[Yersinia pseudotuberculosis mutant OmpF porins with deletions of the external loops: genetic constructions design, expression, isolation and refolding].

Yersinia pseudotuberculosis outer membrane (OM) recombinant mutant OmpF porins with deletions of the external loops L1, L6 and L8 were obtained using site-directed mutagenesis of the recombinant plasmid including ompF gene. Heterologeous expression of the mutant proteins was carried out in strain Rosetta of Escherichia coli (Novagen, USA), porins with the deletions were isolated from the inclusion bodies. Mutant proteins in oligomeric form were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was demonstrated to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. Lacking of the loops L1, L6 and L8 didn't affect the conductivity level of Y pseudotuberculosis porin channel as shown using bilayer lipid membrane (BLM) technique. Lacking of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated with use of immunoblotting technique and ELISA.

[Effect of lantibiotic warnerin on lipid bilayer membranes].

The effect of the lantibiotic warnerin on the ionic permeability of artificial membranes has been studied. Membranes were composed of different lipid fractions, including lipids isolated from warnerin-sensitive cells of Staphylococcus epidermidis. It was shown that warnerin selectively interacts with artificial membranes of different lipid composition, which leads, in some cases, to the formation of ionic channels. A computer model of the spatial structure of warnerin has been coustructed, which supports a high probability of the membranotropic activity of this peptide.

[Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane].

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins].

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6-8 degrees C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the beta-structural proteins (they have 59-96% total beta structures and 0-17% alpha helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl beta-D-glucoside for SDS: the content of beta structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.

[Actinoporins from the Sea of Japan anemone Oulactis orientalis: isolation and partial characterization].

Two cytolytic toxins (cytolysins Or-A and Or-G) were isolated from the Sea of Japan anemone Oulactis orientalis and characterized. Their purification scheme involved a hydrophobic chromatography on Polychrom 1, a gel filtration on Akrilex P-4, a cation-exchange chromatography on CM-32 cellulose, and a reversed-phase HPLC on a Nucleosil C18 column. The molecular masses of Or-A and Or-G were determined by SDS-PAGE in 14% PAG to be ca. 18 kDa. The absence of Cys residues and a high content of basic amino acid residues are characteristic of their amino acid compositions. The hemolytic activities of Or-A and Or-G were found to be 295.86 and 322.58 HU/mg, respectively; these are by three orders of magnitude lower than those of sphingomyelin-inhibitable cytolysins from the tropic sea anemones. The amino acid sequences of the N-terminal fragments of Or-A and Or-G were determined to be ATFRVLAK and GAIIAGAA, respectively. Action of the cytolysins on the erythrocyte membrane is inhibited by exogenous sphingomyelin. They form ion channels in bilayer lipid membranes with the conductivity of 16, 32, and 40 pSm in 0.1 M NaCl and 168, 240, and 320 pSm in 1 M NaCl at pH 7.2. Therefore, they were attributed to the group of actinoporins.

[Effect of extractive substances and total glycoside fraction from Panax ginseng C. A. Meyer on root growth of Cucumis sativus L. seedlings]. / Vliianie ékstraktivnykh veshchestv i summarnoi glikozidnoi fraktsii Panax ginseng C. A. Meyer na rost kornia prorostkov Cucumis sativus L.

We studied the effect of extractive substances (ES) and total glycoside fraction (TGF) from Panax ginseng on plant cell growth. The seeds of cucumber Cucumis sativus L., KIT variety, were used for the biological assay. The tested substances inhibited the primary root growth in Cucumis sativus seedlings. Actively growing seedlings were most sensitive to this effect.

[Effect of a lipopolysaccharide on the conformational state and functional activity of a Yersinia pseudotuberculosis porin]. / Vliianie lipopolisakharida na konformatsionnoe sostoianie i funktsional'nuiu aktivnost' porina iz Yersinia pseudotuberculosis.

Changes in the structure and functional activity of porin, a protein from Yersinia pseudotuberculosis, resulting from the removal of lipopolysaccharide (LPS) normally bound with the protein were studied. The treatment of LPS-containing porin with a 30% SDS solution led to an LPS-free protein that, according to the SDS-PAGE, remained to be a trimer. It was shown by CD and UV spectroscopies and intrinsic protein fluorescence that the removal of LPS caused only conformational changes in the porin secondary and tertiary structures. The LPS-free porin folded into a completely beta-structured protein aggregate. The bilayer lipid membrane technique showed that the pore-forming activity of the LPS-free porin decreased, and its concentration should be increased by two orders of magnitude to achieve the same effect. Incubation of the LPS-free porin with LPS led to a porin-LPS complex and affected the character of the protein functional activity. The treatment of the LPS-free porin by octyl glucoside, a nonionic detergent, resulted in the restoration of the protein pore-forming activity. It was suggested that the LPS and detergent provide a definite protein conformation necessary for its functioning.

[Effect of the method of extracting the pore-forming protein from Yersinia pseudotuberculosis on its macromolecular organization]. / Vliianie sposoba ékstrktsii poroobrazuiushchego belka iz Yersinia pseudotuberculosis na ego makromolekuliarnuiu organizatsiiu.

By means of physico-chemical methods, lipid bilayer reconstitution and immunoenzyme assay, macromolecular organization of porin oligomers from Yersinia pseudotuberculosis, isolated by two extraction methods, was studied. Use of SDS and high temperature in the course of the extraction led to a partial denaturation of porin trimers at the level of the tertiary structure, these conformational changes affecting the porin's pore-forming activity and antigenic structure. At the same time, the partially denatured trimers are as stable under the treatment of urea and guanidine hydrochloride as the native protein.

[Features of a protein component of the endotoxin from Yersinia pseudotuberculosis]. / Kharakteristika belkovogo komponenta éndotoksina iz Yersinia pseudotuberculosis.

The protein moiety of endotoxin from Yersinia pseudotuberculosis was found to consist of two polypeptides with apparent molecular masses 40 and 14.5 kDa (4:1 w/w). The major protein (40 kDa) was isolated from the endotoxin pretreated with sodium deoxy cholate by gel chromatography on the Sephadex G-200 column. Comparative study of this protein and oligomeric form of porin from the outer membrane of Y. pseudotuberculosis using SDS--PAGE, velocity sedimentation, lipid bilayer experiments, chemical and serological analyses revealed their identity. The deoxycholate treatment of the endotoxin does not affect complexes of the major protein and LPS.

[The mechanism of the interaction of the pH-dependent cytostatic cauloside C with the membranes of tumor cells and liposomes]. / Mekhanizm vzaimodeistviia pH-zavisimogo tsitostatika kaulozida C s memranami opukholevykh kletok i liposom.

The effect of the medium pH on accumulation of [3H]-cauloside C by tumor cells, its intracellular localization, and interaction of the glicoside with membranes of tumor cells and liposomes has been studied. The shift towards weakly acids pH leads to the increase in the amount of cauloside C accumulated by tumor cells and changes the pattern of interaction of cauloside C with the membranes.

[Formation of the complex of the triterpene glycoside holothurin A with cholesterol in liposomal membranes]. / Obrazovanie kompleksa triterpenovogo glikozida goloturina A s kholesterinom v liposomal'nykh membranakh.

Interaction between holothurin A triterpene glycoside and lipid-cholesterol liposomes was studied by differential scanning microcalorimetry. Partial restoration of the peak of basic phase transition of dipalmitoyl phosphatidyl choline was shown to be related to the formation of holothurin A (in the membrane)-cholesterol complex. The data obtained are in favor of "sterol" hypothesis of the mechanism of membrane-tropic action of holothurin A.

[Effect of triterpene glycosides on the stability of bilayer lipid membranes, containing different sterols]. / Vliianie triterpenovykh glikozidov na stabil'nost' bisloinykh lipidnykh membran, soderzhashchikh razlichnye steriny.

Low concentrations of triterpene glycosides: holothurin A, stichoposide A and cauloside C sharply change the stability of bilayer lipid-sterine membranes. The glycosides activity decreases in the line: holothurin A, stichoposide A, cauloside C. The effective doses of glycosides are to a great extent determined by structural peculiarities of sterines which compose the films. A correlation was observed between the effect of triterpene glycosides on the stability of model bilayer membranes and their physiological activity. The model lipid-sterine membranes can be used in the primary screening of triterpene glycosides to estimate their physiological activity.

[Effect of pH on the properties of planar lipid bilayers of cholesterol and alpha-monolaurine]. / Vliianie pH na svoistva ploskikh bisloinykh lipidnykh membran iz kholesterina i alpha-monolaurina.

It is discovered that pH of liquid electrolyte influences the structure, stability and conductance of bilayer lipid membranes (BLM) formed from cholesterol-alpha-monolaurine mixture in n-octane. The most stable BLM are formed at cholesterol-alpha-monolaurine weight ratio 1:1, BLM specific resistance being maximal and their thickness minimal (2.9 nm). A sharp reduction of BLM stability is noted when pH is deviated in acidic or alkaline medium. The obtained data are discussed from the viewpoint of adsorbed charge influence on the structure of BLM formed of uncharged lipids.