Photodynamic inactivation of gramicidin channels: a flash-photolysis study

AbstractPhotosensitized inactivation of ionic channels formed by gramicidin in the planar bilayer lipid membrane (BLM) has been studied upon exposure of the BLM to single flashes of visible light in the presence of tetrasulphonated aluminium phthalocyanine. The gramicidin photoinactivation is inhibited by the addition of unsaturated phospholipids to the membrane-forming solution as well as by the addition of azide to the bathing solution, consistent with involvement of singlet oxygen. The characteristic time of the photoinactivation (T) does not change markedly under these conditions. Moreover, T remains nearly constant upon alteration of the flash energy and the photosensitizer concentration. The value of T appears to be sensitive to the gramicidin concentration and to the factors affecting the open time of the gramicidin channels, namely the temperature and the solvent used in the membrane-forming solution. The photoinactivation is not observed with covalent gramicidin dimers. The equations derived from the model of Bamberg and Laeuger (J. Membrane Biol. (1973) 11, 177–194), describing the relaxation of the gramicidin-induced conductance after a sudden distortion of the dimer-monomer equilibrium, are shown to explain consistently the time course of the photoinactivation provided that the damage of the gramicidin molecules leads to deviation from the equilibrium

SPECIAL ASPECTS OF THE SECOND FOREIGN LANGUAGE TEACHING AT THE BASIC COURSE

The aim of this work is to define special aspects of the second foreign language (SFL) grammar teaching at the basic course, because at Ukrainian universities the number of course hours for studying of the SFL is less than for the first one. This leads to more superficial learning of grammar material. In addition, it was believed that the grammar of the SFL can be taught in the context of the general course. The integration of vocabulary and grammar teaching hampered the thorough understanding of grammatical constructions by the students, as the study of the SFL came against the background of the first foreign language enhanced studying. For the scientific analysis of this issue, the methods of causality analysis and generalization were applied, which helped identify the priority of the communicative approach in the SFL grammar teaching, as this approach develops the oral and written skills of students. Nevertheless, there are some factors that impede the fluency of grammar material at the basic course of education. The article reveals problems and ways to solve them during the training. The authors consider that the communicative approach in the SFL grammar teaching will help master the language more quickly and efficiently, which meets the needs of modern society

The interaction of phthalocyanine with planar lipid bilayers Photodynamic inactivation of gramicidin channels

AbstractThe effect of phthalocyanines, the potent photodynamic sensitizers, on the electric properties of the bilayer lipid membrane (BLM) is studied. It is shown, that tetrasulfonated, as well as trisulfonated, aluminium phthalocyanine do not alter the conductance of BLM, but elicit certain changes in the boundary potential difference, which points in favor of dye adsorption on BLM. Under the conditions of intense visible light irradiation, the phthalocyanines cause an increase in the conductance, resulting in the irreversible breakdown of BLM, formed from soy bean phosphati-dylcholine, but fail to change the conductance of BLM, formed from diphytanoilphosphatidylcholine. The phthalocyanine-sensitized inactivation of gramicidin channels incorporated into BLM is observed under the conditions of weak visible light irradiation using an He-Ne laser. The photodynamic blockage of model ionic channels is considerably suppressed after oxygen depletion. The phenomenon consists of a marked reduction of a number of open channels, probably due to photomodification of tryptophan residues, essential for gramicidin functioning. The mechanism of the channel inactivation, involving the photosensitized reaction of the II type, and the relevance to the interaction of sensitizers with biomembranes, is discussed

Tandem Gramicidin Channels Cross-linked by Streptavidin

The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053–13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization

Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin

AbstractMitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria

Technological mineralogy: development of a comprehensive assessment of titanium ores (exemplified by the Pizhemskoye deposit)

Technological mineralogy of titanium ores is the basis for assessing their complexity. It enables, from a unified standpoint, to trace the entire course of changes in mineral matter through operating procedures, including beneficiation, processing, and obtaining target industrial products. The study targets are Pizhemskoye ilmenite-leucoxene sandstones, which are distinguished by a complex polymineral composition. Along with the main ore components, there are other metals with different speciation (isomorphic admixture, independent mineral phases). The optimal set of mineralogical analysis methods for the predictive assessment of their further use is substantiated exemplified by titanium ores of the Pizhemskoye deposit, which are complex, noted for a variable content of iron oxides and contain rare earth metals. Examinations by X-ray phase analysis and scanning electron microscopy confirm that the main titanium phases of sandstones are pseudorutile and a polymineral aggregate, “leucoxene”. Considering the granulometric peculiarities of the magnetic and non-magnetic fractions of the gravity concentrate, the prospects of technologies for processing titanium raw materials are discussed. Along with the problems of obtaining high-quality raw materials, the transformations of mineral phases as a result of extreme impacts and their physicochemical properties as a consequence of isomorphic substitution of a part of Ti atoms with natural modifier agents (Fe and V) in the synthesis of titanium oxide nanostructures for industrial applications are considered (photocatalytic nanoreactor)

Abstract P-28: Structural Dynamics of DNA-Associated Chaperon Facilitates Chromatine Transcription

Background: Histone chaperon FACT (“FAcilitates Chromatin Transcription”) is a multifunctional and conserved eukaryotic protein involved in DNA transcription, replication and repair; which can reversibly unfold nucleosomes in presence of ATP. FACT is necessary for the viability and growth of breast tumor cells meanwhile in normal cells it can be knocked out without loss of vitality. Human FACT (hFACT) is a target for promising anticancer drug curaxins, which causes FACT trapping in chromatin of cancer cells and destabilizes the nucleosome. The nucleosome-unfolding activity is an important function of hFACT in vivo; however, the mechanism of FACT-dependent nucleosome unfolding remains unknown. Methods: Here, we studied negative stained hFACT structure using single particle electron microscopy using JEOL 2100 TEM. Micrographs were captured with 25k magnification, and 4.1 Å pixel size. EM images pre-processing and single particles collection were performed in EMAN2.3, followed by 2D-particles analysis in RELION2.0. Final 2D-classes included ~70 000 single particles images. Results: Based on 2D-classess data analysis we evaluated several states of hFACT reflecting its conformational flexibility: the “closed” complex is characterized by four domains localized close to each other and forming a compact structure; “intermediate” state represented by classes with identified three domains having compact structure and more disordered fourth domain, and the “open” complex, represented by three domains forming almost linear structure. The “closed” and “open” states are present in comparable amounts and significantly outnumber the “intermediate” state. It has been shown that hFACT domains are connected through flexible linkers and SPT16 and SSRP1 dimerization domains (DDs) form the “joint”-like connection between the two subunits. In the “сlosed” conformation, the DNA-binding surface of FACT is covered by its two C-terminal and middle domains (MDs). The N-terminal domain (NTD) of SPT16 was not resolved previously, but it is the best candidate for the forth domain that is clearly visible only in the “closed” conformation of hFACT, based on its dimensions and the longest linker length. Conclusion: We propose that during conversion to the “open” complexes SPT16 NTD is moving away from the other subunits leading to formation of the first intermediate state with the NTD domain poorly resolved or not resolved, while less mobile DDs and MDs maintain more compact structure and the DNA-binding site is still protected by the CTDs. In the “open” state SPT16/SSRP1 visible MDs and DDs form almost linear structure, unmasking the DNA-binding sites and making them accessible for the interaction with a nucleosome

The Importance of Clinical and Biochemical Markers in the Diagnosis of Disorders of the Psychomotor and Physical Development of Children who Underwent Perinatal CNS Lesions

The purpose of this study was to evaluate the blood level of neurotrophic factors (S100 protein, nerve growth factor, L- homocysteine and angiotensin II) in the diagnosis of disorders of the psychomotor and physical development of children who underwent perinatal CNS lesions. A comprehensive approach to assessing the blood levels of neurotrophic factors, along with methods of neuroimaging in children in the first 6 months of the postnatal period, allows identification of the total result of multidirectional degenerative-reparative processes in the neurovascular components of CNS and construction of the diagnostically significant criteria of the severity of neuropathology, which determines the physical development of children in the first year of life