Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.     

Biological and polymeric self-assembled hybrid systems: structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Sodium channel activators: model of binding inside the pore and a possible mechanism of action

Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state.     

Potentialities of modern clinical X-ray radiology in the differential diagnosis of tumor and other obstructive diseases of the large bowel

The misdiagnosis rate in defining the cause of obstructive colonic disease is 8.2-24.4%. This is consistent with the fact that every 5 patients with colonic obstruction present difficulties in establishing the nature of a pathological process. The paper provides the results of analysis of clinical and X-ray symptoms in 350 patients with difficult differentially diagnosed cases of narrowing of the rectum and colon. Based on the analysis, the authors identified the basically important X-ray signs that might differentiate tumor stenoses from other obstructive diseases. They also defined the specific X-ray signs of such diseases as infiltrative cancer; extraintestinal cancer involved in the large bowel; inflammatory strictures in ulcerative colitis, diverticulosis, actinomycosis, tuberculosis, intestinal endometriosis, invagination, and other obstructive diseases. The developed differentiated diagnostic criteria could enhance the overall accuracy of X-ray study in this difficult group of patients from 72.7-80% to 93%.     

Biological factors underlying regularity and chaos in aquatic ecosystems: Simple models of complex dynamics

This work is focused on the processes underlying the dynamics of spatially inhomogeneous plankton communities. We demonstrate that reaction-diffusion mathematical models are an appropriate tool for searching and understanding basic mechanisms of complex spatio-temporal plankton dynamics and fractal properties of planktivorous fish school walks.     

Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains

The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels (n-SASL) and their methylated derivatives (n-MeSASL), labelled at different positions of their doxyl group (n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from -50 to +20 degrees C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.     

Kinetics of photoinduced oxidative-reductive transformation of the reaction center P700 in leaves of C3- and C4-plants

The light-induced changes in the kinetics of the oxidation of photosystem I reaction center P700 in leaves of C3 and C4 plants, depending on the time of leaf adaptation to dark before the illumination were studied. The EPR 1 signal from oxidized centers P+700 was used to monitor the kinetics of P700 redox transients. Similar dependences of the light-induced changes in the kinetics of P700 versus the adaptation time were observed in leaves of three kinds of plants: bean, orange and maize. These data suggest that the deactivation of the Calvin cycle enzymes, which occurs 1-3 min after ceasing the illumination, is the main factor that causes the retardation of the light-induced oxidation of P700.     

Changes in induction of slow fluorescence in leaves of maple located near urban highways

The slow fluorescence induction parameter FM/FT was measured for green leaves from maple trees grown in the park, depending on the distance (5-65 m) between the tree and the highway cross. We concluded that the decrease in the value of FM/FT for trees grown in the vicinity of the road cross was caused by exhaust gases.     

Electron and proton transport in chloroplasts taking into account lateral heterogeneity of thylakoids. Mathematical model]

A mathematical model of a chloroplast was constructed, which takes into account the inhomogeneous distribution of complexes of photosystems I and II between granal and intergranal thylakoids. The structural and functional complexes of photosystems I and II, which are localized in intergranal and granal thylakoids, respectively, and the b/f complex, which is uniformly distributed in thylakoid membranes, are assumed to be immobile. The interactions between spatially distant electron transport complexes are provided by plastoquinone and plastocyanine, which diffuse in the thylakoid membrane and intrathylakoid space, respectively. The main stages of proton transport associated with the functioning of photosystem II and oxidation-reduction transformations of plastoquinone are considered. The model takes into account the interactions of protons with membrane-bound buffer groups, the lateral diffusion of hydrogen ions in the intrathylakoid space and in the lumen between adjacent granal thylakoids, and the transmembrane proton transport associated with the function of ATP synthase and passive leakage of protons from thylakoids outside. The numerical integration of two systems of differential equations describing the behavior of some variables in two different regions: granal and intergranal thylakoids was performed. The model describes adequately the kinetics of processes being studied and predicts the occurrence of inhomogeneous lateral profiles of proton potentials and redox state of electron carriers. Modeling the electron and proton transport with allowance for the topological features of chloroplasts (lateral heterogeneity of thylakoids) is important for correct interpretation of "power-flux" interactions and the experimentally measured kinetic parameters averaged over the entire spatially inhomogeneous thylakoid system.     

Structural domains and matrix attachment regions along colinear chromosomal segments of maize and sorghum

Although a gene's location can greatly influence its expression, genome sequencing has shown that orthologous genes may exist in very different environments in the genomes of closely related species. Four genes in the maize alcohol dehydrogenase (adh1) region represent solitary genes dispersed among large repetitive blocks, whereas the orthologous genes in sorghum are located in a different setting surrounded by low-copy-number DNAs. A specific class of DNA sequences, matrix attachment regions (MARs), was found to be in comparable positions in the two species, often flanking individual genes. If these MARs define structural domains, then the orthologous genes in maize and sorghum should experience similar chromatin environments. In addition, MARs were divided into two groups, based on the competitive affinity of their association with the matrix. The "durable" MARs retained matrix associations at the highest concentrations of competitor DNA. Most of the durable MARs mapped outside genes, defining the borders of putative chromatin loops. The "unstable" MARs lost their association with the matrix under similar competitor conditions and mapped mainly within introns. These results suggest that MARs possess both domain-defining and regulatory roles. Miniature inverted repeat transposable elements (MITEs) often were found on the same fragments as the MARs. Our studies showed that many MITEs can bind to isolated nuclear matrices, suggesting that MITEs may function as MARs in vivo.