Membrane Lipid Composition Influences the Hydration of Proton Half-Channels in FoF1-ATP Synthase.

The membrane lipid composition plays an important role in the regulation of membrane protein activity. To probe its influence on proton half-channels' structure in FoF1-ATP synthase, we performed molecular dynamics simulations with the bacterial protein complex (PDB ID: 6VWK) embedded in three types of membranes: a model POPC, a lipid bilayer containing 25% (in vivo), and 75% (bacterial stress) of cardiolipin (CL). The structure proved to be stable regardless of the lipid composition. The presence of CL increased the hydration of half-channels. The merging of two water cavities at the inlet half-channel entrance and a long continuous chain of water molecules directly to cAsp61 from the periplasm were observed. Minor conformational changes in half-channels with the addition of CL caused extremely rare direct transitions between aGlu219-aAsp119, aGlu219-aHis245, and aGln252-cAsp61. Deeper penetration of water molecules (W1-W3) also increased the proton transport continuity. Stable spatial positions of significant amino acid (AA) residue aAsn214 were found under all simulation conditions indicate a prevailing influence of AA-AA or AA-W interactions on the side-chain dynamics. These results allowed us to put forward a model of the proton movement in ATP synthases under conditions close to in vivo and to evaluate the importance of membrane composition in simulations.

Application of a multicomponent model of convectional reaction-diffusion to description of glucose gradients in a neurovascular unit.

A supply of glucose to a nervous tissue is fulfilled by a cerebrovascular network, and further diffusion is known to occur at both an arteriolar and a microvascular level. Despite a direct relation, a blood flow dynamic and reaction-diffusion of metabolites are usually considered separately in the mathematical models. In the present study they are coupled in a multiphysical approach which allows to evaluate the effects of capillary blood flow changes on near-vessels nutrient concentration gradients evidently. Cerebral blood flow (CBF) was described by the non-steady-state Navier-Stokes equations for a non-Newtonian fluid whose constitutive law is given by the Carreau model. A three-level organization of blood-brain barrier (BBB) is modelled by the flux dysconnectivity functions including densities and kinetic properties of glucose transporters. The velocity of a fluid flow in brain extracellular space (ECS) was estimated using Darcy's law. The equations of reaction-diffusion with convection based on a generated flow field for continues and porous media were used to describe spatial-time gradients of glucose in the capillary lumen and brain parenchyma of a neurovascular unit (NVU), respectively. Changes in CBF were directly simulated using smoothing step-like functions altering the difference of intracapillary pressure in time. The changes of CBF cover both the decrease (on 70%) and the increase (on 50%) in a capillary flow velocity. Analyzing the dynamics of glucose gradients, it was shown that a rapid decrease of a capillary blood flow yields an enhanced level of glucose in a near-capillary nervous tissue if the contacts between astrocytes end-feet are not tight. Under the increased CBF velocities the amplitude of glucose concentration gradients is always enhanced. The introduced approach can be used for estimation of blood flow changes influence not only on glucose but also on other nutrients concentration gradients and for the modelling of distributions of their concentrations near blood vessels in other tissues as well.

MITODYN: An Open Source Software for Quantitative Modeling of Mitochondrial and Cellular Energy Metabolic Flux Dynamics in Health and Disease.

Mitochondrial respiratory chain (RC) transforms the reductive power of NADH or FADH2 oxidation into a proton gradient between the matrix and cytosolic sides of the inner mitochondrial membrane, that ATP synthase uses to generate ATP. This process constitutes a bridge between carbohydrates' central metabolism and ATP-consuming cellular functions. Moreover, the RC is responsible for a large part of reactive oxygen species (ROS) generation that play signaling and oxidizing roles in cells. Mathematical methods and computational analysis are required to understand and predict the possible behavior of this metabolic system. Here we propose a software tool that helps to analyze individual steps of respiratory electron transport in their dynamics, thus deepening understanding of the mechanism of energy transformation and ROS generation in the RC. This software's core is a kinetic model of the RC represented by a system of ordinary differential equations (ODEs). This model enables the analysis of complex dynamic behavior of the RC, including multistationarity and oscillations. The proposed RC modeling method can be applied to study respiration and ROS generation in various organisms and naturally extended to explore carbohydrates' metabolism and linked metabolic processes.

Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model.

Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter's transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate's attenuation of oxaloacetate's inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells.

NMDA and GABA receptor presence in rat heart mitochondria.

The purpose of this study is to demonstrate the presence of three more receptors in mitochondria. Two N-methyl-d-aspartate receptor (NMDAR) subunits (NR1 and NR2B) are found by protein immunoblot and immunogold labeling in mitochondria fraction isolated from rat heart. These data allow supposing NMDAR presence and functioning in the inner mitochondrial membrane. There are no signs of receptor presence obtained in heart tissue lysate, that indicates the receptor localization exactly in mitochondria. The possible receptor functions discussed are its participation in calcium transport and in excitation-metabolism coupling. Besides, preliminary evidence is obtained of GABAA and GABAB receptors presence in heart mitochondria. One can surmise their role in metabolism regulation and their possible co-operation with NMDAR just as in the nervous system.

Geometries of vasculature bifurcation can affect the level of trophic damage during formation of a brain ischemic lesion.

Ischemic lesion is a common cause of various diseases in humans. Brain tissue is especially sensitive to this type of damage. A common reason for the appearance of an ischemic area is a stop in blood flow in some branch of the vasculature system. Then, a decreasing concentration gradient results in a low mean level of oxygen in surrounding tissues. After that, the biochemical ischemic cascade spreads. In this review, we examine these well-known events from a new angle. It is stressed that there is essential evidence to predict the formation of an ischemic micro-area at the base of vascular bifurcation geometries. Potential applications to improve neuroprotection are also discussed.

Fundamental principles of vascular network topology.

The vascular system is arguably the most important biological system in many organisms. Although the general principles of its architecture are simple, the growth of blood vessels occurs under extreme physical conditions. Optimization is an important aspect of the development of computational models of the vascular branching structures. This review surveys the approaches used to optimize the topology and estimate different geometrical parameters of the vascular system. The review is focused on optimizations using complex cost functions based on the minimum total energy principle and the relationship between the laws of growth and precise vascular network topology. Experimental studies of vascular networks in different species are also discussed.

On the regulative role of the glutamate receptor in mitochondria.

The purpose of this work was to study the regulative role of the glutamate receptor found earlier in the brain mitochondria. In the present work a glutamate-dependent signaling system with similar features was detected in mitochondria of the heart. The glutamate-dependent signaling system in the heart mitochondria was shown to be suppressed by γ-aminobutyric acid (GABA). The GABA receptor presence in the heart mitochondria was shown by golding with the use of antibodies to α- and ß-subunits of the receptor. The activity of glutamate receptor was assessed according to the rate of synthesis of hydrogen peroxide. The glutamate receptor in mitochondria could be activated only under conditions of hypoxic stress, which in model experiments was imitated by blocking Complex I by rotenone or fatty acids. The glutamate signal in mitochondria was shown to be calcium- and potential-dependent and the activation of the glutamate cascade was shown to be accompanied by production of hydrogen peroxide. It was discovered that H2O2 synthesis involves two complexes of the mitochondrial electron transfer system - succinate dehydrogenase (SDH) and fatty acid dehydrogenase (ETF:QO). Thus, functions of the glutamate signaling system are associated with the system of respiration-glycolysis switching (the Pasteur-Crabtree) under conditions of hypoxia.

Assessment of microcirculatory effects of glycine by intravital microscopy in rats.

Experimental studies using laboratory animal models have shown a potential vasoactive effect of natural metabolites such as glycine. The present study used intravital microscopy in laboratory rat models to study the microcirculation in the brain pial and mesentery vessels. To investigate the pial microvasculature, a stereotaxis-like animal fixing device was used. The intravital microscopy unit consisted of a binocular microscope equipped with a digital photo-video camera, processor, monitor and printer. Using reflected light, a special contact lens with an amplified focus depth provided high-resolution images of nontransparent tissue objects that typically have insufficient light exposure. Glycine had a vasodilatory effect on microvessels in the rat brain and mesenterium. The diameter of pial arterioles increased after glycine application especially markedly (up to 250% of initial size). These changes were not observed when physiological saline was used. Even a very small amount of glycine (a drop on the needle) was sufficient to stop the early stages of histamine-induced blood stasis development in 3-5 s in mesenterial microvessels. The vasodilatory effect of glycine on the pial microcirculation correlates with its reported positive therapeutic effect in cerebral ischemic stroke. The ability of glycine to avoid or prevent histamine-induced microcirculatory alterations in mesenterial microvessels may have potential clinical applications.

Development of multi-compartment model of the liver using image-based meshing software.

Computer simulation of biological systems for in silico validation has the potential of increasing the efficiency of pharmaceutical research and development by expanding the number of parameters tested virtually. Then only the most interesting subset of these has to be probed in vivo. By focusing on variables with the greatest influence on clinical end points, valuable drug targets can be advanced more quickly. A large number of methods have been developed to rebuild a three-dimensional (3D) model of a liver, mostly to prepare a liver surgery. These models are often not accurate and most of the them don't take into account the fluidics inside the vessels. The aim of this work is to provide an accurate computational multi-compartement model of the healthy and the pathological liver with their network of blood vessels (vasculature) using a finite-element-modeling software. Computed tomography (CT) slices, in DICOM format, from two different patients were used to provide the datasets of transverse images for the modeling. Each dataset of images was segmented in order to extract the liver's shape and define the vein and artery networks. On CT images, the contrast between the liver and the nearby organs (background) is very low because all these structures are a similar density. Thus, we used semi-automatic tools to determine liver contours. Manual segmentation was used as a last resort. Then, strong filtering (bilateral filter) and confidence-connected-region-growing algorithm were applied to rebuild from each - healthy and pathological - liver a multicompartment model including parenchyma, arteries and veins. The precision of the obtained vasculature model allowed anatomical classification of hepatic segments and the quantification of their volumes. Although our study demonstrated the difficulties in use of CT images for computational modeling of the liver, it also confirmed that semi-automatic tools can be used to develop anatomically accurate models of hepatic vasculature.

Diversity of neurodegenerative processes in the model of brain cortex tissue ischemia.

Stroke is known to induce massive cell death in the ischemic brain. Either necrotic or apoptotic types of cell death program were observed in neurons in zone of ischemia. We suggest that spatial heterogeneity of glucose and oxygen distribution plays a crucial role in this phenomenon. In order to elucidate the role of glucose and oxygen in ischemic neurons choice of cell death pathway, conditions corresponding to different areas of insult were reproduced in vitro in the model of surviving brain cortex tissue slices. Three zones were modeled in vitro by varying glucose and oxygen concentration in surviving slices incubation media. Modeled ischemic area I (MIA I) was corresponded to the center of suggested ischemic zone where the levels of glucose and oxygen were considered to be extremely low. MIA II was assigned as intermediate area where oxygen concentration was still very low but glucose was present (this area was also divided into two sub-areas MIA IIa and MIA IIb with physiologically low (5mM) and normal (10mM) level of glucose respectively). MIA III was considered as a periphery area where glucose concentration was close to physiological level and high level of ROS production had been induced by reoxygenation after anoxia. Analysis of molecular mechanisms of cell death in MIA I, IIa, IIb and III was carried out. Cell death in MIA I was found to proceed by necrotic manner. Apoptosis characterized by cyt c release, caspase 3 activation and internucleosomal DNA fragmentation was observed in MIA III. Cell death in MIA II was accompanied by several (not all) hallmarks of apoptosis. Mechanisms of cell death in MIA IIa and MIA IIb were found to be different. Internucleosomal DNA fragmentation in MIA IIa but not in MIA IIb was sensitive to glycine (5mM), inhibitor of NMDA receptor MK-801 (10microM) and PTP inhibitor cyclosporine A (10microM). Activation of caspase 3 was detected in MIA IIb but not in MIA IIa. However cytochrome c release was observed neither in MIA IIa nor in MIA IIb. In MIAs II-III apoptosis was accompanied by uncoupling of oxidative phosphorylation, which was induced by rise of intracellular Ca(2+) and intensive ROS production. Results obtained in present study allow us to propose existence of at least four molecular pathways of cell death development in brain ischemic zone. The choice of cell death pathway is determined by oxygen and glucose concentration in the particular area of the ischemic zone.

A computational model of rat cerebral blood flow using non-uniform rational B-splines.

Non-Uniform Rational B-splines (NURBS) surfaces can be used for a computer simulation of shapes. Some anatomical models of human or animal structures have been recently developed on that basis. We used positron-emission tomography (PET) and computed tomography (CT) data for NURBS modeling of anatomical structures and isotope uptake in the rat brain. Our simplified model of the rat cerebral blood flow is the first step in a larger project aiming a simulation of PET scans in small animals followed by its validation in vivo.

Application of rigid body mechanics to theoretical description of rotation within F0F1-ATP synthase.

ATP synthase catalyses the formation of ATP from ADP and P(i) and is powered by the diffusion of protons throughout membranes down the proton electrochemical gradient. The protein consists of a water-soluble F(1) and a transmembrane F(0) proton transporter part. It was previously shown that the ring of membrane subunits rotates past a fixed subunit during catalytic cycle of the enzyme. However, many parameters of this movement are still unknown. In the present study the mutual protein movement in the membrane part of F(0)F(1)-ATP syntase has been analysed within the framework of rigid body mechanics. On the base of available experimental data it was shown that electrostatic interaction of two charged amino acids residues is able to supply quite enough energy for the rotation. The initial torque, which caused the rotation, was estimated as 3.7 pN nm and for this pattern the angular movement of c subunits complex could not physically have a period less than 10(-9)s. If membrane viscosity and elastic resistance were taken into account then the time of a whole turnover could rise up to 6.3 x 10(-3)s. It is remarkable that rotation will take place only under condition when the elasticity (Young's) module of the central stalk (gamma subunit and other minor subunits) is less than 5.0 x 10(7)N/m(2). Thus, for generally accepted structural parameters of ATP synthase, two-charge electrostatic interaction model does not permit rotation of the rotor if elastic properties of the central stalk are tougher than mentioned above. In order to explain the rotation under that condition one should either suppose a shorter distance between subunit a and c subunits complex or assume interaction of more than two charged amino acids residues.