NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel.

Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na+ channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of 13C,15N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na+ channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn2+ titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. 15N relaxation data revealed characteristic pattern of µs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K+ channels. These results validate structural studies of isolated VSDs of Na+ channels and show possible pitfalls in application of this 'divide and conquer' approach.

Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors.

Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3ß2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved ß-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, ß2, and ß4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4ß2 and α3ß2-nAChRs (IC50 ~0.17 and >3 µM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 µM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3ß2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3ß2-nAChRs.

Human SLURP-1 and SLURP-2 Proteins Acting on Nicotinic Acetylcholine Receptors Reduce Proliferation of Human Colorectal Adenocarcinoma HT-29 Cells.

Human secreted Ly-6/uPAR related proteins (SLURP-1 and SLURP-2) are produced by various cells, including the epithelium and immune system. These proteins act as autocrine/paracrine hormones regulating the growth and differentiation of keratinocytes and are also involved in the control of inflammation and malignant cell transformation. These effects are assumed to be mediated by the interactions of SLURP-1 and SLURP-2 with the α7 and α3ß2 subtypes of nicotinic acetylcholine receptors (nAChRs), respectively. Available knowledge about the molecular mechanism underling the SLURP-1 and SLURP-2 effects is very limited. SLURP-2 remains one of the most poorly studied proteins of the Ly-6/uPAR family. In this study, we designed for the first time a bacterial system for SLURP-2 expression and a protocol for refolding of the protein from cytoplasmic inclusion bodies. Milligram quantities of recombinant SLURP-2 and its 13C-15N-labeled analog were obtained. The recombinant protein was characterized by NMR spectroscopy, and a structural model was developed. A comparative study of the SLURP-1 and SLURP-2 effects on the epithelial cell growth was conducted using human colorectal adenocarcinoma HT-29 cells, which express only α7-nAChRs. A pronounced antiproliferative effect of both proteins was observed. Incubation of cells with 1 µM SLURP-1 and 1 µM SLURP-2 during 48 h led to a reduction in the cell number down to ~ 54 and 63% relative to the control, respectively. Fluorescent microscopy did not reveal either apoptotic or necrotic cell death. An analysis of the dose-response curve revealed the concentration-dependent mode of the SLURP-1 and SLURP-2 action with EC50 ~ 0.1 and 0.2 nM, respectively. These findings suggest that the α7-nAChR is the main receptor responsible for the antiproliferative effect of SLURP proteins in epithelial cells.

[Endoscopic hemostasis in patients with portal hypertension tumor genesis].

AIM OF THE STUDY: to assess the results of using endoscopic techniques to achieve hemostasis for the control of bleeding and the prevention of recurrent bleeding from esophageal and gastric varices in the patients with primary liver cancer and metastatic liver lesions. MATERIALS AND METHODS: in the period of 2009-2011,7 patients with primary liver cancer or metastatic liver lesions complicated by gastro-esophageal bleeding were treated in the Sklifosovsky Clinical and Research Institute for Emergency Medicine. The sources of bleeding were esophageal varices in 6 patients, gastric varices in 1. RESULTS: a diagnostic esophagogastroduodenoscopy (EGDS) revealed, besides the varices, an erosive hemorrhagic gastritis in 4 patients, a ruptured mucosa of cardio-esophageal transition in 1, an erosive hemorrhagic gastritis and a liver tumour invasion in the stomach antrum in 1. The hemostasis achieved by means of paravasal injections with a 20% glucose solution was efficient in 2 of 3 patients. The endoscopic ligations of esophageal varices were performed in 3 patients. Five patients died from the underlying disease progressing. In one of them, the cause of death was a recurrent bleeding from esophageal varices. CONCLUSIONS: in the patients with primary liver cancer and metastatic liver lesions, the use of mini-invasive endoscopic techniques is more appropriate, because the surgical treatment of this patient population carries a very high risk.

The role of chain rigidity in lipid self-association: comparative study of dihexanoyl- and disorbyl-phosphatidylcholines.

In the course of structure-function investigations of lipids a phosphatidylcholine molecule with short and rigid tails, di-2,4-hexadienoylphosphatidylcholine (DiSorbPC), was synthesized and studied in comparison with its saturated analog, dihexanoylphosphatidylcholine (DHPC). Conjugated double bonds in the acyl chains in DiSorbPC reduce considerably the number of possible conformers of the lipid within an aggregate. This leads to impaired packing of unsaturated acyl chains and thus, to a surprisingly high (115 Å(2)) area per molecule for DiSorbPC at the air-water interface and failure to form micelles of regular size and shape. Details on DiSorbPC aggregation and packing provided by a set of experimental techniques combined with molecular dynamics simulations are presented.

[Effect of risk factors in colorectal cancer hepatic metastases].

The risk factors, influencing late results of surgical treatment, were analyzed in 69 patients, suffering a colorectal cancer hepatic metastases. The significance of clinico-morphological (the metastases maximal size, quantity, localization, the tumor grade, the hepatic affection synchronicity with primary tumor appearance, the hepatoduodenal ligament lymph nodes affection, microvascular invasion, the resection edges) and molecular (CK 20, beta-cat, Ki 67, Muc 2 and 5A) factors of prognosis was studied up. The hepatic resection variant was chosen, depending on the largest metastasis size present, the tumor nodes quantity and localization, the extrahepatic foci present, the lymph nodes affection and hepatic functional reserve secured. Basing on estimation of the level and type of expression for molecular factors there was determined correlation with the tumor recurrence rate. High degree correlation was established for the CK 20, beta-cat, Ki 67 expression and the recurrence rate, but it was not true for the Muc 2 and 5A expression. One, three and five years have survived 85, 50 and 31% of patients accordingly, and without recurrences--68, 27 and 14% accordingly. No one of clinico-morphological factors, but the metastasis grade, have had influenced the disease prognosis.

[The early rehabilitation of the patients following extensive liver resection and transplantation].

This study included 500 patients who had undergone extensive liver resection and transplantation of whom 250 were treated with the use of remedial gymnastics, physiotherapeutic factors, and massage. The positive dynamics of objective characteristics of the patients' condition. None of the treated patients showed a negative response to the proposed program of medical rehabilitation and activation throughout the early postoperative period.

[Volume of surgical resection by liver focal lesions].

Results of surgical treatment of 109 patients with focal lesions of the liver were analyzed. Volume resection by malignant and benign liver lesions was defined, depending on size, localization, number of foci and functional liver reserve. Postoperative lethality of patients with colorectal cancer metastases was 4.3%. Method of surgical treatment depended directly on the tumor stage. Overall 5-year survival by stage IVA hepatocellular cancer was 48%, 3.5-year survival was 62%. Overall and disease-free survival by colorectal cancer metastases was 35.1 and 22.4%, respectively.

[Prediction of the spatial structure of proteins: emphasis on membrane targets].

Knowledge of the spatial structure of proteins is a prerequisite for both awareness of their functional mechanisms and the framework for rational drug discovery and design. Meanwhile, direct structural determination is often hampered or impractical due to the complexity, expensiveness, and limited capabilities of experimental techniques. These issues are especially pronounced for integral membrane proteins. On numerous occasions, the theoretical prediction of protein structures may facilitate the process by exploiting physical or empirical principles. This paper surveys modern techniques for the prediction of the spatial structure of proteins using computer algorithms, and the main emphasis is placed on the most "complex" targets - membrane proteins (MPs). The first part of the review describes de novo methods based on empirical physical principles; in the second part, a comparative modeling philosophy, which accounts for the structure of related proteins, is described. Special focus is made regarding pharmacologically relevant classes of G-coupled receptors, receptor tyrosine ki-nases, and other MPs. Algorithms for the assessment of the models quality and potential fields of application of computer models are discussed.

A novel method for packing quality assessment of transmembrane alpha-helical domains in proteins.

Here we present a novel method for assessment of packing quality for transmembrane (TM) domains of alpha-helical membrane proteins (MPs), based on analysis of available high-resolution experimental structures of MPs. The presented concept of protein-membrane environment classes permits quantitative description of packing characteristics in terms of membrane accessibility and polarity of the nearest protein groups. We demonstrate that the method allows identification of native-like conformations among the large set of theoretical MP models. The developed "membrane scoring function" will be of use for optimization of TM domain packing in theoretical models of MPs, first of all G-protein coupled receptors.

[Mode of surgery completion in peritonitis]. / Sposoby zaversheniia operatsii pri peritonite.

1310 patients with various forms of peritonitis were operated during 1989-1998. The tactics of the treatment was determined depending on bacterial contamination of the abdominal cavity. In abscesses of the abdominal cavity with massive bacterial contamination (6-7 CFU/g) drainage procedure was used. Mortality rate made up 4.8%. In local extended and diffuse peritonitis with a slight bacterial contamination of the abdominal cavity (3-5 CFU/g) and in absence of fibrinous deposition fixed on peritoneum, the drainage of the abdominal cavity was not used, and laparoscopy was performed in postoperative period for the control of the course of infectious process. Mortality rate was 0.6%. In extended peritonitis with massive bacterial contamination (6-8 CFU/g) the method of repeated explorations and sanitations of the abdominal cavity was used, mortality rate being 17.8%. The overall lethality made up 7.8%. Postoperative wound infection occurred in 6.7%, intraabdominal infection as abscesses or progressing peritonitis--in 2.1% of cases.

[The efficacy of different antimicrobial preparations in treating patients with disseminated peritonitis]. / Izuchenie éffektivnosti razlichnykh antimikrobnykh preparatov pri lechenii bol'nykh s rasprostranennym peritonitom.

The results of our investigations revealed that in 68% of cases the treatment of patients with diffuse peritonitis with ampiox, gentamicin and metronidazole led to arresting the infectious process and to a considerable decrease in microbial contamination of the abdominal cavity. The use of cephalosporins and metronidazole proved to be effective in 85% of cases. In this treatment a considerable decrease in the amount of aerobic and anaerobic bacteria in peritoneal exudate was registered. In the process of treatment with cyprophloxacin positive clinical and microbiological dynamics were observed in all patients.

[The effect of different antimicrobial preparations on the normalization of the intestinal microflora in patients with disseminated peritonitis]. / Vliianie razlichnykh antimikrobnykh preparatov na normalizatsiiu mikroflory kishechnika u bol'nykh s rasprostranennym peritonitom.

These investigations revealed that the treatment of patients with generalized peritonitis with ampiox, gentamicin and metronidazole, as well as with cephalosporins and metronidazole, led to the aggravation of dysbiotic disturbances in normal intestinal microflora, observed in these patients before treatment. Ciprofloxacin had a selective decontaminating effect on the intestine, which facilitated the decrease of suppuration in postoperative wounds.

[Ciprofloxacin and other antimicrobial drugs in the treatment of disseminated peritonitis]. / Primenenie tsiprofloksatsina i drugikh antimikrobnykh preparatov pri lechenii rasprostranennogo peritonita.

Forty two patients with general peritonitis were treated with ciprofloxacin and combinations of various antimicrobial drugs. Ciprofloxacin had the highest effect on aerobic and anaerobic microbes in the peritoneal cavity which defined the favourable clinical results in all the patients. Moreover, there were observed no disorders in the composition of the normal intestinal microflora which in the end promoted a decrease in the number of the patients with suppuration of the surgical wound.