Чинники ефективності антикризового управління суб'єктами господарювання в економіці України

У статті розглядаються проблеми формування підходів організації антикризового управління суб'єктами господарювання в економіці України. На ґрунті вітчизняного та зарубіжного досвіду й результатів власних досліджень автора запропоновано психологічний тип антикризового менеджера. (The article is devoted to the problems of forming of approaches of organization of anticrisis management by the subjects of menage in the economy of Ukraine. On the base of domestic and foreign experience and results of own researches of author the psychological type of anticrisis manager is offered.

The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis

Background/Aims: The conformation, folding and lipid binding properties of the intestinal fatty acid binding proteins (IFABP) have been extensively investigated. In contrast, the functional aspects of these proteins are not understood and matter of debates. In this study, we aim to address the deleterious effects of FA overload on cellular components, particularly mitochondria; and how IFABP helps in combating this stress by restoring the mitochondrial dynamics. Methods: In the present study the functional aspect of IFABP under conditions of lipid stress was studied by a string of extensive in-cell studies; flow cytometry by fluorescence-activated cell sorting (FACS), confocal imaging, western blotting and quantitative real time PCR. We deployed ectopic expression of IFABP in rescuing cells under the condition of lipid stress. Again in order to unveil the mechanistic insights of functional traits, we arrayed extensive computational approaches by means of studying centrality calculations along with protein-protein association and ligand induced cluster dissociation. While addressing its functional importance, we used FCS and in-silico computational analyses, to show the structural distribution and the underlying mechanism of IFABP’s action. Results: Ectopic expression of IFABP in HeLa cells has been found to rescue mitochondrial morphological dynamics and restore membrane potential, partially preventing apoptotic damage induced by the increased FAs. These findings have been further validated in the functionally relevant intestinal Caco-2 cells, where the native expression of IFABP protects mitochondrial morphology from abrogation induced by FA overload. However, this native level expression is insufficient to protect against apoptotic cell death, which is rescued, at least partially in cells overexpressing IFABP. In addition, shRNA mediated IFABP knockdown in Caco-2 cells compromises mitochondrial dynamics and switches on intrinsic apoptotic pathways under FA-induced metabolic stress. Conclusion: To summarize, the present study implicates functional significance of IFABP in controlling ligand-induced damage in mitochondrial dynamics and apoptosis

The Presence of the Iron-Sulfur Motif Is Important for the Conformational Stability of the Antiviral Protein, Viperin

Viperin, an antiviral protein, has been shown to contain a CX3CX2C motif, which is conserved in the radical S-adenosyl-methionine (SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α -β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational stability of the mutant proteins may have direct relevance to the absence of their antiviral activity

Effects of Arginine and Other Solution Additives on the Self-Association of Different Surfactants: An Investigation at Single-Molecule Resolution

Fluorescence correlation spectroscopy is used to monitor the self-association of SDS and DTAB monomers at single-molecule resolution. Tetramethylrhodamine-5-maleimide (TMR) has been chosen as a probe because rhodamine dyes have been shown to bind surfactant micelles. Correlation functions obtained by FCS experiments have been fit using conventional discrete diffusional component analysis as well as the more recent maximum entropy method (MEM). Hydrodynamic radii calculated from the diffusion time values increase with surfactant concentration as the monomers self-associate. Effects of several solution additives on the self-association property of the surfactants have been studied. Urea and glycerol inhibit self-association, and arginine shows a dual nature. With SDS, arginine favors self-association, and with DTAB, it inhibits micelle formation. We propose surfactant self-association to be a “supersimplified” model of protein aggregation

Studies of Protein Folding and Misfolding Using Fluorescence Correlation Spectroscopy (FCS) and other Biophysical Methods

Parkinson‟s disease (PD) is a multifactorial movement disorder (resting of tremor, rigidity, Bradykinesia), in which both genetic as well as environmental factors play important roles. Several evidences have been accumulated which implies that the aggregation of α-synuclein is a critical factor in PD manifestation. Rare familial cases of PD have been associated with mutations in α-synuclein even though the function of α-synuclein is still completely unknown. The primary structure of purified α-synuclein, exists as a “natively unfolded” protein although a recent report raises some controversies over the native structure of protein. A number of different conformational states of α-synuclein including partially-folded (key intermediate in aggregation and fibrillation), oligomeric species, as well as fibrillar and amorphous aggregates have been observed. A number of factors that either accelerate or inhibit the rate of fibrillation have been observed in vitro. Accelerators include environmental factors (certain pesticides and metals), molecular crowding and various natural and synthetic charged polymers. α‑synuclein (α‑syn) aggregation can take place either in the cytoplasm or when in association with the cellular membrane. In the cytosol, unfolded monomers interact to form oligomers of varying morphologies that eventually gets converted in to fibrils. The accumulation of these amyloid fibrils leads to the formation of intracellular inclusions called Lewy bodies. Membrane bound monomeric α‑syn adopts a predominantly α‑helical conformation and it undergoes a conformational change via oligomerization to eventually form membrane bound β‑sheet‑rich structures that self‑associate to form trans‑membrane amyloid pores and fibrils. During α‑syn fibrillogenesis and aggregation, the intermediate species (oligomers and amyloid fibrils) are highly toxic, affecting mitochondrial function, endoplasmic reticulum–Golgi trafficking, protein degradation and/or synaptic transmission, all of which are thought to induce the process of neurodegeneration. We have aimed to establish the aggregation mechanism of alpha synuclein in membrane as well as in cytosol. With the help of mainly single molecule fluorescence spectroscopy, a number of heterogeneous intermediates are indentified in the presence of membrane or membrane mimics. In this case, the secondary and tertiary structural changes, dynamic fluctuations (extended and compact), and accumulation of aggregation prone species are observed in order to understand the structural features and contribution of such conformations in the formation of amyloid aggregates. Interestingly, the aggregation mechanism and the structural features of amyloid fibrils in WT and familial mutants of alpha synuclein are different. This indicates that there is a correlation between conformational changes in intermediates and the rate of aggregation. But in the case of cytosol, the stability and aggregation mechanism of a protein cannot be measured with the help of conventional methods as the viscosity and available space (i.e.; excluded volume) severely alters and hence affects the single molecule detection. Here we have developed a strategy that employs „two-diffusion fit‟ and „effective medium model‟ to evaluate the conformation of proteins in the presence of viscous medium like polyacrylamide gel. With the help of this model, we were able to measure the conformation of proteins with accuracy even under viscous conditions. We could also explain the stability of protein as a function of excluded volume effect. The structural deviation of a-syn from that of globular proteins have been shown to deviate more under viscous conditions than under solution conditions, which could be explained more clearly by using this method. The proper monitoring of heterogeneous conformations that are generated during the aggregation process of alpha synuclein in membrane and cytosol could be enabled which may pave way for newer therapeutics applications and may help in revealing the mystery of protein stability in cellular environments

Nanoparticle-Protein Interaction Studies Using Spectroscopic and Biophysical Methods

The interaction of nanoparticles with biological interfaces has been studied extensively, where it has been found that proteins form the most abundant class of biomolecules interacting with nanoparticles. Also, nanoparticles due to the huge potential that they offer are being constantly scrutinized for their role in various aspects of biomedical, therapeutic and biotechnological applications. As a matter of fact, the exposure of such particulate matter to living systems is increasing exponentially, raising serious concerns related to their safety. The toxicity due to nanoparticles may arise due to their potential to interact with any biological system, even crossing the most protected blood brain barrier. In addition, they can induce conformational changes in protein, can alter the fluidity of membranes, and may release reactive oxygen species (ROS) and cause severe damage to DNA. Since the potential of nanoparticles are luring, such deleterious manifestations need to be alleviated in order to tap the resource that nanoparticles provide. This is a huge challenge, since nanoparticles are as complex in structure as proteins. Here, we have developed a novel synthetic approach for the surface modification of one of the most biocompatible magnetite nanoparticles with a more biocompatible surface modifying agent, poly(ethylene) glycol. The stability and the efficiency of the new procedure have been extensively analysed. The interaction of magnetite nanoparticles with different protein systems has been taken in to account. It was observed that bare magnetite nanoparticles affect the proteins differently than that of the surface modified ones. In one of the protein systems, the bare magnetite nanoparticles affect the redox state of the protein; induce intermediate formation and leads to aggregation. The surface modification of nanoparticles maintains the redox state and prevents the aggregation of the protein. In another protein system, the early aggregation kinetics was analysed in the presence of bare and surface modified nanoparticles. Whereas, bare nanoparticles speeds up the aggregation rate, surface modified nanoparticles are found to delay the rate of aggregation

Osmolytes ameliorate the effects of stress in the absence of the heat shock protein Hsp104 in Saccharomyces cerevisiae.

Aggregation of the prion protein has strong implications in the human prion disease. Sup35p is a yeast prion, and has been used as a model protein to study the disease mechanism. We have studied the pattern of Sup35p aggregation inside live yeast cells under stress, by using confocal microscopy, fluorescence activated cell sorting and western blotting. Heat shock proteins are a family of proteins that are produced by yeast cells in response to exposure to stressful conditions. Many of the proteins behave as chaperones to combat stress-induced protein misfolding and aggregation. In spite of this, yeast also produce small molecules called osmolytes during stress. In our work, we tried to find the reason as to why yeast produce osmolytes and showed that the osmolytes are paramount to ameliorate the long-term effects of lethal stress in Saccharomyces cerevisiae, either in the presence or absence of Hsp104p