Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that - at a subsequent stage of transfection into yeast cells - it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein's secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 µg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

Correction to: Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

The original publication has been updated. The acknowledgment was omitted from the original article and is published below.

Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

Non-glycosylated, recombinant human granulocyte colony-stimulating factor (rhG-CSF), produced by Escherichia coli (filgrastim, leukostim) is widely used to treat a number of serious human diseases and aids in the recovery post bone marrow transplantation. Although glycosylation is not required for the manifestation of the biological activity of G-CSF, a number of studies have shown that the carbohydrate residue significantly increases the physicochemical stability of the G-CSF molecule. Therefore, the aim of the present study was to design a Pichia pastoris strain capable of producing glycosylated rhG-CSF, and to study its effects on rat bone marrow cells. The nucleotide sequence of the rhG-CSF gene has been optimized for expression in P. pastoris, synthesized, cloned into the pPICZαA vector and expressed under the control of the AOX promoter in P. pastoris X33. One of the selected clones secreting rhG-CSF, produced 100-120 mg/l of rhG-CSF three days post-induction with methanol. The recombinant cytokine was purified using two-step, ion-exchange chromatography. The final yield of purified G-CSF was 35 mg/L of culture medium. The biological activity of rhG-CSF was examined in rat bone marrow cells. The P. pastoris strain was designed to produce relatively high levels of rhG-CSF. The rhG-CSF protein had a strong stimulating effect on the growth of rat bone marrow cells, which was comparable to that of the commercial drug leukostim, but showed a more persistent effect on granulocyte cells and monocyte sprouts, enabling the enhanced maintenance of the viability of the cells into the 4th day of incubation.

Energy surface and minimum energy paths for Fréedericksz transitions in bistable cholesteric liquid crystals.

The multidimensional energy surface of a cholesteric liquid crystal in a planar cell is investigated as a function of spherical coordinates determining the director orientation. Minima on the energy surface correspond to the stable states with particular director distribution. External electric and magnetic fields deform the energy surface and positions of minima. It can lead to the transitions between states, known as the Fréedericksz effect. Transitions can be continuous or discontinuous depending on parameters of the liquid crystal which determine an energy surface. In a case of discontinuous transition when a barrier between stable states is comparable with the thermal energy, the activation transitions may occur, and it leads to the modification of characteristics of the Fréedericksz effect with temperature without explicit temperature dependencies of liquid crystal parameters. A minimum energy path between stable states on the energy surface for the Fréedericksz transition is found using the geodesic nudged elastic band method. Knowledge of this path, which has maximal statistical weight among all other paths, gives the information about a barrier between stable states and configuration of director orientation during the transition. It also allows one to estimate the stability of states with respect to the thermal fluctuations and their lifetime when the system is close to the Fréedericksz transition.

Anisotropy of electrostatic interaction in smectic-C

The contribution to the free energy of distortion of the ferroelectric smectic-C^{*} due to the electrostatic interaction of polarization charges is calculated. These calculations are performed by accounting for the anisotropy of the permittivity, which is essential for smectic-C^{*}. Fluctuations of the c director in an external electric field are considered. It is shown that the anisotropy of the permittivity strongly affects the interaction of the polarization charges, the spectrum orientation fluctuations, and the angular dependence of the light scattering intensity.

Simulation of radiation transfer and coherent backscattering in nematic liquid crystals.

We consider the multiple scattering of light by fluctuations of the director in a nematic liquid crystal. Using methods of numerical simulation the peak of the coherent backscattering and the coefficients of anisotropic diffusion are calculated. The calculations were carried out without simplifying assumptions on the properties of the liquid crystal. The process of multiple scattering was simulated as a random walk of photons in the medium. We investigated in detail the transition to the diffusion regime. The dependence of the diffusion coefficients on the applied magnetic field and the wavelength of light were studied. The results of simulation showed a nonmonotonic dependence of the diffusion coefficients on the external magnetic field. For calculation of the peak of the coherent backscattering we used the semianalytical approach as long as in nematic liquid crystals this peak is extremely narrow. The parameters of the backscattering peak and of diffusion coefficients which were found in numerical simulations were compared with the experimental data and the results of analytical calculation.

First-order and continuous Fréedericksz transitions in cholesteric liquid crystals.

The Fréedericksz transition in twist cells of cholesteric liquid crystals with a finite surface energy is considered. It is shown that this transition can be either of the second order or of the first order depending on the values of the Frank constants, pitch, surface energies, and the cell thickness. A simple criterion that determines the order of the phase transition is obtained. By numerically minimizing the free energy of the liquid-crystal pattern the distribution of the director in the presence of the external electric field is calculated. For this purpose the polar angle of the director was presented as a partial sum of the Fourier series and of the appropriate function. The azimuthal angle was eliminated using Euler-Lagrange equations. Calculations were performed for different sets of liquid-crystal parameters which provide the phase transition of the first and of the second order. The numerical results are in a good agreement with theoretical formulas based on the Landau-type theory.

[Isolation of expressed in E. coli human interferon beta1b (Ser17) by ion-exchange chromatography].

A method for isolation of interferon beta1b (Serl7) from inclusion bodies, comprising the steps of solution and reduction of protein from the inclusion bodies, refolding, chromatography on DEAE-Sepharose, chromatography on SP-Sepharose, concentrating, desalting and addition of stabilizers. The solution of reduced protein was diluted with pH 8.0 buffer of 50 mM Tris-HCl, 25 microM CuCl2 and 0.5% Twin 20 for refolding. We used gradient of pH (from 9.3 upto 11.3) for elution of interferon-beta from cation-exchange column. We concentrated of eluate and then desalted on the Sephadex G-50 column with 1 mM NaOH. Then the protein solution was neutralized with mannitol and Na-phosphate. Obtained preparation of interferon-beta was pure by gel-electrophoresis and by HPLC analysis, and had practically indentical level of antiproliferative activity with well-known preparation of Betaferone. Thus we show the possibility of isolation and obtaining of pure and active interferone-beta by ion-exchange chromatography in the presence of non-ion detergent Twin 20. We believe this method for interferon betalb preparation is perspective for scaling and using in the develop of industrial technology for production of this preparation.

[Molecularly-genetic aspects of metabolic syndrome].

The practical value of metabolic syndrome is defined by it's great popularity, which becomes an epidemy, sometimes--by fatal complications sideways cardiovascular system and by capability of their prevention in conditions of forehanded diagnostics and individual correction of metabolic disorders. The authors, investigated 76 patients with indications of complete metabolic syndrome, for the first time analyzed genetic factors of risk of cardiovascular diseases and proposed the ways of optimization of diagnostics, prophylaxis and treatment of metabolic syndrome and syndrome diseases.

[Paraneoplastic membranous nephropathy].

The most frequent clinical manifestation of renal lesion in patients with paraneoplastic syndrome is nephrotic syndrome, which is associated with a special morphological type of renal lesion, membranous nephropathy (MN). The latter accompanies various types of cancer. The article contains case reports of patients with paraneoplastic MN, which was coped with after radical antitumoral treatment.

Scattering of light in cholesteric liquid crystals with large pitch.

We consider the problem of light scattering in a slab of cholesteric liquid crystal with the pitch which significantly exceeds the wavelength of light. The electromagnetic wave propagation and the Green's function are investigated for this medium basing on geometric optics approximation. The correlation function of the director fluctuations is calculated with the aid of the vector analog of the WKB approximation. A general approach to treatment of single light scattering in a stratified medium with smoothly varying properties based on the Kirchhoff method is developed. Angular and polarization dependencies of the single light scattering intensity as well as extinction of the mean field are analyzed. Unusual dependence of the light scattering intensity on the size of the system is found.

Description of dynamic x-ray scattering from freely standing smectic-A films.

The consequent description of the x-ray scattering from the free standing smectic-A films is suggested. Calculations are based on the discrete model for the film dynamics. The scattering intensity temporal autocorrelation function is obtained within the framework of this approach neglecting the multiple scattering and refraction effects. It is shown that the behavior of this function crucially depends on the film thickness. In particular, in thin films containing less than 10(3) layers the time dependence of has damping oscillation character. This behavior is determined by an acoustic mode that describes the film motion caused by the action of the surface tension. For thick films containing more than 10(4) layers the dynamics of the intensity temporal autocorrelation function is determined either by an acoustic mode or by a wide spectrum of modes depending on the x-ray geometry. In both the cases the autocorrelation function is a relaxation one. The results obtained are compared with the experiments on the coherent soft and hard x-ray scattering.

Multiple scattering temporal correlation function in a half space with finite-size heterogeneities.

An exact solution for the boundary problem of temporal correlations of light multiply scattered from a medium occupying a half space is found by means of the Wiener-Hopf method, taking into account single-scattering anisotropy. Within the P1 approximation a universal initial decay rate of the temporal correlation function is obtained. For larger time intervals a higher single-scattering anisotropy yields a higher decay rate contrary to predictions of the diffusion approximation. Within the P2 approximation, which takes account of the first- and second-order Legendre polynomials, the solution obtained becomes universal in an expanded temporal range and agrees rather well with the known measurement data.

Dynamic and correlation properties of solid supported smectic-A films.

Dynamic properties and layer displacement-layer displacement correlation functions of the smectic-A films on a substrate are investigated. The eigenfrequencies spectrum and eigenmotions of the film are calculated within the framework of a discrete model. It was found that the static as well as dynamic properties of freely standing and solid supported smectic-A films differ significantly. In particular, the acoustic mode is absent in the films on a substrate and the surface tension is not essential for the film dynamics. The correlation length of the spatial layer displacement correlation functions is finite for films on a substrate in contrast to the free standing films. An effect of thermal fluctuations on the specular and diffuse x-ray scattering was analyzed.

Dynamics of free-standing smectic-A films.

The eigenvibrations and time-dependent layer displacement-layer displacement correlation functions are analyzed in a free-standing thin smectic-A films with the help of a discrete layer model. The film motions are described using the Chebyshev polynomials of second kind, U(n)(x). The eigenfrequencies problem is essentially simplified within the framework of this approach since the numerical solution of the high degree algebraic characteristic equation is replaced by the analytical solution of a rather simple trigonometrical equation. The dependences of eigenmodes on wave number q(perpendicular) were analyzed. For small q(perpendicular) one mode is a low frequency acoustic wave and other modes are high frequency optical oscillations. As the wave number q(perpendicular) increases all modes successively turn into relaxation when starting with the acoustic mode. The rather simple expression for susceptibility matrix and for spectral densities of layer displacement correlation functions were obtained using the Chebyshev polynomials. It was shown that the frequency dependences of spectral densities are sensitive to wave number q(perpendicular). For small q(perpendicular) the spectral densities of displacement-displacement correlation functions have a sharp peak and for large q(perpendicular) they turn into a contours of Lorentzian type.

[Photoinactivation of microorganisms in the presence of photosensitizers]. / Fotoinaktivatsiia mikroorganizmov v prisutstvii fotosensibilizatorov.

Two photosensitizing preparation for the photodynamic therapy of oncological diseases have been obtained. As revealed in this study, under experimental conditions their action suppresses the growth of Enterococcus faecalis and Staphylococcus aureus, which may be of great importance for the treatment of infections. And in contrast to antibiotic therapy, their use may not affect the development of normal intestinal microflora. The study has shown that their derivatives with the residues of carbonic (or muramic) acids more intensively penetrate microorganisms through their cell walls. This makes in possible to expand the spectrum of action of the preparations. The proposed method permits the rapid analysis of the photochemical activity of preparation for photodynamic therapy in the process of their development.

[The blood-simulating properties of a recombinant colony-stimulating factor and glycyram during cytostatic myelosuppression]. / Gemostimuliruiushchie svoistva rekombinantnogo koloniestimuliruiushchego faktora i glitsirama v usloviiakh tsitostaticheskoi mielosupressii.

The hemostimulating activity of the drug glycyrram and the recombinant granulocytic-macrophagic colony-stimulating factor (CSF) was compared on models of myelosuppression induced by injection of an alkylating agent cyclophospane (CF) or the antimetabolite 5-fluorouracil (5-FU). It was found that glycerram stimulated hematopoiesis more effectively after 5-FU injection but to a lesser extent (in relation to CSF) in CF injection.

[Cloning and expression of a plasmid pBS195 gene, determining oxygenase activity, in Escherichia coli cells]. / Klonirovanie i ékspressiia v kletkakh Escherichia coli gena plazmida pBS195, determiniruiushchego oksigenaznuiu aktivnost'.

Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a 32P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli mini-cells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa.

[Structure-functional characteristics of pBS195 plasmids from a broad range of gram-positive and gram-negative hosts]. / Strukturno-funktsional'naia kharakteristika plazmidy pBS195 s shirokim krugom grampolozhitel'nykh i gramotritsatel'nykh khoziaev.

The ability of plasmid pBS195 to be inherited by a broad host range including both gram-positive and gram-negative bacteria attracts special attention to its molecular genetic organization. Physical map of the plasmid has been constructed and the gene for kanamycin resistance and oriV homologous to the one of pUC19 have been localized. The determinants for kanamycin-resistance have been shown to share no homology with the genes for the I and II type phosphotransferases and type II nucleotide-transferase. The minicells of Escherichia coli x925 harboring pBS195 produce two plasmid-encoded proteins with mol. masses 26 and 34 kD found in SDS-PAGE. The plasmid determines an oxygenase activity and shares homology with a nahC gene in the EcoRI-BamHI region (0 bp-1.6 kbp). The enzyme was identified as a methylpyrocatehase possessing high activity in Escherichia coli cells and substrate specificity uncharacteristic of this type enzymes.

[The effectiveness of the health resort treatment of patients with diseases of the locomotor apparatus]. / Effektivnost' kurortnogo lecheniia bol'nykh s zabolevaniiami oporno-dvigatel'nogo apparata.

A study of 80 patients with spinal osteochondrosis and 27 patients with osteochondrosis deformans revealed high efficacy of treatment at the "Marcial Waters" health results (Karelian SSR). The authors developed methods of analgesiometry permitting to evaluate objectively treatment results.