxic action of hydroxytamoxifen. The effects of hydroxytamoxifen on the physical properties of bacterial lipid membrane preparations were also evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Cultures of B. stearothermophilus grown in a complex medium containing hydroxytamoxifen in the concentration range of 1 to 7 microM exhibited progressively longer lag adapting periods, decreased specific growth rates and lower growth yields, as compared to control cultures. Hydroxytamoxifen also affected the electron redox flow of B. stearothermophilus protoplasts and induced significant perturbation of the structural order of bacterial lipid dispersions. We concluded that the bacterial model provides useful information about the nature and repercussion of membrane physical interactions of this lipophilic drug, on the basis of an easy and economic methodology.

Vitreous humor in the pathologic scope: Insights from proteomic approaches

The vitreous humor (VH) is the largest component of the eye. It is a colorless, gelatinous, highly hydrated matrix that fills the posterior segment of the eye between the lens and retina in vertebrates. In VH, a diversity of proteins that can influence retinal physiology is present, including growth factors, hormones, proteins with transporter activity, and enzymes. More importantly, the protein composition of VH has been described as being altered in a number of disease states. Therefore, attempts aiming at establishing a map of VH proteins and detecting putative biomarkers for ocular illness or protein fluctuations with putative physiologic significance were conducted over the last two decades, using proteomic approaches. Proteomic strategies often involve gel‐based or LC techniques as sample fractioning approaches, subsequently coupled with MS procedures. This set of studies resulted in the proteomic characterization of a range of ocular disease samples, with particular incidence on diabetic retinopathy. However, practical therapeutic applications arising from these studies are scarce at the moment. A pertinent example of therapeutic targets arising from VH proteomics has emerged concerning vasoproliferative factors present in the vitreous, which should be involved in neovascularization and subsequent fibrovascular proliferation of the retina, in ocular disease context. Therefore, this review attempts to sum up the information acquired from the proteomic approaches to ocular disease conducted in VH samples, highlighting its clinical potential for disclosing ocular disease mechanisms and engendering pharmacological therapeutic treatments.

On the use of Irwin and Preston wall shear stress probes in turbulent incompressible flows with pressure gradients

Abstract A set of wall shear stress pressure probes, of the type developed by Irwin and Preston, was tested in a duct flow under various positive and negative pressure gradients. A hot wire wall shear stress probe with temperature compensation was employed as the reference instrument. In the present study, the parameter Δ used by Patel to quantify the severity of the pressure gradient was made to vary between −0.035 and 0.05, thus extending the range of results available in the literature. The experimental facility constructed allowed the probes to be calibrated and tested without changing of position, as well as obtaining the same value of Δ with different types of flow. It was found that both pressure probes studied had broadly the same behaviour, and that, in the case of positive pressure gradients, the measuring errors produced by the Irwin and Preston probes depended very strongly on the conditions of the flow development. The use of an integral parameter to complement Δ is suggested in order to try to predict the errors produced by the pressure probes under study.

A biophysical approach to menadione membrane interactions: Relevance for menadione-induced mitochondria dysfunction and related deleterious/therapeutic effects

Menadione (MEN), a polycyclic aromatic ketone, was shown to promote cell injury by imposing massive oxidative stress and has been proposed as a promising chemotherapeutic agent for the treatment of cancer diseases. The mechanisms underlying MEN-induced mitochondrial dysfunction and cell death are not yet fully understood. In this work, a systematic study was performed to unveil the effects of MEN on membrane lipid organization, using models mimicking mitochondrial membranes and native mitochondrial membranes. MEN was found to readily incorporate in membrane systems composed of a single phospholipid (phosphatidylcholine) or the lipids dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and tetraoleoylcardiolipin at 1:1:1 molar ratio, as well as in mitochondrial membranes. Increased permeability in both membrane models, monitored by calcein release, seemed to correlate with the extent of MEN incorporation into membranes. MEN perturbed the physical properties of vesicles composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine plus tetraoleoylcardiolipin (at 7:3 molar ratio), as reflected by the downshift of the lipid phase transition temperature and the emergence of a new transition peak in the mixed lipid system, detected by DSC. (31)P NMR studies revealed that MEN favored the formation of non-lamellar structures. Also, quenching studies with the fluorescent probes DPH and TMA-DPH showed that MEN distributed across the bilayer thickness in both model and native mitochondrial membranes. MENs ability to promote alterations of membrane lipid organization was related with its reported mitochondrial toxicity and promotion of apoptosis, predictably involved in its anti-carcinogenic activity.