Polysaccharide Galactan Inhibits Pseudomonas aeruginosa Biofilm Formation but Protects Pre-formed Biofilms from Antibiotics.

Microorganisms residing within a biofilm become more tolerant to antibiotics and other types of adverse impact, and biofilm formation by pathogenic bacteria is an important problem of current medicine. Polysaccharides that prevent biofilm formation are among the promising candidates to help tackle this problem. Earlier we demonstrated the ability of a potato polysaccharide galactan to inhibit biofilm formation by a Pseudomonas aeruginosa clinical isolate. Here we investigate the effect of potato galactan on P. aeruginosa biofilms in more detail. Microscopic analysis indicated that the galactan did not interfere with the adhesion of bacterial cells to the substrate but prevented the build-up of bacterial biomass. Moreover, the galactan not only inhibited biofilm formation, but partially destroyed pre-formed biofilms. Presumably, this activity of the galactan was due to the excessive aggregation of bacterial cells, which prohibited the formation and maintenance of proper biofilm architecture, or due to some other mechanisms of biofilm structure remodeling. This led to an unexpected effect, i.e., P. aeruginosa biofilms treated with an antibiotic and the galactan retained more viable bacterial cells compared to biofilms treated with the antibiotic alone. Galactan is the first polysaccharide demonstrated to exert such effect on bacterial biofilms.

Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities.

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.

Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

[PHARMACOLOGICAL POSTCONDITIONING BY SEVOFLURANE DURING CARDIAC SURGERY.]

BACKGROUND: Optimization of myocardial protection during cardiac surgery with a long period of anoxia infarction using sevoflurane postconditioning of myocardium. THE AIM: to develop the optimal pharmacological postconditioning protocol with sevoflurane for infarction patients ,undergoing cardiac surgery. MATERIALS AND METHODS: Two groups were formedfor this study: CON] 00 (n-32) with aortic cross-clamping time 114±15 min and SEV100 group (n-34), where the myocardium anoxia was 119±22 minutes. According to previously developed in the pilot study Protocol, we added sevofturane in the circuit of extracorporeal circulation in a dose of 2.0 vol. % 20 minutes before removing the clamp from the aorta and the first 20 min of reperfusion in the group SE V100. In the group CON1 00 pharnacological postconditioning wasn't conducted. To assess the adequacy of the cardioprotection against ischemic damage in operated patients, we used the following clinical and laboratory parameters: changing the level of troponin T; the concentration of lactate and glucose as a marker of severity of anaerobic metabolism; concentration of proinflammatory cytokines IL-6, IL-8, TNF-alpha in blood serum as reperfusion injury markers. Also we used the registration of central hemodynamics data: measuring the mean invasive blood pressure; central venous pressure; Cardiac output was measured by the method of transesophageal echocardiography TEEcho-CG, calculated left ventricular ejection fraction by Simpson. We evaluated the clinical course of the perioperative period: incidence ofperioperative myocardial ischemia; the need and the duration ofuse of cardiotonic drugs in the perioperative period; the incidence of reperfusion arrhythmias; the frequency of self-recovery heart rate. RESULTS: According to the results of anaerobic metabolism markers, we can conclude that the period of the myocardium anoxia ofpatients in both groups experienced no significant difference. However; a completely different pattern was observed when comparing the proinflammatory cytokines, such as IL-6, IL-8, TNF-a. This confirms that the group SEV] 00 survived the reperfusion is much better than the group CON100. Instrumental examination also showed that the group ofpatients in which pharmacological postconditioning with sevofturane was held signficantly better suffered ischemia and reperfiision injury compared to control group. Self-recovery heart rate after removing the aorta clamp in the group CON100 was observed in 81%, in group SEV100 same - 93%. Similarly, the frequency of myocardial ischemia episodes on the ECG in reperfusion period was two times lower in the group SEV100 compared with group CON100 - 5.8% and 12.5% respectively. Reperfusion arrhythmia is almost 3 times more frequent in the group CON100 - 21,8%, in the group SEV100, where he conducted pharmacological postconditioning with sevoflurane is 8.8%.. CONCLUSION: Combined with sevoflurane cardioprotection FPC has a much better resistance to myocardial ischemia-reperfusion injury in patients with myocardial infarction time over 100 minutes than monoprotection with cardioplegic solution "Console ". This method can be recommended as an additional method ofprotection against myocardial ischemia-reperfusion injury.

[Surgery of pancreatic injuries]. / Khirurgiya povrezhdenii podzheludochnoi zhelezy.

AIM: To estimate different approaches to treatment of victims with pancreatic trauma with pancreatic trauma. MATERIAL AND METHODS: It was analyzedthe results of treatment of 342 victims with pancreatic trauma in N.V. Sklifosovskiy Research Institute of Emergency Care for the period 1991-2012. RESULTS: It was concluded that for the las decade curative and diagnostic tactics for pancreatic injury in victims with combined abdominal trauma has been changed; current diagnostic markers of pancreatic lesion and adequate intraoperative diagnosis are used. All of this together with timely specific therapy and adherence to guidelines of surgical treatment decreased mortality rate from 17.0 to 11.1% and suppurative complications incidence from 43.8 to 19.9%.

[The comparative characteristic of coefficients of endogenic intoxication under severe acute pancreatitis].

The sample included 43 patients with severe acute pancreatitis. Two coefficients of endogenic intoxication were applied: Kei1 = (AMP/ECA)x 100, AMP -average molecular peptides, ECA--effective concentration of albumin and Kei2 = (Kplp/aos/ECA) x 100, PLP--products of lipid peroxidation, AOS--indicators of antioxidant system. ECA--effective concentration of albumin. The comparative characteristic of both coefficients is given. It is established that Kei2 provides more informative indicators of endogenic intoxication in patients died at third day after operation. The study proved that both Kei can be applied for evaluation of endogenic intoxication and prognosis of generalized pancreonecrosis depending on resources of laboratory service.

[Sevoflurane optimal dosage estimation for myocardium pharmacological postconditioning: an experimental study].

We estimated the optimal dosage of inhalation anesthetic sevoflurane, for the maximum cardioprotective effect with minimal angioparalytic action. 25 pigs were included in this study, they were divided into 5 groups, depending on the sevoflurane dosage used for pharmacological postconditioning (PPC): control group - PPC has't been conducted, a group of PPC 0.5 - sevoflurane PPC in a dose of 0.5 V%, a group of PPC 1.0 - sevoflurane PPC in a dose of 1.0 V%, a group of PPC 1.5 - sevoflurane PPC in a dose of 1.5 V%, a group of PPC 2.0 - sevoflurane PPC in a dose of 2.0 V%, a group of PPC 2.5 - sevoflurane PPC in a dose of 2.5 V%. Ischemia was simulated by left coronary artery crossclamping. Further PPC was held according to the following Protocol: 20 min before left coronary artery clamp off and first 20 min of reperfusion sevoflurane was given into CPB circuit. Myocardial ischemia period was 60 min in all groups. It was found and experimentally proved that the optimal sevoflurane dosage for PPC is 2 V%

[Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations].

Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

[Tuberculosis morbidity among military personnel in modern conditions].

Among identified in the army for one year of active tuberculosis patients constitute 38.7% of the number of soldiers from young recruits, including 59.7% of infiltrative pulmonary tuberculosis. At the same time during the examination of these individuals revealed only about half of patients. Timely delivery of fluorographic examination recruitment plays a crucial role in preventing the spread of tuberculosis in the Armed Forces. From the military doctors need more work in groups at increased risk of tuberculosis, early identification of need in the dispensary dynamic observation and conducting a full range of therapeutic and preventive measures in foci of tuberculous infection.

[The choice of the targets and the search for their inhibitors by means of computer simulation for the development of innovative preparations to treat of chronic bacterial infections].

Persistence is a form of interaction of pathogenic bacteria with a host aimed to promote their long-term survivalby means of inactivation of the host's protective systems via modulation of intracellular signal pathways. Persistent forms of a pathogen are refractory to traditional antibiotic therapy and cause chronic infectious diseases. Directed search for protein targets and new antibacterial drugs using computer simulation and experimental testing for the development of innovative preparations to treat chronic bacterial infections appears to have good prospects as a method for the management of persistent infections. A stepwise strategy for realization of such approach is exemplified by the search of preparations against chlamydial infection.

[Conserved structural features of ETS domain--DNA complexes].

ETS proteins comprise a family of widespread transcription factors regulating expression of many animal genes. Structurally ETS proteins are characterized by conserved DNA-binding ETS domain, recognizing DNA sequences with trinucleotide GGA. Comparative analysis of structural features of ETS domain-DNA complexes was carried out and conserved contacts, important in terms of interaction stability and specificity were identified. The analysis revealed 9 conserved hydrogen bonds with DNA backbone phosphates and 2 conserved bidentate hydrogen bonds with DNA major groove atoms, one conserved hydrophobic cluster on protein-DNA interface, important for binding site recognition, and 12 conserved water molecules, possibly mediating ETS domain-DNA interaction. The results are represented in specialized data bank of protein-DNA complexes NPIDB.

Actin cytoskeleton organization regulated by the PAK family of protein kinases.

Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton.

Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.

[Pathogenetic approach to diagnosis and treatment of acute pancreatitis].

Results of examination and treatment of 958 patients with acute pancreatitis are analyzed. The study group consisted of 372 patients treated in accordance with special clinical protocols (2001-2004). The control group consisted of 586 patients treated before these protocols extensive use (1995-2001). The two groups were similar by the main clinical characteristics. It was demonstrated that the treatment according standard protocols permitted to reduce general lethality from 3.9% (control group) to 2.7% (study group), postoperative lethality - from 14.3 to 11.5%. General lethality at severe acute pancreatitis decreased from 18.5 to 12.7%, postoperative lethality - from 26 to 19%.

[Laparoscopy in urgent abdominal surgery].

The use of videolaparoscopic methods for the treatment of penetrating stomach and duodenal ulcers, acute cholecystitis, acute pancreatitis, acute appendicitis, intestinal obstruction, acute gynecological diseases and abdominal trauma is analyzed. Laparoscopic methods at urgent abdominal surgery improves the quality of diagnosis and treatment, decrease the rate of postoperative complications and lethality, reduce the hospital stay.

[The incidence of sarcoidosis in the Armed Forces: diagnosis, treatment and regular medical check-up].

Sarcoidosis is a chronic multi-system granulomatous disease of unknown etiology, which is characterized by development of epitheliocellular granulomas. Its therapy is not substantiated enough. The incidence of sarcoidosis among the called up servicemen is 1,1 and among the contract servicemen--3,5. According to the structure of contingents with initial diagnosis the called up servicemen constitute 18,6%, officers and praporschiks--57,7% and the contract servicemen without commissioned ranks--23,7%. The comparative analysis has shown that among the contract servicemen suffering from sarcoidosis the number of women was 2,1 times more than among the patients with tuberculosis. The medical personnel in the sarcoidosis group is observed more often (1,5 times more) than in the tuberculosis.

[Transcription factor ZF5 regulates expression of mammalian gene containing GCC-triplet repeats in 5'-regulatory region in human hepatoma HepG2 cells].

Some nuclear proteins of human HeLa and HepG2 cells are capable of binding to GCC-triplet repeats--(GCC)n > 3 in 5'-regulatory regions of a number of mammalian genes--G-C-elements. According to our previous data, nucleotide sequence (GCC)4 in promoter of mouse ribosomal protein L32 gene (rpL32) between 17 and 6 bp upstream of transcription start site interacts to nuclear proteins from HepG2 cells, and may be considered as a GCC-element. We suggest that one of those proteins, with molecular weight about 52 kDa, which may interact with rpL32 GCC-element, is a known conservative mammalian transcription factor ZF5. DNA-binding domain of ZF5 contains a few Kruppel-like Zn-fingers (Cys2His2-type) interacting with the GC-rich nucleotide sequences in 5'-regulatory regions of a number of mammalian genes. Our results (obtained by EMSA) showed that recombinant GST-ZF5 fused protein containing ZF5 DNA-binding domain specifically binds a few GS-rich sequences: (GCC)g-9riplet repeats, 5'-GCGCGC-3' (known ZF5 consensus binding site) and (more preferable) the fragment (-24...+1 bp) of rpL32 promoter. The high affinity of ZF5 DNA-domain binding with the latter may be explained by the presence in this fragment of two overlapped subsequences, each being capable of binding to ZF5: (GCC)4 and 5'-GCGCGC- 3'. Zf5 cDNA was cloned from HepG2 cells by RT-PCR method, and then used for construction of the gene expression vector. It has been shown that Zf5 cDNA expression vector specifically down-regulates (in luciferase assays) the activity of rpL32 promoter (-155...+159) including the above mentioned GC-rich subsequences by cotransfection of HepG2 cells. Therefore, our results enable us to consider GCC-elements as a novel class of ZF5 targets in 5'-regulatory regions of mammalian genes.

[Complex of immunological characteristics for the diagnostics of pancreatogenic immunodeficiency].

The results of the immunological study of blood in 89 patients with severe acute pancreatitis (SAP) were analyzed. Immune reactions in SAP were characterized by the presence of moderate leukocytosis, an increase in the phagocytic and metabolic activity of neutrophils, relative lymphopenia, a decrease in the number of B-lymphocytes. The average values of the parameters of the immunogram of patients with the favorable course of the disease on days 2 - 3 were analogous to the average population values in 89 examinees. The direction and the degree of manifestation were slightly variable and could be registered within one standard quadratic deviation. Such heterogeneity in the character of immune reactions made it possible to regard the average values of the immunogram parameters of these patients as "the norm". For the objective evaluation of the immune status of SAP patients a scale for the evaluation, in points, of disturbances in immune reactions to the destructive process in the pancreas was developed. This scale for the evaluation, in points, of the severity of disturbances in the immune status of SAP patients made it possible to prognosticate the development of complications.

[Fistulography in destructive pancreatitis in postoperative period].

Results of different x-ray methods applied in 151 patients early after operations for destructive pancreatitis were analyzed. The technique of fistulography is described. Plain roentgenography of the chest demonstrated changes in 77.4% cases, most often occurred disk-shaped atelectases and pleural effusion. Examination of the abdominal cavity revealed functional disorders in 69.8% cases, direct symptoms of purulent process -- in 52.9%, radiographic signs of aseptic sequestration -- in 47.1% patients. X-ray signs of purulent process in the retroperitoneal fat and aseptic sequestration of the infiltrate or fat are outlined. The value of fistulography for localization and determination of the shape of drained cavities, position of the drainage tubes in the cavity, condition of the adjacent tissue, progression of purulent process, adequacy of drain of the purulent cavities is shown. Based on the results of complex x-ray examination with ultrasound investigation and CT data, the indications to repeated procedure (surgery or additional drainage) were formulated in 82 (54.3%) of 151 patients.

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.