The activity of isolated neurons and the modulatory state of an isolated nervous system represent a recent behavioural state.

Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrated that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we showed that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accordance with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger-induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which has an impact on the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia.

The activity of isolated snail neurons controlling locomotion is affected by glucose.

The involvement of serotonin in mediating hunger-related changes in behavioral state has been described in many invertebrates. However, the mechanisms by which hunger signals to serotonergic cells remain unknown. We tested the hypothesis that serotonergic neurons can directly sense the concentration of glucose, a metabolic indicator of nutritional state. In the snail Lymnaea stagnalis, we demonstrate that completely isolated pedal serotonergic neurons that control locomotion changed their biophysical characteristics in response to glucose application by lowering membrane potential and decreasing the firing rate. Additionally, the excitatory response of the isolated serotonergic neurons to the neuroactive microenvironment of the pedal ganglia was significantly lowered by glucose application. Because hunger has been reported to increase the activity of select neurons and their responses to the pedal ganglia microenvironment, these responses to glucose are in accordance with the hypothesis that direct glucose signaling is involved in the mediation of the hunger-related behavioral state.

Direct and decarboxylation-dependent effects of neurotransmitter precursors on firing of isolated monoaminergic neurons.

To elucidate mechanisms that underlie the profound physiological effects of the monoamine precursors 5-hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA), we examined their action on single monoaminergic neurons isolated from the ganglia of the gastropod snail Lymnaea stagnalis. In isolated serotonergic PeA motoneurons, 5-HTP produced excitation. The effect was mimicked by serotonin at 0.5-1 microM, masked by pretreatment with serotonin at higher concentrations, and abolished by the inhibitor of aromatic amino acid decarboxylase (AAAD) m-hydroxybenzylhydrazine (NSD-1015), the inhibitor of the vesicular monoamine transporter reserpine or the serotonin receptor antagonist mianserin. Exposure of the dopaminergic interneurons RPeD1 to L-DOPA caused a biphasic effect composed of a depolarization followed by a hyperpolarization. AAAD inactivation with NSD-1015, as well as the blockade of dopamine receptors with sulpiride, resulted in the enhancement of the excitatory effect, and the abolition of the inhibitory effect. Dopamine caused hyperpolarization and masked the inhibitory phase of L-DOPA action. The results show that precursors affect the rate of firing of isolated monoaminergic neurons and that their effect is completely or partially mediated by the enhanced synthesis of the respective neurotransmitter, followed by extrasynaptic release of the latter and activation of extrasynaptic autoreceptors.

Force fields including charge transfer and local polarization effects: Application to proteins containing multi/heavy metal ions.

The question whether molecular dynamics (MD) simulations can yield reliable structural and dynamical properties of metalloproteins depend on the accuracy of the force field, i.e., the potential energy function (PEF) and associated parameters modeling the interactions of the metal ion of interest with water and protein ligands. Previously, we had developed a CTPOL PEF for protein simulations of Zn(2+) bound to Cys(-) and/or His(0) that includes charge transfer and local polarization effects as well as metal van der Waals parameters that reproduce the structural and thermodynamical properties of 22 dications. Here, we evaluate if the CTPOL PEF and the new metal parameters (referred to as the CTPOLa force field) can be applied to proteins containing polynuclear metal-binding sites and heavy toxic metal ions, using the CdZn(2)-Cys(9) beta-domain of rat liver metallothionein-2 and the Hg(2+)-bound 18-residue peptide from MerP as test systems. Using the CTPOLa force field, simulations of the beta-domain of rat liver metallothionein-2 totaling 19 ns could preserve the experimentally observed CdZn(2)-Cys(9) complex geometry and overall protein structure, whereas simulations neglecting charge transfer and local polarization effects could not. However, the CTPOLa force field cannot reproduce the experimentally observed linear bicoordination of Hg(2+) in the MerP peptide without adding an angular restraint to the CTPOL PEF to correct the angle distribution about Hg(2+). Thus, the force fields presented herein for the group IIB metal ions can be applied to simulation studies of proteins containing polynuclear metal-binding sites and heavy metal ions in aqueous solution. PEF neglecting charge transfer and local polarization effects in conjunction with vdW parameters adjusted to reproduce the structural and thermodynamical properties of only the metal ion in question could not yield an accurate representation of the metal-binding site and overall protein structure.

Immediate protein targets of photodynamic treatment in carcinoma cells.

Oxidative stress induced in tumor cells undergoing photodynamic treatment (PDT) leads to extensive modification of many proteins in these cells. Protein oxidation mainly gives rise to formation of carbonyls and oxidized thiols. The immediate targets of PDT-induced protein oxidation in A431 tumor cells have been identified using a proteomic approach involving selective biotinylation, affinity purification and mass spectrometric identification of modified proteins. In all, 314 proteins were shown to undergo PDT-mediated oxidative modifications. While abundant structural proteins and chaperones represented a significant fraction of the carbonylated proteins, labeling of proteins containing oxidized thiols allowed identification of many proteins at low abundance and those involved in signaling and redox homeostasis. On the basis of the identification of these proteins, several likely mechanisms of PDT-induced triggering of apoptosis were put forward. This may not only lead to a further understanding of the complex network of cellular responses to oxidative stress, but it may also help in detailed targeting of photodynamic treatment applied to cancer.

Peripheral and axial substitution of phthalocyanines with solketal groups: synthesis and in vitro evaluation for photodynamic therapy.

Phthalocyanines (Pcs) are a class of photosensitizers (PSs) with a strong tendency to aggregate in aqueous environment, which has a negative influence on their photosensitizing ability in photodynamic therapy. Pcs with either peripheral or axial solketal substituents, that is, ZnPc(sol)8 and Si(sol)2Pc, respectively, were synthesized and their tendency to aggregate as well as their photodynamic properties in 14C and B16F10 cell lines were evaluated. The results were compared to more hydrophilic silicon Pcs, that is, Si(PEG750)2Pc and Pc4. The order of cellular uptake was Pc4 > ZnPc(sol)8 > Si(PEG750)2Pc > Si(sol2)Pc. In contrast, Si(sol2)Pc showed the highest photocytotoxicity, while ZnPc(sol)8 did not show any photocytotoxicity up to a concentration of 10 microM in both cell types. UV/vis spectroscopy showed that Si(sol)2Pc is less prone to aggregation than ZnPc(sol)8, which can explain the lack of photoactivity of the latter. Si(sol)2Pc was predominantly located in lipid droplets, whereas Si(PEG750)2Pc was homogeneously distributed in the cytosol, which is probably the main cause of their difference in photoactivity. The very high photodynamic efficacy of Si(sol)2Pc makes this PS an interesting candidate for future studies.

Zn protein simulations including charge transfer and local polarization effects.

Nearly half of all proteins contain metal ions, which perform a wide variety of specific functions associated with life processes. However, insights into the local/global, structural and dynamical fluctuations in metalloproteins from molecular dynamics simulations have been hampered by the "conventional" potential energy function (PEF) used in nonmetalloprotein simulations, which does not take into the nonnegligible charge transfer and polarization effects in many metal complexes. Here, we have carried out molecular dynamics simulations of Zn(2+) bound to Cys(-) and/or His(0) in proteins using both the conventional PEF and a novel PEF that accounts for the significant charge transfer and polarization effects in these Zn complexes. Simulations with the conventional PEF yield a nontetrahedral Cys(2)His(2) Zn-binding site and significantly overestimate the experimental Zn-S(Cys(-)) distance. In contrast, simulations with the new PEF accurately reproduce the experimentally observed tetrahedral structures of Cys(2)His(2) and Cys(4) Zn-binding sites in proteins, even when the simulation started from a nontetrahedral Zn(2+) configuration. This suggests that simulations with the new PEF could account for coordinational changes at Zn, which occurs during the folding/unfolding of Zn-finger proteins and certain enzymatic reactions The strategy introduced here can easily be applied to investigate Zn(2+) interacting with protein ligands other than Cys(-) and His(0). It can also be extended to study the interaction of other metals that have significant charge transfer and polarization effects.

Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers.

Fibril formation of islet amyloid polypeptide (IAPP) is associated with cell death of the insulin-producing pancreatic beta-cells in patients with Type 2 Diabetes Mellitus. A likely cause for the cytotoxicity of human IAPP is that it destroys the barrier properties of the cell membrane. Here, we show by fluorescence confocal microscopy on lipid vesicles that the process of hIAPP amyloid formation is accompanied by a loss of barrier function, whereby lipids are extracted from the membrane and taken up in the forming amyloid deposits. No membrane interaction was observed when preformed fibrils were used. It is proposed that lipid uptake from the cell membrane is responsible for amyloid-induced membrane damage and that this represents a general mechanism underlying the cytotoxicity of amyloid forming proteins.

Clot penetration and fibrin binding of amediplase,a chimeric plasminogen activator (K2 tu-PA).

Amediplase (K(2) tu-PA) is a hybrid plasminogen activator, consisting of the kringle 2 domain of alteplase and the protease domain of urokinase. The objective of this study was to determine the in vitro clot penetration of amediplase in relation to its fibrin binding and to compare the properties with those of alteplase. The clot lysis activity of amediplase in internal clot lysis models (both purified system and plasma system) was about 10 times less than that of alteplase. The clot lysis activity of amediplase in an external clot lysis model (plasma system) was similar to that of alteplase at therapeutic concentrations around 1 micro g/ml. The fibrin-clot binding properties of amediplase and alteplase were studied in a purified system as well as in a plasma system. In both systems amediplase bound to fibrin although to a significantly lower extent than alteplase. The binding of amediplase or alteplase did not increase during plasmin-mediated degradation of fibrin. The binding of amediplase was fully inhibited by epsilon-aminocaproic acid, indicating that the observed binding was specific and occurred via the lysine binding site in the kringle of amediplase. Clot penetration was studied during pressure-driven fluid permeation using syringes containing plasma clots. Amediplase was able to enter the clot without significant hindrance, while alteplase was concentrated on the top of the plasma clot and hardly entered into the inner parts of the clot. Diffusion-driven clot penetration was studied during clot lysis using confocal microscopy. Alteplase was detected on or close to the clot surface, while two-chain urokinase, which has no affinity to fibrin, was also detected deep inside the clot. Amediplase showed a penetration behaviour, which was distinct from that of alteplase and similar to that of two-chain urokinase. We concluded that the fibrin binding of amediplase is moderate and does not hinder clot penetration under permeation-driven or diffusion-driven transport conditions. Enhanced clot penetration, especially in large clots, could allow a more efficient lysis during thrombolytic therapy.

Photodynamic treatment and H2O2-induced oxidative stress result in different patterns of cellular protein oxidation.

Photodynamic treatment (PDT) is an emerging therapeutic procedure for the management of cancer, based on the use of photosensitizers, compounds that generate highly reactive oxygen species (ROS) on irradiation with visible light. The ROS generated may oxidize a variety of biomolecules within the cell, loaded with a photosensitizer. The high reactivity of these ROS restricts their radius of action to 5-20 nm from the site of their generation. We studied oxidation of intracellular proteins during PDT using the ROS-sensitive probe acetyl-tyramine-fluorescein (acetylTyr-Fluo). This probe labels cellular proteins, which become oxidized at tyrosine residues under the conditions of oxidative stress in a reaction similar to dityrosine formation. The fluorescein-labeled proteins can be visualized after gel electrophoresis and subsequent Western blotting using the antibody against fluorescein. We found that PDT of rat or human fibroblasts, loaded with the photosensitizer Hypocrellin A, resulted in labeling of a set of intracellular proteins that was different from that observed on treatment of the cells with H2O2. This difference in labeling patterns was confirmed by 2D electrophoresis, showing that a limited, yet distinctly different, set of proteins is oxidized under either condition of oxidative stress. By matching the Western blot with the silver-stained protein map, we infer that alpha-tubulin and beta-tubulin are targets of PDT-induced protein oxidation. H2O2 treatment resulted in labeling of endoplasmic reticulum proteins. Under conditions in which the extent of protein oxidation was comparable, PDT caused massive apoptosis, whereas H2O2 treatment had no effect on cell survival. This suggests that the oxidative stress generated by PDT with Hypocrellin A activates apoptotic pathways, which are insensitive to H2O2 treatment. We hypothesize that the pattern of protein oxidation observed with Hypocrellin A reflects the intracellular localization of the photosensitizer. The application of acetylTyr-Fluo may be useful for characterizing protein targets of oxidation by PDT with various photosensitizers.

Activated neutrophils oxidize extracellular proteins of endothelial cells in culture: effect of nitric oxide donors.

Protein oxidation of human umbilical-vein endothelial cells (HUVEC) in culture was examined under various conditions of oxidative stress. Extracellular protein (ECP) oxidation was assessed by determining dityrosine bond formation, which is indicated by the covalent coupling of the membrane-impermeable tyramine-fluorescein conjugate (TyrFluo) to HUVEC proteins. The acetylated membrane-permeable form of TyrFluo (acetylTyrFluo) was used for the determination of intracellular protein (ICP) oxidation. Oxidative stress was induced by exposing the HUVEC to PMA-activated human neutrophils, to a horseradish peroxidase/hydrogen peroxide (HRP/H(2)O(2)) system or to H(2)O(2) alone. Coupling of the probes was determined by confocal laser scanning microscopy and by Western blotting using anti-fluorescein antibody. Diethylamine nitric oxide (DEANO) was used to determine the effect of NO on the tyrosyl radical formation in proteins. The oxidative burst generated by activated neutrophils for 15 min, resulted in inducing dityrosine formation in ECP of HUVEC. Similar results were obtained with HRP/H(2)O(2), but H(2)O(2) alone did not have any effect on ECP. In the presence of DEANO (0.1 mM or higher), ECP oxidation was almost completely inhibited. This indicates that NO may protect endothelial cells against protein oxidation by activated neutrophils under pro-inflammatory conditions. Activated neutrophils did not oxidize ICP of HUVEC, which strongly suggests that the effect of the oxidative burst was restricted to the proteins exposed to the medium.

Numerical simulation of local pharmacokinetics of a drug after intravascular delivery with an eluting stent.

We use mathematical modelling to delineate the influence of two important factors on local pharmacokinetics of a drug delivered via an eluting stent, namely: (1) diffusional resistance of a stent coating, and (2) reversible binding of a drug to the vascular tissue. A system of differential equations that describes diffusion of the drug out of the polymeric coating of the stent into the vascular tissue and into the bloodstream, as well as reversible binding of the drug within the vascular tissue, was solved numerically and the spatial profiles of the concentration of the drug at various points of time were produced and analysed. Also, kinetic curves of the spatial average concentration of the drug within the wall were constructed, and the areas under those curves (AUC) were calculated. The simulations showed that AUC might be enhanced, if the stent is coated with a continuous layer of a drug-releasing medium with a high diffusional resistance. Both the residence time and the average concentration of the drug within the vascular wall increase in this case mainly because the coating imposes a diffusional barrier between the vascular tissue and the bloodstream, thereby reducing the wash-out. If the drug reversibly binds to the tissue, the residence time increases greatly, but the AUC for the free (unbound) drug remains unchanged, implying that the presence of the drug in the vessel is prolonged at the expense of a proportional reduction in concentration of a free drug within the tissue. These findings justify the design of eluting stents with continuous coatings with enhanced diffusional resistance and the engineering of drugs with enhanced affinity to the vascular matrix. Reversible binding to tissue may be beneficial for prolonging the presence of the drug in the target tissue, and for avoiding potential toxic peak effects of high concentrations of the free (unbound) drug.