An artificial neural network model for predicting the subcellular localization of photosensitisers for photodynamic therapy of solid tumours.

Photodynamic therapy (PDT) is a promising modality for the treatment of tumours based on the combined action of a photosensitiser (PS), visible light and molecular oxygen, which generates a local oxidative damage that leads to cell death. The site where the primary photodynamic effect takes place depends on the subcellular localization of the PS and affects the mode of action and efficacy of PDT. It is therefore of prime interest to develop structure-subcellular localization prediction models for a PS from its molecular structure and physicochemical properties. Here we describe such a prediction method for the localization of macrocyclic PSs into cell organelles based on a wide set of physicochemical properties and processed through an artificial neural network (ANN). 128 2D-molecular descriptors related to lipophilicity/hydrophilicity, charge and structural features were calculated, then reduced to 76 by using Pearson's correlation coefficient, and finally to 5 using Guyon and Elisseeff's algorithm. The localization of 61 PSs was compiled from literature and distributed into 3 possible cell structures (mitochondria, lysosomes and "other organelles"). A non-linear ANN algorithm was used to process the information as a decision tree in order to solve PS-organelle assignment: first to identify PSs with mitochondrial and/or lysosomal localization from the rest, and to classify them in a second stage. This sequential ANN classification method has permitted to distinguish PSs located into two of the most important cell targets: lysosomes and mitochondria. The absence of false negatives in this assignation, combined with the rate of success in predicting PS localization in these organelles, permits the use of this ANN method to perform virtual screenings of drug candidates for PDT.

Incorporation of hydrophobic porphyrins into liposomes: characterization and structural requirements.

The ability of photosensitisers to give reactive oxygenated products is considered decisive for photodynamic applications, but the hydrophobic nature of many porphyrins makes necessary to obtain suitable pharmaceutical formulations. This paper reports the structural photosensitiser features that allow the preparation of stable liposomal formulations. Metallated and non-metallated TPPs and TPyPs and different lipid/porphyrin ratios were considered in order to procure liposomal preparations containing porphyrin concentrations adequate to necessary doses. The results show that the incorporation of porphyrins into liposomes can be related with their ability to form aggregates in a watery media. Thus, ZnTPP, which structural properties avoid the formation of aggregates, was efficiently incorporated into stable liposomes. Moreover, the efficient generation of singlet oxygen by ZnTPP liposomal suspensions has been shown. Because of this, the synthesis of hydrophobic porphyrin derived structures or other sensitisers, which do not aggregate in a watery media and with Q-bands shifted to higher lambda values than ZnTPP, will be efficiently incorporated into liposomes and useful for clinical applications.

Effect of germinated seeds extract on the respiratory activity of human skin fibroblasts and sheep liver mitochondria. Influence on cell viability and proliferation and their uselfulness as active cosmetic ingredient.

The present work studies the ability of a germinated seeds extract (alfalfa, radish, wheat, soy) to influence cell respiration of skin fibroblasts and the respiratory activity of sheep liver isolated mitochondria, and the cytotoxicity of the product on cultured skin fibroblasts. The content of free and total amino acids present in this extract was determined. Aspartic acid represented 37.52% of free amino acids in the extract, but 48.07% glutamic acid was found when total amino acids obtained by acid hydrolysis were quantified. The extract contains more than 6.5% free serine, glutamic acid and alanine. Arginine, serine, aspartic acid and threonine are the other amino acids that represent more than 4.5% total amino acids. Respiration of sheep liver mitochondria and human skin fibroblasts (Foreskin ATCC CRL-1635) was checked polarographically. The results obtained show the ability of this extract to stimulate cellular respiration of both in vitro models, but the effect is more marked on cell cultured fibroblasts, and its dose dependency. The role of acidic amino acids, aspartic and glutamic acids, on the increase of oxygen consumption by mitochonria is suggested. Cytotoxicity of the germinated seeds extract was tested by the assessment of the viability, the morphological changes and the proliferation of cell cultured human skin fibroblasts. It has been shown that germinated seeds extract are non-toxic to cell cultures at doses of up to 10 mg mL(-1); neither morphological changes nor membrane integrity alterations or changes in cell proliferation were observed.

Aggregation and fusion of vesicles composed of N-palmitoyl derivatives of membrane phospholipids.

N-Acylphosphatidylethanolamines and N-acylphosphatidylserines have been isolated from mammalian cells and have been associated with some tissue degenerative changes, although the relationship between their synthesis and the uncontrolled sequence of events that ends in irreversible tissue damage is not completely established. Our results show that monovalent and divalent cations induce aggregation and fusion of liposomes constituted by N-palmitoylphosphatidylethanolamine (NPPE) and N-palmitoylphosphatidylserine (NPPS). The effectiveness of cations to induce the aggregation of NPPE and NPPS liposomes is Ca2+ > Mg2+ >> Na+. NPPS liposomes aggregate at lower concentrations of divalent cations than NPPE liposomes, but with sodium NPPE liposomes aggregate to a higher extent than NPPS liposomes. The reaction order for the aggregation processes depends on the lipid and the cation nature and range from 1.04 to 1.64. Dynamic light scattering shows an irreversible increase of the size of the aggregates in the presence of all cations tested. The irreversibility of the aggregation process and the intermixing of bilayer lipids, as studied by resonance energy transfer assay, suggest that fusion, rather than aggregation, occurs. The existence of a real fusion was demonstrated by the coalescence of the aqueous contents of both NPPS and NPPE liposomes in the presence of either monovalent or divalent cations. The different binding sensitivity of Ca2+ to NPPS and NPPE liposomes, determined by zeta potential measurements, agrees with the results obtained in the aggregation and fusion assays. Our results suggest that the synthesis in vivo of N-acylated phospholipids can introduce important changes in membrane-mediated processes.

Fatty acid composition of platelet membrane lipids after administration of two different fat emulsions in critically ill patients.

OBJECTIVE: To determine the effects on platelet membrane fatty acid composition following administration of two different fat emulsions. DESIGN: Prospective, randomized, double-blind study. SETTING: Intensive care unit in a university-affiliated hospital. PATIENTS: 12 adult critically ill patients in need of total parenteral nutrition. INTERVENTIONS: Patients were treated with total parenteral nutrition (TPN) for 7 days, receiving for fat intake either a long-chain triglyceride (20% LCT) emulsion (group 1, n=6) or a medium-chain triglyceride-LCT (20% MCT/LCT) emulsion (group 2, n=6). MEASUREMENTS AND RESULTS: High-performance liquid chromatography of membrane fatty acids was carried out before and after 7 days of TPN. In the LCT group, an increase in C18:2n-6 and a decrease in caprylic acid and docosahexaenoic acid, which resulted in a decreased ratio of n-3/n-6 fatty acid content, was observed. In the MCT/LCT group, a reduced percentage of palmitoleic acid and arachidonic acid was shown. CONCLUSIONS: The observed changes in fatty acid composition are in agreement with the lipid composition of the fat emulsions used. Because the C18:2n-6/C18:3n-3 ratio in both emulsions is close (approximately 9.0), the observed changes in the fatty acid composition of platelets may not be relevant for platelet function.

N-stearoyl-phosphatidylserine: synthesis and role in divalent-cation-induced aggregation and fusion.

N-Acylphosphatidylserines have been isolated from intact and injured tissues, but the participation of such acidic phospholipids in membrane aggregation and fusion has not been demonstrated. We have synthesized N-stearoylphosphatidylserine (NSPS) and examined divalent-cation-induced aggregation of NSPS-liposomes, which leads to membrane destabilization and fusion. The purified lipid was characterized by its chromatographic and spectroscopic (infrared and 1H nuclear magnetic resonance) properties and by its chemical degradation pattern. Aggregation of unilamellar NSPS-liposomes was studied as a function of calcium and magnesium concentration. The ability of calcium and magnesium to induce vesicle aggregation is higher for phosphatidylserine (PS)-liposomes (threshold concentration 1.5 mM for calcium and 4.6 mM for magnesium) than for NSPS-liposomes (threshold concentration 2.8 mM for calcium and 6.6 mM for magnesium). The irreversibility of the aggregation reactions after adding EDTA suggests that vesicle fusion might occur in the presence of calcium and magnesium. Preliminary studies, based on mixing of both lipid and internal aqueous contents, show that fusion rather than aggregation of NSPS-liposomes occurs in the presence of calcium ions. The tendency of NSPS-liposomes to aggregate at higher cation concentrations than PS-liposomes suggests that N-acylation of phosphatidylserine protects the membrane against degenerative damage caused by aggregation and fusion.

Inhibition of rat hepatic mitochondrial aldehyde dehydrogenase isozymes by repeated cyanamide administration: pharmacokinetic-pharmacodynamic relationships.

The inhibition of rat hepatic mitochondrial aldehyde dehydrogenase (ALDH) isozymes was studied in apparent steady-state conditions after repeated intra-peritoneal cyanamide administration. The low-Km mitochondrial ALDH isozyme was more susceptible to cyanamide-induced inhibition (DI50 = 0.104 mg kg-1) than the high-Km isozyme (DI50 = 8.52 mg kg-1), with almost complete inhibition occurring at 0.35 mg kg-1 total cyanamide administered for the low-Km isozyme. The relationships between plasma and liver cyanamide concentrations and the inhibition of high-Km ALDH were established by means of the sigmoid Imax model. The effect of dosing rate on the plasma concentration of cyanamide at apparent steady-state showed non-linearity, indicating that clearance or first-pass metabolism of cyanamide during its absorption after intraperitoneal administration did not remain constant throughout the range of doses studied.

Inactivation mechanism of low-KM rat liver mitochondrial aldehyde dehydrogenase by cyanamide in vitro.

The inactivation of low-KM rat liver mitochondrial aldehyde dehydrogenase (ALDH) by the alcohol-sensitizing agent cyanamide (H2NCN) has been studied in vitro. The effect of the concentrations of NAD+ at different concentrations of catalase on the inactivation of ALDH by cyanamide (20 and 200 microM) in vitro point to an ALDH-NAD(+)-catalase complex prior to the binding to cyanamide to form the holoenzyme-inhibitor complex. Cyanamide itself could be responsible for the inactivation of ALDH. The possibility that both irreversibly inactivated ALDH and cyanamide remain free at the end of the inactivation process is discussed. The effects of pH and ionic strength on the inactivation process are also described. The pseudo-first order rate constants for inactivation of low-KM ALDH depends on both effects, suggesting that electrostatic forces are involved in the process and that a group with pK approximately 6.8, presumably a histidine residue, at the active site of ALDH could be involved. A representative equation for the inactivation process of low-KM ALDH by cyanamide in vitro has been fitted to experimental kinetic data, involving both catalase and inhibitor concentrations.

Kinetic behaviour of soluble and mitochondrial bound lactate dehydrogenase.

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The kinetic behaviour of mitochondrial bound enzyme fits a bibi sequential type mechanism as well as the cytosolic rabbit liver lactate dehydrogenase. The bound enzyme has greater values of Km(NADH) and Km(pyruvate) than the soluble one, suggesting that binding induces a decrease in the affinity of both substrates. The behaviour of the free and the mitochondrial-bound enzyme is of the Michaelis-Menten type, but the kinetics of a mixture of rabbit liver cytosolic and mitochondrial-bound lactate dehydrogenase is sigmoidal, suggesting that a cooperative phenomenon takes place.

Comparative binding of lactate dehydrogenase to mitochondrial fractions.

Beef liver mitochondrial fraction showed LDH activity (1.76 +/- 0.25 U/g pellet). Sixty seven% of the initial mitochondrial pellet LDH activity (almost M4 isoenzyme) was released when suspended in NaCl 0.15 M. When the washed particles were sonicated in a 0.15 M NaCl medium, the solubilized LDH activity (all five isoenzymes as cytosoluble fraction) was 5-fold higher than the initial pellet activity. The different isoenzymatic composition of intramitochondrial and externally bound forms of the enzyme should be taken into account when investigating the physiological role of intramitochondrial LDH. Beef liver cytosoluble LDH (very little content of M4 isoenzyme) showed no affinity for the beef liver mitochondrial fraction but purified M4-LDH isoenzyme was able to bind to the particulate fraction from the same source. This suggests an isoenzyme specificity for the interaction. The maximum amount of cytosoluble LDH bound to the mitochondrial fraction depends on the enzyme and the particulate fraction source. Therefore, binding capacity to the mitochondrial fraction depends not only on the net charge of LDH isoenzymes, which play a predominant role in the binding, but also on individual characteristics of the LDH isoenzymes and mitochondrial fractions from different sources. This suggests that electrostatic forces are not the only ones involved in the binding process.

Inactivation of low-Km rat liver mitochondrial aldehyde dehydrogenase by cyanamide in vitro. A catalase-mediated reaction.

The inactivation of the affinity chromatography purified low-Km rat liver mitochondrial aldehyde dehydrogenase (ALDH)--free of catalase activity--by the alcohol sensitizing agent cyanamide was studied in vitro. This ALDH-purified preparation was not susceptible to cyanamide inactivation at concentrations up to 2.5 mM. On the other hand, ALDH activity appears to be irreversibly inhibited when the incubation mixture contained ALDH, catalase, NAD+ and cyanamide. Influence of catalase, NAD+ and cyanamide concentrations in the incubation mixtures on the ALDH activity were also established. The time course of the concentration of cyanamide in an incubation mixture when ALDH activity was inhibited by cyanamide in the presence of catalase and NAD+, was evaluated by HPLC. No disappearance of cyanamide was observed for a period of time up to 24 hr. This result suggests that no metabolic conversion of cyanamide to an active inhibitory form takes place, as has been suggested recently.

Modulation of lactate dehydrogenase activity by enzyme-protein interaction.

Some lactate dehydrogenase modulator proteins have been isolated from the lactate dehydrogenase-free crude mitochondrial fraction of rabbit muscle, beef liver and chicken liver. It was shown that beef and chicken liver mitochondrial extracts exhibited activatory capacity in contrast to the inhibitory capacity of rabbit muscle mitochondrial extracts. All modulators can be precipitated by 80% ammonium sulphate saturation and show high anodic electrophoretic mobility and heat stability. Modulators have higher affinity for alkaline pI lactate dehydrogenase isoenzymes, independent of whether the M and H subunits are predominant. The inhibitor and the activator molecules compete for lactate dehydrogenase since their modulatory capacity was nullified when similar relative amounts were used. This study shows the existence of analogous proteins with an acidic pI in the different mitochondrial fractions which modify lactate dehydrogenase activity.

Lactate and malate dehydrogenase binding to the microsomal fraction from chicken liver.

Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.

Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme.

Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.