Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (Translocator Protein of 18 kDa (TSPO)).

Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances.

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide.

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig.

Shedding light on the mitochondrial permeability transition.

The mitochondrial permeability transition is an increase of permeability of the inner mitochondrial membrane to ions and solutes with an exclusion size of about 1500Da. It is generally accepted that the permeability transition is due to opening of a high-conductance channel, the permeability transition pore. Although the molecular nature of the permeability transition pore remains undefined, a great deal is known about its regulation and role in pathophysiology.

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor).

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor.

Photodynamic action of Tri-meso (N-methyl-pyridyl), meso (N-tetradecyl-pyridyl) porphine on Staphylococcus epidermidis biofilms grown on Ti6Al4V alloy.

Staphylococcus epidermidis is a leading cause of nosocomial infections, and its virulence is attributable to formation of biofilm, especially on implanted devices. Photodynamic treatment (PDT) has been actively investigated for the eradication of bacterial biofilm growing on dental plaques and oral implants. In this study, we used Tri-meso (N-methyl-pyridyl), meso (N-tetradecyl-pyridyl) porphine (C14) for inactivation of two structurally distinct S.

Inclusion of 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenylporphine in supramolecular aggregates of cationic amphiphilic cyclodextrins: physicochemical characterization of the complexes and strengthening of the antimicrobial photosensitizing activity.

Recent findings suggest that visible light-promoted photooxidative processes mediated by sensitizers of appropriate chemical structure could represent a useful tool for properly addressing the problem of the increasing occurrence of infectious diseases caused by multiantibiotic-resistant microbial pathogens. The monocationic meso-substituted porphyrin 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphine (TDPyP) complexed into supramolecular aggregates of cationic amphiphilic beta-cyclodextrin (SC(6)NH(2)) (mean diameter = 20 nm) appeared to be endowed with favorable properties to act as a photosensitizing agent, including a very high quantum yield (Phi(Delta) = 0.90) for the generation of the highly reactive oxygen species, singlet oxygen ((1)O(2)).

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation.

Mutual stimulation of beta-amyloid fibrillogenesis by clioquinol and divalent metals.

As reported by some authors, clioquinol (CQ), a 8-hydroxyquinoline derivative, has produced very encouraging results in the treatment of Alzheimer's disease (AD). Its biological effects are most likely ascribed to complexation of specific metal ions, such as copper (II) and zinc (II), critically associated with beta-amyloid (A beta) aggregation/fibrillogenesis and degeneration processes in the brain. The present study was aimed at assessing the in vitro effects of CQ on the aggregation/fibrillogenesis properties of human A beta either alone or complexed with Cu(2+) and Zn(2+).

New cationic liposomes as vehicles of m-tetrahydroxyphenylchlorin in photodynamic therapy of infectious diseases.

Antimicrobial photodynamic therapy is emerging as a promising therapeutic modality for bacterial infections. For optimizing the antibacterial activity of the photosensitizer m-tetrahydroxyphenylchlorin, it has been encapsulated in mixed cationic liposomes composed of different ratios of dimyristoyl- sn-glycero-phosphatidylcholine and any of four cationic surfactants derived from l-prolinol. The delivery efficiency of the different liposomes formulations has been evaluated on a methicillin-resistant Staphylococcus aureus bacterial strain (MRSA), and one of the tested formulations shows a biological activity comparable to that of the free chlorin.

Potential pathogenic role of beta-amyloid(1-42)-aluminum complex in Alzheimer's disease.

The etiopathogenesis of Alzheimer's disease is far from being clearly understood. However, the involvement of metal ions as a potential key factor towards conformational modifications and aggregation of amyloid is widely recognized. The aim of the present study is to shed some light on the relationship between metal ions, amyloid conformation/aggregation, and their potential relationship with the conformational aspects of AD.

Assessing the molecular basis for rat-selective induction of the mitochondrial permeability transition by norbormide.

It was recently demonstrated that the rat-selective toxicant norbormide also induces rat-selective opening of the permeability transition pore (PTP) in isolated mitochondria. Norbormide is a mixture of endo and exo stereoisomers; however, only the endo forms are lethal to rats. In the present study we tested both endo and exo isomers as well as neutral and cationic derivatives of norbormide to: (i) verify if the PTP-regulatory activity by norbormide is stereospecific; (ii) define the structural features of norbormide responsible for PTP-activation, (iii) elucidate the basis for the drug species-specificity.

Efficient photoinactivation of methicillin-resistant Staphylococcus aureus by a novel porphyrin incorporated into a poly-cationic liposome.

Antimicrobial photodynamic therapy is emerging as a promising therapeutic modality for bacterial infections. Our studies aim at identifying strategies for optimizing the antibacterial activity of porphyrin-type photosensitisers. The photoinactivation properties of a novel, positively charged meso-substituted porphyrin, namely 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphyrin were tested against a typically antibiotic-resistant pathogen, such as methicillin-resistant Staphylococcus aureus.

Comparative effects of Abeta(1-42)-Al complex from rat and human amyloid on rat endothelial cell cultures.

Metal ions are widely recognized as a key factor for the conformational changes and aggregation of the Alzheimer's disease amyloid (Abeta). So far Al(3+) has received much less attention than other biometals in terms of interaction with Abeta. Brain endothelial cells have been identified as important regulators of the neuronal microenvironment, including Abeta levels.

Synthesis and activity studies of analogues of the rat selective toxicant norbormide.

Norbormide [5-(alpha-hydroxy-alpha-2-pyridylbenzyl)-7-(alpha-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. A series of NRB-related analogues were prepared to investigate the structural features responsible for, and the in vitro biological markers indicative of, in vivo lethality of the parent molecule in rats. Their synthesis and biological evaluation (vasoconstriction, vasodilation, mitochondrial dysfunction, cardiotoxicity and lethality) is described.

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis.

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e.

Aggregation/fibrillogenesis of recombinant human prion protein and Gerstmann-Sträussler-Scheinker disease peptides in the presence of metal ions.

In this study we investigated the role of Cu(2+), Mn(2+), Zn(2+), and Al(3+) in inducing defective conformational rearrangements of the recombinant human prion protein (hPrP), which trigger aggregation and fibrillogenesis. The research was extended to the fragment of hPrP spanning residues 82-146, which was identified as a major component of the amyloid deposits in the brain of patients affected by Gerstmann-Sträussler-Scheinker (GSS) disease. Variants of the 82-146 wild-type subunit [PrP-(82-146)(wt)] were also examined, including entirely, [PrP-(82-146)(scr)], and partially scrambled, [PrP-(82-146)(106)(-)(126scr)] and [PrP-(82-146)(127)(-)(146scr)], peptides.

Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by liposome-delivered photosensitising agents.

The uptake of two photosensitising agents (hematoporphyrin and chlorophyll a) by a highly pathogenic bacterium, namely methicillin-resistant Staphylococcus aureus (MRSA), has been studied by using unilamellar liposomes of different size, fluidity and electric charge as carriers. Optimal results are obtained by using hematoporphyrin embedded in fluid cationic vesicles composed by the monocationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), which yield an endocellular concentration of photosensitiser much higher than that obtained by incubation of the cells with the free porphyrin, yet promote a tighter binding and a more efficient photoinactivation of MRSA. Apparently, the photosensitiser is successfully transferred from the liposome to the bacterial cells when the presence of the tetrapyrrolic derivative does not appreciably perturb the native three-dimensional organisation of the lipid vesicle, such as it occurs with hematoporphyrin.

Species-specific modulation of the mitochondrial permeability transition by norbormide.

In the present study, we show that norbormide stimulates the opening of the permeability transition pore (PTP) in mitochondria from various organs of the rat but not of guinea pig and mouse. Norbormide does not affect the basic parameters that modulate the PTP activity since the proton electrochemical gradient, respiration, phosphorylation and Ca(2+) influx processes are only partially affected. On the other hand, norbormide induces rat-specific changes in the fluidity of the lipid interior of mitochondrial membranes, as revealed by fluorescence anisotropy of various reporter molecules.

Discrimination between two steps in the mitochondrial permeability transition process.

It is well known that a lag phase generally elapses between the addition of inducers of the mitochondrial permeability transition and the opening of the pore. To advance our present understanding as regards the significance of this phenomenon, we used experimental approaches which are sensitive to different aspects of the permeability transition process. The pore conformation was sensed by the fluorescence anisotropy changes of hematoporphyrin-labelled mitochondria.

Meso-substituted tetra-cationic porphyrins photosensitize the death of human fibrosarcoma cells via lysosomal targeting.

In this paper we present a study on the intracellular localisation and the efficiency of cell photoinactivation of a series of derivatives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphine (C1), whose degree of lipophilicity was varied through replacement of one methyl group with an alkyl chain of various length. Human HT1080 fibrosarcoma cells exposed to the various C1 derivatives (0.25 microM) for 24 h and irradiated with increasing doses of red-light (0.

Fluorescence spectroscopy of the tryptophan microenvironment in Carcinus aestuarii hemocyanin.

The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate.

Structural modifications of the permeability transition pore complex in resealed mitochondria induced by matrix-entrapped disaccharides.

Mitochondrial resealing after the opening of the permeability transition (PT) pore was studied in saline- and sugar-based media by following the fluorescence anisotropy changes of mitochondria-bound hematoporphyrin (HP), a probe sensitive to conformational variations of the pore complex [Biochemistry 38 (1999) 9300]. The HP anisotropy changes correlated well with complete mitochondrial resealing in saline media and suggested that the pore complex regained the native structure after closure. Rebuilding of the pore complex structure was also achieved in monosaccharide-based media, thus ruling out a major influence of the swollen state of mitochondria on the reconstitution properties of the pore components.