Membrane Barrier Function Research Articles

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L

Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X Sterol = 0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol > lanosterol ≥ ergosterol, and cholesterol > lanosterol > ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a gamma-zein related sequence. Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-gamma/TNF-alphaa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-gamma/TNF-alphaa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery.

Dexamethasone delivery to posterior segment of the eye

Due to anatomic barriers and lacrimal drainage it is difficult to obtain therapeutic drug concentrations in the posterior part of the eye after topical drug administrations. Lipophilic cyclodextrins, such as randomly methylated β-cyclodextrin (RMβCD), are known to act both as solubilizers of water-insoluble drugs in aqueous solutions and as penetration enhancers that reduce the barrier function of lipophilic membranes. The purpose of this study was to investigate the effects of RMβCD on dexamethasone delivery from aqueous eye drop solution into rabbit eyes. Dexamethasone (0.5 and 1.5% w/v) drops (50 μl) were administered to the left eye of rabbits (n = 6) and the drug levels measured in different eye tissues 2 h after administration. In aqueous humor dexamethasone levels were 1,190 ± 110 and 1,670 ± 630 ng/g (mean ± SD) after administration of the 0.5 and 1.5% dexamethasone eye drops, respectively. In the retina the levels were 33 ± 7 and 66 ± 49 ng/g, and in optic nerve 41 ± 12 and 130 ± 50 ng/g, respectively. In a previous study the dexamethasone concentration in aqueous humor after topical administration of 1.3% (w/v) dexamethasone eye drops in aqueous 2-hydroxypropyl-β-cyclodextrin (HPβCD) solution was determined to be 320 ± 230 ng/g and 66 ± 20 ng/g after administration of Maxidex® eye drops. Both the hydrophilic HPβCD and the lipophilic RMβCD enhance topical dexamethasone delivery into the eye, but of the two, the lipophilic RMβCD results in higher dexamethasone concentrations.

The Detection of UV‐induced Membrane Damages by a Combination of Two Biosensor Techniques

Ketoprofen is an important anti-inflammatory drug, but its dermal application is limited because of the photosensitizing properties causing phototoxic reactions of the skin when exposed to UV light. We have recently investigated the peroxide formation of ketoprofen in solutions of linoleic acid during UV irradiation. To continue these studies and focus on UV-induced changes in membrane integrity and barrier function we established an in vitro model using two biosensor techniques simultaneously. Support-fixed bilayers were irradiated with different doses of UV-B up to damaging intensities with or without ketoprofen. Cyclic voltammetry was carried out to detect alterations in membrane permeability; quartz crystal microbalance (QCM) measurements were helpful in analyzing whether a permeability increase was caused by depletion of membrane components. In absence of ketoprofen, increasing UV-B doses induce membrane permeabilities of both unsaturated and saturated bilayers. QCM measurements could not reveal a significant loss of membrane components as a reason for the permeability. In contrast, 0.3 mM ketoprofen induced a dose-dependent increase in membrane permeability. QCM results indicated a mass loss. Although this model does not explain all molecular mechanisms of membrane damage by ketoprofen, the combined application of both QCM and CV is a novel and powerful tool to investigate functional mechanisms of UV-induced membrane damages.

Poloxamer 188 prevents acute necrosis of adult skeletal muscle cells following high-dose irradiation

Acute cellular necrosis occurring minutes to hours after massive ionizing radiation exposure (IR) results from rapid membrane lipid peroxidation, blebbing and membrane breakdown. We have shown, previously, that certain polymer surfactants can restore structural integrity and transport barrier function of cell membranes following high-dose IR. We now investigate, specifically, the efficacy of the amphiphilic surfactant Poloxamer 188 (P188) in preventing acute necrosis of adult rat skeletal muscle cells after high-dose IR. Explanted cells were treated with 60 Co IR doses of 10, 40 or 80 Gy and their viability was determined using fluorometric probes at 4 and 18 h post-IR. IR of 10 Gy did not cause acute necrosis. Significant acute cell necrosis was observed after 40 and 80 Gy doses in a dose-dependent manner. Post-IR treatment with P188 significantly enhanced the cells’ viability post-IR treatment. By comparison 10 kDa neutral dextran, a hydrophilic polymer, was found to be ineffective. Despite progressive cell death over 18 h after high-dose IR, cells treated with P188 manifested greater survival than media or dextran-treated cells. It appears that use of P188 or similar multi-block copolymers to prolong viability of irradiated cells in vitro through membrane sealing is an important step in development of effective interventional therapy for extreme IR exposure. Not only can repairing the membrane prevent acute necrosis, but it also can provide a critical time opportunity to address other mechanisms of cell death, such as apoptosis or mitotic arrest, which manifest over a longer time frame.

Mechanism of delayed resuscitation in promoting loss of intestinal mucous membrane barrier function after rats scalding

Mechanism of delayed resuscitation in promoting loss of intestinal mucous membrane barrier function after rats scalding

Biointerfacing polyelectrolyte microcapsules.

Imitating nature: The authors attempted to assemble lipids on polyelectrolyte (PE) capsules (see graphic) to reduce the permeability of the capsules towards ions and small neutral molecules, thus imitating the barrier function of biological membranes. The lipid may form bilayer structures on the capsule surface, which provides the possibility to incorporate membrane-specific components such as channels and receptors for specific permeation and recognition.

The Effect of Fructan on the Phospholipid Organization in the Dry State

Fructans, a family of oligo- and polyfructoses, are implicated to play a drought-protecting role in plants. Inulin-type fructan is able to preserve the membrane barrier during dehydration. However, whether other fructans would be able to perform this function is unknown. In addition, almost nothing is known about the organization of these systems, which could give insight into the protective mechanism. To get insight into these questions the effect of different fructans on phosphatidylcholine-based model systems under conditions of dehydration was analyzed. Using a vesicle leakage assay, it was found that both levan- and inulin-type fructans protected the membrane barrier. This suggests that fructans in general would be able to protect the membrane barrier function. Furthermore, both fructan-types inhibited vesicle fusion to a large extent as measured using a lipid-mixing assay. Using x-ray diffraction, it was found that in the presence of both inulin- and levan-type fructans the lamellar repeat distance increased considerably. From this it was concluded that fructans are present between the lipid bilayers during drying. Furthermore, they stabilize the L α phase. In contrast to fructans, dextran did not increase the lamellar repeat distance and it even promoted L β phase formation. These data support the hypothesis that fructans can have a membrane-protecting role during dehydration, and give insight into the mechanism of protection.

On Adaptative Changes of Rates of Oxygen Consumption and Lipid Metabolism in Littorina saxatilis at Parasitic Invasions

Effects of parasites as a biotic factor on physiological and some biochemical characteristics of gastropod molluscs Littorina saxatilis are considered. The individuals infected at young age and incapable for reproduction due to parasitic castration have a lower intensity of respiration as compared with non-infected individuals of the same size. Large infected individuals that had time to realize their reproductive potential before pathological changes in hepatopancreas do not differ in respiration from the normal individuals. Comparative analysis of the lipid fraction of liver, particularly of the fatty acid (FA) composition of total lipids and phospholipids, allowed revealing essential differences between the control and infected individuals, as well as between infected individuals of different age groups. The absence of glycogen in the liver of infected L. saxatilis, which is revealed using thin-layer chromatography, indicates functional disturbances, including those in glycogen synthesis. We suggest that the reduction of intensity of metabolism in infected individuals is connected with peculiarities of digestive process at structural changes in the hepatopancreas damaged by sporocysts. In infected molluscs the FA composition relates mainly to parasite tissues than to the liver itself. In this connection, the role of lipids in regulation of enzyme activity and permeability of cell membranes is directed first of all to maintenance of parasite metabolism. The revealed elevated synthesis of stearic acid in infected individuals can be connected with its accumulation in parasite adipose cells. The FA composition of phospholipids in infected individuals had changes that can be directed to realization of barrier function of cell membranes, specifically to restriction of the rate of oxygen diffusion into sporocysts owing to condensation of membranes. Together with adaptive changes in FA composition the ratios saturated FA : unsaturated FA and ω3 : ω6 acids in control and infected individuals were preserved at the constant level, which in any case is connected with maintenance of normal functioning of cell membranes.

The peptide antibiotic clavanin A interacts strongly and specifically with lipid bilayers.

In this study the interaction of the antimicrobial peptide clavanin A with phosphatidylcholine bilayers is investigated by DSC, NMR, and AFM techniques. It is shown that the peptide interacts strongly and specifically with the lipids, resulting in increased order-disorder phase transition temperatures, phase separation, altered acyl chain and headgroup packing, and a drastically changed surface morphology of the bilayer. These results are interpreted in terms of clavanin-specific interactions with lipids and are discussed in the light of the different mechanisms by which clavanin A can destroy the barrier function of biological membranes.

The bactericidal activity of lectins from nitrogen-fixing bacilli

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 micrograms/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobium and Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.

Structural characterization of plasmenylcholine photooxidation products.

Oxidative damage to plasmenyl-type lipids contributes to decreased membrane barrier function, loss of membrane structure and formation of nonlamellar defects in membrane bilayers. Previous results from this laboratory have shown that membrane-soluble sensitizers (e.g. zinc phthalocyanine and bacteriochlorophyll a) mediate the photooxidation of palmitoyl plasmenylcholine (1-O-alk-1'-Z-enyl-2-palmitoyl-sn-glycero-3-phosphocholine; PPlsC) vesicles with the subsequent creation of lamellar defect structures, vesicle contents leakage and membrane-membrane fusion. Because plasmalogen lipids are significant components of sarcoplasma and myelin membranes, we sought to characterize the products of their photooxidation. This study focuses on the photooxidation of PPlsC vesicles in the presence of the water-soluble sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS4(4-)). Attack of photogenerated singlet oxygen on the 1-O-alkenyl ether linkage of PPlsC lipids was expected to generate dioxetane- and ene-type photoproducts. The products formed during continuous aerobic irradiation (28 mW/cm2, (610 nm) of PPlsC vesicles in the presence of AlPcS4(4-) were separated via reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) or evaporative light-scattering detection (ELSD). Photooxidized dipalmitoyl-phosphatidylcholine-cholesterol vesicles (control) were used to optimize the HPLC-ECD conditions, using 7 alpha-hydroperoxy-cholesterol as standard. HPLC-ECD was found to be most sensitive for PPlsC hydroperoxides, whereas HPLC-ELSD was more sensitive for nonhydroperoxide photoproducts. The three major photoproducts formed during vesicle irradiation were isolated via preparative HPLC and then characterized by 1H-nuclear magnetic resonance and mass spectrometry. 1-Formyl-2-palmitoyl-sn-glycero-3-phosphocholine and 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine were identified as dioxetane cleavage products that coeluted at approximately 3 min. The second fraction (retention time [RT] = 48 min) was identified as a PPlsC allylic hydroperoxide. The third photoproduct, eluting at RT = 64 min, is tentatively identified as an oxidation product arising from allylic hydroperoxide degradation via Hock rearrangement or free radical decomposition.

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6±0.9 to 9.1±0.5 mF/m 2 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp( O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.

Nitric oxide directly impairs intestinal barrier function.

Excess production of nitric oxide (NO) has been implicated in endotoxin-induced loss of gut barrier function in vivo. Thus, we tested the direct effect of NO on the barrier function of intestinal mucosal membranes suspended ex vivo in Ussing chambers and on IEC-6 enterocyte monolayers. In these experiments, ex vivo-mounted ileal membranes or IEC-6 cell enterocyte monolayers were exposed to the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) over a dose range (10 microm to 2 mM) or medium. SNAP at concentrations of 1 or 2 mM, but not 10 or 100 microM, increased the rates of bacterial translocation (BT) across both the ileal membranes and the IEC-6 monolayers by >1 log (P < 0.05), as well as the permeability of the IEC-6 monolayers to phenol red (P < 0.05). The ileal membranes exposed to 1 or 2 mM SNAP for 3 h manifested histologic evidence of mucosal injury and decreases in electrical resistance and potential difference values (P < 0.05), while the IEC-6 cells exposed to SNAP for 18 h had increased levels of cell death (P < 0.05). Since NO produced locally by stimulated enterocytes could contribute to barrier dysfunction, NO production, iNOS mRNA levels, and monolayer permeability were measured in enterocytes (IEC-6 and Caco-2) exposed to medium, endotoxin (lipopolysaccharide [25 microg/mL]) or a cytokine mixture (IL-1beta 10 ng/mL, TNF-alpha 10 ng/mL, and INF-gamma 250 U/mL) for 6 or 24 h. Endotoxin increased NO production, iNOS mRNA expression, and monolayer permeability in the IEC-6, but not the Caco-2 cells, while exposure to the cytokine mixture increased both NO production, iNOS mRNA expression, and monolayer permeability in both the IEC-6 and Caco-2 cell lines. Based on the results of these studies it appears that NO can directly increase ileal mucosal membrane and enterocyte monolayer permeability and BT and that increased NO production and iNOS mRNA expression is associated with endotoxin- and/or cytokine-induced loss of enterocyte monolayer barrier function.

Isolation of toxigenic Nocardiopsis strains from indoor environments and description of two new Nocardiopsis Species, N. exhalans sp. nov. and N. umidischolae sp. nov.

Nocardiopsis strains were isolated from water-damaged indoor environments. Two strains (N. alba subsp. alba 704a and a strain representing a novel species, ES10.1) as well as strains of N. prasina, N. lucentensis, and N. tropica produced methanol-soluble toxins that paralyzed the motility of boar spermatozoa at <30 microg of crude extract (dry weight) x ml(-1). N. prasina, N. lucentensis, N. tropica, and strain ES10.1 caused cessation of motility by dissipating the mitochondrial membrane potential, Deltapsi, of the boar spermatozoa. Indoor strain 704a produced a substance that destroyed cell membrane barrier function and depleted the sperm cells of ATP. Indoor strain 64/93 was antagonistic towards Corynebacterium renale. Two indoor Nocardiopsis strains were xerotolerant, and all five utilized a wide range of substrates. This combined with the production of toxic substances suggests good survival and potential hazard to human health in water-damaged indoor environments. Two new species, Nocardiopsis exhalans sp. nov. (ES10.1T) and Nocardiopsis umidischolae sp. nov. (66/93T), are proposed based on morphology, chemotaxonomic and physiological characters, phylogenetic analysis, and DNA-DNA reassociations.

Molecular Mechanisms of Water and Solute Transport across Archaebacterial Lipid Membranes

Archaebacteria thrive in environments characterized by anaeobiosis, saturated salt, and both high and low extremes of temperature and pH. The bulk of their membrane lipids are polar, characterized by the archaeal structural features typified by ether linkage of the glycerol backbone to isoprenoid chains of constant length, often fully saturated, and with sn-2,3 stereochemistry opposite that of glycerolipids of Bacteria and Eukarya. Also unique to these bacteria are macrocyclic archaeol and membrane spanning caldarchaeol lipids that are found in some extreme thermophiles and methanogens. To define the barrier function of archaebacterial membranes and to examine the effects of these unique structural features on permeabilities, we investigated the water, solute (urea and glycerol), proton, and ammonia permeability of liposomes formed by these lipids. Both the macrocyclic archaeol and caldarchaeol lipids reduced the water, ammonia, urea, and glycerol permeability of liposomes significantly (6-120-fold) compared with diphytanylphosphatidylcholine liposomes. The presence of the ether bond and phytanyl chains did not significantly affect these permeabilities. However, the apparent proton permeability was reduced 3-fold by the presence of an ether bond. The presence of macrocyclic archaeol and caldarchaeol structures further reduced apparent proton permeabilities by 10-17-fold. These results indicate that the limiting mobility of the midplane hydrocarbon region of the membranes formed by macrocyclic archaeol and caldarchaeol lipids play a significant role in reducing the permeability properties of the lipid membrane. In addition, it appears that substituting ether for ester bonds presents an additional barrier to proton flux.

Toxic-metabolite-producing bacteria and fungus in an indoor environment.

Toxic-metabolite-emitting microbes were isolated from the indoor environment of a building where the occupant was suffering serious building-related ill-health symptoms. Toxic substances soluble in methanol and inhibitory to spermatozoa at <10 microg (dry weight) ml(-1) were found from six bacterial isolates and one fungus. The substances from isolates of Bacillus simplex and from isolates belonging to the actinobacterial genera Streptomyces and Nocardiopsis were mitochondriotoxic. These substances dissipated the mitochondrial membrane potential (Deltapsi) of boar spermatozoa. The substances from the Streptomyces isolates also swelled the mitochondria. The substances from isolates of Trichoderma harzianum Rifai and Bacillus pumilus damaged the cell membrane barrier function of sperm cells.

The putative pore-forming domain of Bax regulates mitochondrial localization and interaction with Bcl-X(L).

Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action. Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions. A hairpin pair of amphipathic alpha-helices (alpha5-alpha6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax. We mutagenized several charged residues in the alpha5-alpha6 domain of Bax, changing them to alanine. These substitution mutants of Bax constitutively localized to mitochondria and displayed a gain-of-function phenotype when expressed in mammalian cells. Furthermore, substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells. The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X(L), through a BH3-independent mechanism. These observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (i) regulation of mitochondrial targeting and (ii) modulation of binding to antiapoptotic Bcl-2 proteins.

Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator.

Bcl-2 family protein including anti-apoptotic (Bcl-2) or pro-apoptotic (Bax) members can form ion channels when incorporated into synthetic lipid bilayers. This contrasts with the observation that Bcl-2 stabilizes the mitochondrial membrane barrier function and inhibits the permeability transition pore complex (PTPC). Here we provide experimental data which may explain this apparent paradox. Bax and adenine nucleotide translocator (ANT), the most abundant inner mitochondrial membrane protein, can interact in artificial lipid bilayers to yield an efficient composite channel whose electrophysiological properties differ quantitatively and qualitatively from the channels formed by Bax or ANT alone. The formation of this composite channel can be observed in conditions in which Bax protein alone has no detectable channel activity. Cooperative channel formation by Bax and ANT is stimulated by the ANT ligand atractyloside (Atr) but inhibited by ATP, indicating that it depends on the conformation of ANT. In contrast to the combination of Bax and ANT, ANT does not form active channels when incorporated into membranes with Bcl-2. Rather, ANT and Bcl-2 exhibit mutual inhibition of channel formation. Bcl-2 prevents channel formation by Atr-treated ANT and neutralizes the cooperation between Bax and ANT. Our data are compatible with a ménage à trois model of mitochondrial apoptosis regulation in which ANT, the likely pore forming protein within the PTPC, interacts with Bax or Bcl-2 which influence its pore forming potential in opposing manners.

Pathophysiology of mitochondrial cell death control.

Mitochondria have been recently recognized to play a major role in the control of apoptosis or programmed cell death. Permeabilization of mitochondrial membranes, a decisive feature of early cell death, is regulated by members of the Bcl-2 family which interact with the permeability transition pore complex (PTPC). Thus, the cytoprotective oncoprotein Bcl-2 stabilizes the mitochondrial membrane barrier function, whereas the tumor suppressor protein Bax permeabilizes mitochondrial membranes. The regulation of membrane permeabilization is intertwined with that of the bioenergetic and redox functions of mitochondria. The implications of alterations in the composition of the PTPC and in mitochondrial function for the pathophysiology of cancer (reduced apoptosis) and neurodegeneration (enhanced apoptosis) are discussed.