The Influence of Lipid Matrix Composition on the Microenvironment of Levofloxacin in Liposomal Forms.

We have studied the interaction of the antibacterial drug levofloxacin with lipid bilayers of various compositions: 100% DPPC and with the addition of 20% cardiolipin. For DPPC liposomes, levofloxacin was found to penetrate into the subpolar region at the lipid-water interface. The role of the anionic lipid in the interaction of an active molecule with a bilayer has been established: levofloxacin enters the microenvironment of the phosphate group, displacing water, and does not penetrate into the hydrophobic part of the bilayer. For the first time, the study of the microenvironment of levofloxacin in the liposome by IR and CD spectroscopy was carried out. Such an approach based on a combination of several spectral methods opens up new prospects for the creation of new medicinal properties and the possibility of predicting the nature of the interaction of active molecules with biomembranes in order to predict their efficacy and potential side effects.

[Cyanocobalamin-containing lubricant for topical therapy of LASIK-associated dry eye]. / Tsianokobalaminsoderzhashchii lubrikant v sostave mestnoi terapii LASIK-assotsiirovannogo sindroma sukhogo glaza.

AIM: to study the effect of topical treatment with Artelac Balance, a cyanocobalamin-containing lubricant, in patients with post-LASIK corneal neuropathy and LASIK-associated dry eye. MATERIAL AND METHODS: Patients scheduled for LASIK were enrolled. Inclusion criteria were contact lens wear for 5 years or longer and more than 6 diopters of myopia. The patients were divided into 3 groups. Group 1 was prescribed the cyanocobalamin-containing topical lubricant for 3 months before and 1 month after surgery, group 2 - the same cyanocobalamin-containing lubricant, but only for the first month after surgery, and group 3 received a standard preservative-free lubricant for the first month after surgery. Tear osmolarity and production as well as corneal sensitivity were assessed 90±10 days before and 30±3 days after surgery. The percentage of patients with dry eye symptoms and those who required prolonged use of lubricants were also estimated at day 30±3. RESULTS: Preoperatively, there were no statistically significant differences in demographic characteristics, tear osmolarity, tear production, or corneal sensitivity between the three study groups. At day 30±3 after surgery, tear production and corneal sensitivity were both significantly higher in group 1 than in group 3 (16.8 mm vs. 13.2 mm, p=0.003 and 37.7±3.1 mm vs. 32.4±2.2 mm, p=0.023, respectively). As to the percentage of patients with dry eye symptoms and those who required prolonged use of lubricants, it was lower in group 1 than in group 3 (51.3% vs. 76.3%, p=0.03 and 38.5% vs. 63.2%, p=0.03, respectively). Between the groups 2 and 3, none of the studied parameters differed significantly. CONCLUSION: Combined pre- and postoperative use of Artelac Balance, a cyanocobalamin-containing lubricant, has been proved effective in restoring corneal sensitivity and reducing dry eye symptoms in patients at high risk for developing LASIK-associated dry eye.

[PEG-chitosan branched copolymers to improve the biocatalytic properties of Erwinia carotovora recombinant L-asparaginase].

A new approach to the regulation of catalytic properties of medically relevant enzymes has been proposed using the novel recombinant preparation of L-asparaginase from Erwinia carotovora (EwA), a promising antitumor agent. New branched co-polymers of different composition based on chitosan modified with polyethylene glycol (PEG) molecules, designated as PEG-chitosan, have been synthesized. PEG-chitosan copolymers were further conjugated with EwA. In order to optimize the catalytic properties of asparaginase two types of conjugates differing in their architecture have been synthesized: (1) crown-type conjugates were synthesized by reductive amination reaction between the reducing end of the PEG-chitosan copolymer and enzyme amino groups; (2) multipoint-conjugates were synthesized using the reaction of multipoint amide bond formation between PEG-chitosan amino groups and carboxyl groups of the enzyme in the presence of the Woodward's reagent. The structure and composition of these conjugates were determined by IR spectroscopy. The content of the copolymers in the conjugates was controlled by the characteristic absorption band of C-O-C bonds in the PEG structure at the frequency of 1089 cm-1. The study of catalytic characteristics of EwA preparations by conductometry showed that at physiological pH values the enzyme conjugates with PEG-chitosan with optimized structure and the optimal composition demonstrated 5-8-fold higher catalytic efficiency (kcat/Km) than the native enzyme. To certain extent, this can be attributed to favorable shift of pH-optima in result of positively charged amino-groups introduction in the vicinity of the active site. The proposed approach, chito-pegylation, is effective for regulating the catalytic and pharmacokinetic properties of asparaginase, and is promising for the development of prolonged action dosage forms for other enzyme therapeutics.

[Bacterial recombinant L-asparaginases: properties, structure and anti-proliferative activity].

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.

PEG-chitosan and glycol-chitosan for improvement of biopharmaceutical properties of recombinant L-asparaginase from Erwinia carotovora.

Conjugation with the new branched copolymers, PEG-chitosan and glycol-chitosan, is suggested to improve the therapeutic properties of L-asparaginase from Erwinia carotovora (EwA). The structure and composition of such conjugates were optimized for maximal catalytic efficiency (kcat/KM) under physiological conditions, yielding improvement by a factor of 3-6 compared to the native enzyme. This effect is attributed mainly to the shift of pH activity profile towards lower pH values due to the polycationic nature of the copolymer. The thermostability of EwA conjugates was also considerably improved. Chito-PEGylation, similarly to PEGylation, can be expected to improve pharmacokinetic properties and to reduce immunogenicity of this medically relevant enzyme. It is worth mentioning that a new versatile approach based on IR spectroscopy has been developed to determine PEG-chitosan copolymer composition as well as composition of copolymer-enzyme conjugates. The proposed analytic method is "reagent-free" and allows fast and reliable determination of parameters of interest from the single IR spectrum in contrast to laborious and unreliable methods based on polymer free amino group titration with TNBS and OPA.

Disaccharide 1-phosphate polymers of some representatives of the Bacillus subtilis group.

Disaccharide 1-phosphate polymers as well as teichoic acids of various structures have been found in the cell walls of the representatives of the Bacillus subtilis group, namely Bacillus subtilis subsp. spizizenii VKM B-720 and VKM B-916, B. subtilis VKM B-517, and Bacillus vallismortis VKM B-2653(T). Disaccharide 1-phosphate polymers are composed of repeating units of the following structure: -P-4)-ß-D-GlcpNAc-(1→6)-α-D-Galp-(1-, the N-acetylglucosamine residues are partially acetylated at positions O3 and O6 (VKM B-720 and VKM B-916); -P-4)-ß-D-Glcp-(1→6)-α-D-GlcpNAc-(1-, the glucopyranose residues are partially acetylated at positions O2 or O3 (VKM B-517); -P-6)-α-D-GlcpNH3(+)/α-D-GlcpNAc-(1→2)-α-D-Glcp-(1-, the N-acetylglucosamine residues are partially deacetylated (VKM B-2653(T)). The structures of the two last disaccharide 1-phosphate polymers have not been reported so far for Gram-positive bacteria. The teichoic acids in the studied strains are O-D-alanyl-1,5-poly(ribitol phosphates) substituted with ß-D-glucopyranose (VKM B-517, VKM B-720, VKM B-916) or 2-acetamido-2-deoxy-ß-D-glucopyranose (VKM B-2653(T)). The structures of the phosphate-containing polymers have been studied by chemical methods and by NMR spectroscopy.

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3.

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.

Regulation of acid phosphatase in reverse micellar system by lipids additives: structural aspects.

Reverse micelles system is suggested as a direct tool to study the influence of membrane matrix composition on the activity and structure of membrane-associated enzymes with the use of acid phosphatase (AP) as an example. In reverse micelles the functioning of the monomeric and dimeric forms of AP could be separately observed by variation of the size of the micelles. We found that including the lipids into the micellar system can dramatically affect the enzyme functioning even at low lipid content (2% w/w), and this effect depends on the lipid nature. Structural studies using CD spectroscopy and DLS methods have shown that the influence of lipid composition on the enzyme properties might be caused by the interaction of lipids with the enzyme as well as by the influence of lipids on structure and properties of the micellar matrix.

Heterogeneous set of cell wall teichoic acids in strains of Bacillus subtilis VKM B-760 and VKM B-764.

Cell walls of Bacillus subtilis VKM B-760 and VKM B-764 are characterized by heterogeneous composition of teichoic acids. Polymer I with structure -6)-beta-D-Galp-(1-->1)-sn-Gro-(3-P-, polymer II with structure -6)-alpha-D-Glcp-(1-->1)-sn-Gro-(3-P-, and a small amount of unsubstituted 1,3-poly(glycerol phosphate) were detected in strain VKM B-760. Strain VKM B-764 contains an analogous set of teichoic acids, but a characteristic feature of polymer II is the presence of disubstituted glycerol residue with alpha-glucopyranose localization in the integral chain at C-1 hydroxyl and beta-glucopyranose as a side branch at C-2 hydroxyl (polymer III): -6)-alpha-D-Glcp-(1-->1)-[beta-D-Glcp-(1-->2)]-sn-Gro-(3-P-. The structures of polymer I in bacilli and polymer III in Gram-positive bacteria are described for the first time. Teichoic acids were studied by chemical methods and on the basis of combined analysis of one-dimensional 1H-, 13C-, and (31)P-NMR spectra, homonuclear two-dimensional (1)H/(1)H COSY, TOCSY, and ROESY, and heteronuclear two-dimensional (1)H/(13)C gHSQC- and HMQC-TOCSY experiments. Simultaneous presence of several different structure teichoic acids in the bacillus cell walls as well as chemotaxonomical perspectives of the application of these polymers as species-specific markers for members of the Bacillus genus is discussed.

Anionic polymers of the cell wall of Bacillus subtilis subsp. subtilis VKM B-501(T).

Teichoic acid and disaccharide-1-phosphate polymer were identified in the cell walls of Bacillus subtilis subsp. subtilis VKM B-501(T). The teichoic acid represents 1,3-poly(glycerol phosphate) 80% substituted by alpha-D-glucopyranose residues at O-2 of glycerol. The linear repeating unit of disaccharide-1-phosphate polymer contains the residues of beta-D-glucopyranose, N-acetyl-alpha-D-galactosamine, and phosphate and has the following structure: -6)-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1-P-. The structures of two anionic polymers were determined by chemical and NMR-spectroscopic methods. The 1H- and 13C-NMR spectral data on disaccharide-1-phosphate polymer are presented for the first time.

Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system.

The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.

Double-strand break repair in bacteriophage T4: recombination effects of 3'-5' exonuclease mutations.

The role of 3'-5' exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i x ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3'-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (<100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesis-dependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed.

Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro.

The giant protein titin serves a primary role as a scaffold for sarcomere assembly; however, proteins that mediate this remodeling have not been identified. One potential mediator of this process is the protease calpain 3 (C3), the protein mutated in limb girdle muscular dystrophy type 2A. To test the hypothesis that C3 mediates remodeling during myofibrillogenesis, C3 knockout (C3KO) mice were generated. The C3KO mice were atrophic containing small foci of muscular necrosis. Myogenic cells fused normally in vitro, but lacked well-organized sarcomeres, as visualized by electron microscopy (EM). Titin distribution was normal in longitudinal sections from the C3KO mice; however, EM of muscle fibers showed misaligned A-bands. In vitro studies revealed that C3 can bind and cleave titin and that some mutations that are pathogenic in human muscular dystrophy result in reduced affinity of C3 for titin. These studies suggest a role for C3 in myofibrillogenesis and sarcomere remodeling.

New magnet-sensitive structures in bacterial and archaeal cells.

The objects of the investigation were: distribution of intracellular magnet-sensitive structures among different taxonomic groups of prokaryotes, localisation and organisation of the magnet-sensitive inclusions (MsI) in cells. The MsI were discovered in representatives of both prokaryotic domains (Bacteria and Archaea), 2 kingdoms and 7 orders of bacteria. They were some amorphous or non-crystalline globules with the electron-transparent centre surrounded with an electron-dense homogenous matrix. The magnetic nature of the structures was shown by attraction with an applied magnet both for the cell suspensions and for the MsI isolated and separated from the destroyed cells. The MsI were studied with transparent electron microscopy and with X-ray analyses. When the cells were grown in the iron-containing nutrient medium, the matrix was enriched with iron. It was shown also that some bacteria grown with cobalt or with chromium contained the cobalt- or chromium-enriched magnetic inclusions.

Plasmin-independent gelatinase B (matrix metalloproteinase-9) release by monocytes under the influence of urokinase.

It is shown that the release of matrix metalloproteinase-9 (gelatinase B) by THP-1 and U937 cells into conditioned media is increased under the action of recombinant single-chain urokinase. This effect is not accompanied by proteolytic activation of gelatinase B and is related to release of a pro-form of the enzyme. The action of urokinase on monocytes is time-dependent and becomes significant 12-24 h after the beginning of cell incubation. The dependence of the effect on the concentration of urokinase is characterized by half-maximum at about 20 nM and saturation at about 200 nM. The urokinase-induced gelatinase B release is not dependent on the action of plasmin because plasmin inhibitors aprotinin and alpha2-antiplasmin do not abolish this action. Additionally, tissue type plasminogen activator does not induce gelatinase B release by monocytes as observed under the action of urokinase. Nevertheless, the catalytic activity of urokinase participates in the development of the observed effect because it is significantly depressed by the natural urokinase inhibitor PAI-1. The effect of urokinase is completely abolished by actinomycin D and cycloheximide, indicating the participation of transcription and translation processes in its development.

The chemical modification of alpha-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water-organic media.

We considered alpha-chymotrypsin (CT) in homogeneous water-organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water-ethanol and water-DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water-ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.

Formation of quasi-regular compact structure of poly(methacrylic acid) upon an interaction with alpha-chymotrypsin.

Structure and dynamic properties of free poly(methacrylic acid) (PMA) and PMA complexed with alpha-chymotrypsin (CT) were studied using the time resolved fluorescence anisotropy technique. We have found that the interaction of PMA with CT induces the formation of a quasi-regular structure of PMA. At a CT/PMA weight ratio of 4:1 the interaction with CT leads to formation of approximately four equal segments of polyelectrolyte, each binding one CT molecule and characterized by an independent rotational mobility. Increase of the CT/PMA weight ratio above 8:1 gives rise to the overall rotation of the whole enzyme-polyelectrolyte complex. In water-ethanol mixtures the mobility of PMA segments containing CT decreases and the structure of the complex becomes even more rigid due to enhancement of the electrostatic interaction between CT and PMA. Formation of the compact and quasi-regular structure of the complex is perhaps the main reason behind the enhancement of enzyme stability and suppression of enzyme aggregation in water-organic cosolvent mixtures.

Catalytic activity of thermolysin under extremes of pressure and temperature: modulation by metal ions.

The catalytic activity of thermolysin (TL), a Zn-dependent neutral protease from Bacillus thermoproteolyticus, has been studied over a wide interval of pressures (1 bar to 4 kbar) and temperatures (20 degreesC to 80 degreesC) by monitoring hydrolysis of a low-molecular-mass substrate, 3-(2-furylacryloyl)-glycyl-L-leucine amide. This reaction shows a very large negative value for the activation volume and, because of that, simultaneous increase in temperature and pressure leads to a significant (up to 40-fold) acceleration of the reaction. At pressures higher than 2-2.5 kbar, the reaction rate starts to decrease due to disactivation of TL. This disactivation is explained in part by pressure-promoted dissociation of zinc ion from the active site and can be inhibited by adding exogenous zinc. Thus, this thermostable protease does not specifically show a higher stability at high pressure in comparison with small mesophilic proteases.