Expression of leptin, NGF and adiponectin in metabolic syndrome.

Adipose tissue secretes a variety of adipokines involved in the regulation of energy metabolism and insulin resistance. Metabolic syndrome corresponds to a clinical condition in which white adipose tissue is characterized by an increased production and secretion of inflammatory molecules which may have local effects on adipose tissue physiology but also systemic effects on other organs. The aim of this study was to assess the expression of leptin, NGF and adiponectin in women with metabolic syndrome compared to healthy controls. Plasma leptin, NGF and adiponectin levels were measured by the ELISA method. Leptin and NGF immunohistochemical expression was analyzed in subcutaneous adipose tissue. The results indicated that in women with metabolic syndrome waist circumference, body mass index, HOMA index, glucose, total cholesterol and triglyceride levels were significantly increased in parallel with overxpressed plasma levels of leptin and NGF and decreased adiponectin. The immunohistochemical expression of leptin and NGF was very strong. In conclusion, this is the first study demonstrating a complex of immunochemical and immunohistochemical expression of the key adipokines including leptin, NGF and adiponectin in women with metabolic syndrome. Locally-produced pro-inflammatory adipokines probably contribute to the ethiopathogenic mechanisms ofmetabolic syndrome.

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. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

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. epidermidis biofilms grown on Ti6Al4V alloy and compared its photosensitizing efficiency with that of the parent molecule, tetra-substituted N-methyl-pyridyl-porphine (C1). A more significant reduction in bacterial survival was observed when both bacterial biofilms were exposed to a lower dose of C14, and simultaneously to visible light in comparison with C1. The different responses of both staphylococcal biofilms to C1- or C14-treatment appeared to depend on photosensitizer endocellular concentration. C14 bound to both biofilms to a greater extent than C1. Moreover, C14 penetrates deeper into the bacterial membranes, as determined by fluorescence quenching experiments with methylviologen, allowing for better bacterial killing photoefficiency. Confocal laser scanning microscope (CLSM) analysis indicated damage to bacterial cell membranes in both photodynamically treated biofilms, while disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM). In summary, C14 may be a potential photosensitizer for the inactivation of staphylococcal biofilms for many device-related infections which are accessible to visible light.

Tuning the excited-state dynamics of GFP-inspired imidazolone derivatives.

The excited-state dynamics of five derivatives of the GFP-chromophore, which differ by the position and nature of their substituents, has been investigated in solvents of various viscosity and polarity and in rigid media using femtosecond-resolved spectroscopy. In polar solvents of low viscosity, like acetonitrile or methanol, the fluorescence decays of all compounds are multiexponential, with average lifetimes of the order of a few picoseconds, whereas in rigid matrices (polymer films and low temperature glasses), they are single exponential with lifetimes of the order of a few nanoseconds and fluorescence quantum yields close to unity. Global analysis of the fluorescence decays recorded at several wavelengths and of the transient absorption spectra reveals the presence of several excited-state populations with slightly different fluorescence and absorption spectra and with distinct lifetimes. These populations are attributed to the existence of multiple ground-state conformers. From the analysis of the dependence of the excited-state dynamics on the solvent and on the nature of the substituents, it follows that the nonradiative deactivation of all these excited chromophores involves an intramolecular coordinate with large amplitude motion. However, depending on the solvent and substituent, additional channels, namely, inter- and intramolecular hydrogen bond assisted nonradiative deactivation, are operative. This allows tuning of the excited-state lifetime of the chromophore. Finally, an ultrafast photoinduced intramolecular charge transfer is observed in polar solvents with one derivative bearing a dimethylaminophenyl substituent.

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)). Although the yield of (1)O(2) generation was comparable to that obtained after TDPyP incorporation into cationic unilamellar liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) SC(6)NH(2)-bound TDPyP was more active than DOTAP-bound TDPyP in photosensitizing the inactivation of the Gram-positive methicillin-resistant bacterium Staphylococcus aureus (MRSA). At variance with DOTAP-bound TDPyP, photoactivated SC(6)NH(2)-bound TDPyP was efficient also in photokilling Gram-negative bacterial pathogens, such as Escherichia coli . These observations are in agreement with the well-known photobactericidal effect of positively charged porphyrin derivatives, which can be markedly enhanced after incorporation into carriers with multiple positive charges. In addition, transmission electron microscopy studies revealed that potentiation of the TDPyP-mediated photobactericidal effect by incorporation into SC(6)NH(2) is a consequence of the carrier's ability to promote an efficient crossing of the very tightly organized three-dimensional architecture of the bacterial outer wall by the embedded porphyrin so that a prompt interaction between the short-lived photogenerated (1)O(2) and the nearby targets, whose integrity is critical for cell survival, can take place.

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. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.

Spectroscopic properties and conformational stability of Concholepas concholepas hemocyanin.

The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.

Early excited state dynamics of 6-styryl-substituted pyrylium salts exhibiting dual fluorescence.

A series of 6-styryl-2,4-diphenylpyrylium salts exhibiting dual fluorescence has been investigated by fluorescence up-conversion in conjunction with quantum chemical calculations. The short-wavelength emission is due to an excited state localized on the pyrylium fragment and the long-wavelength emission arises from a charge-transfer state delocalized over the whole molecule. The two fluorescing states do not exhibit a precursor-successor relationship. The rise time of the short-wavelength fluorescence is smaller than 200 fs, and that of the long-wavelength emission depends on the electron-donating property of the styryl group substituent. The rise is almost prompt with the weaker donors but is slower, wavelength and viscosity dependent with the strongest electron-donating group. A model involving a S(2)/S(1) conical intersection is proposed to account for these observations.

Fluorescence properties and conformational stability of the beta-hemocyanin of Helix pomatia.

The beta-hemocyanin (beta-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-beta-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the beta-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-beta-HpH is characterized by a transition temperature (Tm) of 84 degrees C and 63 degrees C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper-dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule.

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. Membrane permeabilization was ascertained by following the matrix swelling consequent to external solute equilibration. We show that the anisotropy changes of mitochondria-bound hematoporphyrin precede both membrane depolarization (proton permeation) and matrix swelling (solute permeation), thus sensing a step of the permeability transition process that involves the pore in its closed state. We suggest that the opening of the pore is preceded by a structural remodelling of mitochondrial domains containing hematoporphyrin-near, pore-regulating histidines. Such a perturbation is strongly inhibited at acidic matrix pH and completely blocked by cyclosporin A. In sucrose-based media the opening of the pore can be strongly delayed, as compared to salt-based media, a fact which probably reflects perturbation of mitochondrial membranes by sugar. We conclude that the mitochondrial permeability transition could be described as an at least two-step process which is mainly regulated by conformational changes of the pore components.

Structure and stability of arthropodan hemocyanin Limulus polyphemus.

In the hemolymph of many arthropodan species, respiratory copper proteins of high molecular weight, termed hemocyanins (Hcs) are dissolved. In this communication, we report on the protein stability of different hemocyanin species (Crustacea and Chelicerata) using fluorescence spectroscopy. Five to seven major electrophoretically separable protein chains (structural subunits) were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography from different hemocyanins with very high sequence homology of the active site regions binding copper ions (CuA and CuB), and especially the relative sequence positions of histidine (His) and tryptophan (Trp) residues of these protein segments are in all cases identical. The conformational stabilities of the native dodecameric aggregates and their isolated structural subunits towards various denaturants (pH and guanidine hydrochloride (Gdn.HCl)) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby both, the oxy- and apo-forms of the protein are considered. These two classes of Crustacea and Chelicerata Hcs have the similar Trp-fluorescence quantum yields, but different values of lambda(max) emission (about 325 and 337 nm, respectively). Differences in the quantum yields are observed of the oxy- and apo-forms, which must be attributed to the fluorescence quenching effect of the two copper ions (CuA and CuB) in the active site. The position of emission maximum indicates tryptophan side chains are situated in a non-polar environment. Denaturation studies of Hcs by Gdn.HCl indicate that the denaturation process consists of two steps: dissociation of the native molecule into its structural subunits and denaturation of the subunits at concentrations >1.5M Gdn.HCl. Two steps of denaturation are also observed after keeping the protein in buffer solutions at different pH values with different pH-stability for holo-oxy and apo-Hc forms.

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 (0.45-27 J/cm2) were inactivated with different efficiencies. The efficiency of cell photoinactivation increased with the increasing length of the hydrocarbon tail and lipophilicity and correlated with the efficiency of the porphyrin accumulation into the cells. Despite the presence of positive charges, these porphyrins did localise rather selectively in lysosomes while mitochondrial localisation was not evident, as demonstrated by fluorescence microscopy studies. Studies on isolated mitochondria provided evidence that the porphyrin uptake and distribution in these organelles were not modulated by the transmembrane potential but were exclusively controlled by partitioning phenomena which might have prevented mitochondria localization in whole cells. Our findings demonstrated that these porphyrins entered the cells through the endocytotic pathway and were transported to lysosomes whose pH increased rapidly upon irradiation. Lysosomal damage did not cause any intracellular redistribution of the porphyrin and represented the primary event causing cell death, very likely via necrosis.

Conformational states of the Rapana thomasiana hemocyanin and its substructures studied by dynamic light scattering and time-resolved fluorescence spectroscopy.

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.

Fluorescence studies on denaturation and stability of recombinant human interferon-gamma.

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol). The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.

Structure-function relationships in the neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis.

The neurotoxic complex Vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic and catalytically inactive protein, stabilizing the enzyme and reducing the activity and toxicity. Structure-function relationships in this complex were studied by spectroscopic methods. A good correlation between the ionization behaviour and accessible surface area (ASA) of the tyrosyl residues was observed. In the toxic PLA2 subunit phenolic groups participate in H-bonding network that stabilizes the catalytically and pharmacologically active conformation. The tryptophan fluorescence decay of Vipoxin is well fitted by two exponentials with lifetimes of 0.1 (54%) and 2.5 (46%) ns. W20P, W31P and W31I are located in the interface between the two subunits and participate in hydrophobic interactions stabilizing the complex. Dissociation of the complex leads to a transition of the tryptophans from hydrophobic to hydrophilic environment, which influences mainly tau2. The longer lifetime is more sensitive to the polarity of the environment. Circular dichroism measurements demonstrate that the two components of the neurotoxin preserve their secondary structure after dissociation of the complex. The results of the spectroscopic studies are in accordance with a mechanism of blockade of transmission across the neuromuscular junctions of the breathing muscles by interaction of a dissociated toxic PLA2 with a membrane. The loss of toxicity is connected with slight changes in the secondary structure of PLA2. CD studies also show a substantial contribution of disulfide bonds to the stability of the neurotoxic complex and its components.

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. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues.