Citrobacter freundii Methionine γ-Lyase: The Role of Serine 339 in the Catalysis of γ- and ß-Elimination Reactions.

Serine 339 of the active site of Citrobacter freundii methionine γ-lyase (MGL) is a conserved amino acid in most pyridoxal 5'-phosphate-dependent enzymes of the cystathionine ß-lyase subclass, to which MGL belongs. The reaction mechanism of the MGL-catalyzed γ-elimination reaction is poorly explored. We replaced serine 339 with alanine using site-directed mutagenesis. The replacement of serine 339 with alanine led to a significant (by two orders of magnitude) decrease in efficiency in the catalysis of the γ- and ß-elimination reactions by the mutant form of the enzyme. The exchange rates of the C-α- and C-ß-protons in the amino acids in complexes consisting of the enzyme and competitive inhibitors decreased by one-two orders of magnitude. The spectral characteristics of the mutant form indicated that the replacement did not lead to significant changes in the conformation and tautomerism of MGL internal aldimine. We crystallized the holoenzyme and determined its spatial structure at 1.7 E resolution. The replacement of serine 339 with alanine did not affect the overall course of the polypeptide chain of the MGL subunit and the tetrameric enzyme structure. An analysis of the obtained kinetic and spectral data, as well as the known spatial structures of C. freundii MGL, indicates that serine 339 is necessary for efficient catalysis of γ- and ß-elimination reactions at the stage of C-α-proton abstraction from the external aldimine, the γ-elimination reaction at the stages of coenzyme C4'-atom protonation, and C-ß-proton abstraction from a ketimine intermediate.

APPLICATION OF BIOLOGICAL TISSUE REPAIR STIMULATOR AND SEALANTS IN SURGICAL TREATMENT OF BRONCHOPLEURAL FISTULAS.

The aim of the study was to increase the effectiveness of surgical treatment of patients with pleural empyema complicated by the development of bronchopleural fistula due to the personification of the surgical approach to treatment. The cohort observation group consisted of 136 patients who were treated at the I. V. Davydovsky State Clinical Hospital No. 23 for pleural empyema complicated by bronchopleural fistula. The main group included patients in whom a biological stimulator of local tissue repair (BSRMT) was used to treat EP with BPS. The comparison group included patients who used the installation of a valvular broncho blocker for the treatment of EP with BPS. Results: the effectiveness of the use of fibrin glue ranged from 57.1% to 86.4%, depending on the etiology of the EP. The effectiveness of broncho location ranges from 80% to 84.2%. The greatest effectiveness was observed with the simultaneous use of fibrin glue and broncho blocking. The use of biological stimulants based on fibrinogen and growth factors may be effective in the treatment of various nosologies of the thoracic profile. Fibrin glue, obtained from its own blood plasma, has good adhesion indicators, is biologically inert, in addition, it has the property of stimulating repair, which is extremely important in the treatment of such pathology as bronchopleural fistulas. However, the issue of the use of fibrin glue and other sealants requires further study.

[Changes in Titin Structure during Its Aggregation].

In this paper, the property of the muscle titin protein to form in vitro specific amyloid-like aggregates is discussed. The main difference from the known amyloid aggregates is the formation of a quaternary structure that resembles cross-ß, with no changes in the secondary structure. Based on the results obtained earlier, as well as the results of this study, we make assumptions about changes in the structure of titin that occur during the formation of amyloid-like aggregates. In particular, our X-ray diffraction data on the titin aggregates suggest that ß-strands in the aggregates of this protein are not located perpendicular to the fibril axis, as described for other amyloid proteins, but in parallel. The distance between the ß-sheets in the aggregates may vary, and the ß-sheets themselves are not strictly oriented along one of the axes, which can lead to the appearance of a diffuse ring reflection of ~8-12 Å. In this regard, the titin aggregates should not be called amyloid, but amyloid-like, with a quaternary structure that resembles cross-ß. It cannot be excluded that the formation of this quaternary structure can also occur due to the partial unfolding of titin domains, followed by the interaction of open ß-strands between neighboring domains and/or domains of neighboring molecules.

Crystal structures and RNA-binding properties of Lsm proteins from archaea Sulfolobus acidocaldarius and Methanococcus vannielii: Similarity and difference of the U-binding mode.

Sm and Sm-like (Lsm) proteins are considered as an evolutionary conserved family involved in RNA metabolism in organisms from bacteria and archaea to human. Currently, the function of Sm-like archaeal proteins (SmAP) is not well understood. Here, we report the crystal structures of SmAP proteins from Sulfolobus acidocaldarius and Methanococcus vannielii and a comparative analysis of their RNA-binding sites. Our data show that these SmAPs have only a uridine-specific RNA-binding site, unlike their bacterial homolog Hfq, which has three different RNA-binding sites. Moreover, variations in the amino acid composition of the U-binding sites of the two SmAPs lead to a difference in protein affinity for oligo(U) RNA. Surface plasmon resonance data and nucleotide-binding analysis confirm the high affinity of SmAPs for uridine nucleotides and oligo(U) RNA and the reduced affinity for adenines, guanines, cytidines and corresponding oligo-RNAs. In addition, we demonstrate that MvaSmAP1 and SacSmAP2 are capable of melting an RNA hairpin and, apparently, promote its interaction with complementary RNA.

Use of Fluorescent Nucleotides to Map RNA-Binding Sites on Protein Surface.

Currently, studies of RNA/protein interactions occupy a prominent place in molecular biology and medicine. The structures of RNA-protein complexes may be determined by X-ray crystallography or NMR for further analyses. These methods are time-consuming and difficult due to the versatility and dynamics of the RNA structure. Furthermore, due to the need to solve the "phase problem" for each dataset in crystallography, crystallographic structures of RNA are still underrepresented. Structure determination of single ribonucleotide-protein complexes is a useful tool to identify the position of single-stranded RNA-binding sites in proteins. We describe here a structural approach that incorporates affinity measurement of a protein for various single ribonucleotides, ranking the RNA/protein complexes according to their stability. This chapter describes how to perform these measurements, including a perspective for the analysis of RNA-binding sites in protein and single-nucleotide crystal soaking.

Structural Aspects of Ribosomal RNA Recognition by Ribosomal Proteins.

This review is focused on the structural aspects of interaction between ribosomal proteins and ribosomal RNA in bacterial ribosomes and complexes of ribosomal proteins with specific fragments of ribosomal RNA. Special attention is given to the recognition of specific spatial architecture of the double-stranded ribosomal RNA by ribosomal proteins and to the role of unstructured protein regions in stabilization of distant ribosomal RNA segments.

Preparation and characterization of metastable trigonal layered MSb2O6 phases (M = Co, Ni, Cu, Zn, and Mg) and considerations on FeSb2O6.

MSb2O6 compounds (M = Mg, Co, Ni, Cu, Zn) are known in the tetragonal trirutile forms, slightly distorted monoclinically with M = Cu due to the Jahn-Teller effect. In this study, using a low-temperature exchange reaction between ilmenite-type NaSbO3 and molten MSO4-KCl (or MgCl2-KCl) mixtures, these five compositions were prepared for the first time as trigonal layered rosiaite (PbSb2O6)-type phases. Upon heating, they irreversibly transform to the known phases via amorphous intermediates, in contrast to previously studied isostructural MnSb2O6, where the stable phase is structurally related to the metastable phase. The same method was found to be applicable for preparing stable rosiaite-type CdSb2O6. The formula volumes of the new phases show an excellent correlation with the ionic radii (except for M = Cu, for which a Jahn-Teller distortion is suspected) and are 2-3% larger than those for the known forms although all coordination numbers are the same. The crystal structure of CoSb2O6 was refined via the Rietveld method: P3[combining macron]1m, a = 5.1318(3) Å, and c = 4.5520(3) Å. Compounds with M = Co and Ni antiferromagnetically order at 11 and 15 K, respectively, whereas the copper compound does not show long-range magnetic order down to 1.5 K. A comparison between the magnetic behavior of the metastable and stable polymorphs was carried out. FeSb2O6 could not be prepared because of the 2Fe2+ + Sb5+ = 2Fe3+ + Sb3+ redox reaction. This electron transfer produces an additional 5s2 shell for Sb and results in a volume increase. A comparison of the formula volume for the stable mixture FeSbO4 + 0.5Sb2O4 with that extrapolated for FeSb2O6 predicted that the trirutile-type FeSb2O6 can be stabilized at high pressures.

Supramolecular organization of Hfq-like proteins.

Bacterial Hfq proteins are structural homologs of archaeal and eukaryotic Sm/Lsm proteins, which are characterized by a 5-stranded ß-sheet and an N-terminal α-helix. Previously, it was shown that archaeal Lsm proteins (SmAP) could produce long fibrils spontaneously, in contrast to the Hfq from Escherichia coli that could form similar fibrils only after special treatment. The organization of these fibrils is significantly different, but the reason for the dissimilarity has not been found. In the present work, we studied the process of fibril formation by bacterial protein Hfq from Pseudomonas aeruginosa and archaeal protein SmAP from Methanococcus jannaschii. Both proteins have high homology with E. coli Hfq. We found that Hfq from P. aeruginosa could form fibrils after substitutions in the conserved Sm2 motif only. SmAP from M. jannaschii, like other archaeal Lsm proteins, form fibrils spontaneously. Despite differences in the fibril formation conditions, the architecture of both was similar to that described for E. coli Hfq. Therefore, universal nature of fibril architecture formed by Hfq proteins is suggested.

Bacterial Small Regulatory RNAs and Hfq Protein.

Small regulatory RNA (sRNA) is a unique noncoding RNA involved in regulation of gene expression in both eukaryotic and bacterial cells. This short review discusses examples of positive and negative translation regulation by sRNAs in bacteria and participation of Hfq in these processes. The importance of structure investigation of nucleotide-protein and RNA-protein complexes for designing a model of Hfq interaction with both mRNA and sRNA simultaneously is demonstrated.

[INFLUENCE OF EXTRACORPOREAL DETOXIFICATION METHODS ON TISSUE PERFUSION IN SEPTIC SHOCK].

OBJECTIVE: To improve the results of abdominal sepsis treatment by comprehensive application of extracorporeal detoxification methods controlled by tissue perfusion. SUBJECT AND METHODS: Fifteen patients with abdominal sepsis were examined, septic shock was diagnosed to all of them. Patients were divided into two groups. The first group (n = 7) consists of patients with acute renal failure, who had undergone adsorption of Lipopolysaccharide. The second group (n = 8) consists of patients with acute renalfailure, who had undergone prolonged hemofiltration regardless of the products of nitrogen metabolism level to terminate systemic inflammatory response. Dynamic monitoring of tissue perfusion was performed using Doppler ultrasound flowmeter methods. RESULTS: According to high frequency Doppler ultrasound results all the patients with abdominal sepsis have significant peripheral circulatory disorders maintaining in volumetric and linear blood flow velocity reduction. As a result of application extracorporeal detoxification methods indexes of tissue perfusion were improved. Performance of selective endotoxine hemosorbtion and hemofiltration provides substantially stabilizing effect on Doppler microcirculation indexes: average volumetric blood flow velocity (Qam) increased 4.5 times, end-diastolic linear blood flow velocity (Vakd)--increased to 85%, peripheral resistance index (RI) reduced 2.8 times. Doppler tissue perfusion indexes monitoring allows directly monitor extracorporeal detoxification methods effectiveness, supplementing system hemodynamic monitoring data. CONCLUSION: Timely application extracorporeal detoxification methods in abdominal sepsis can improve tissue perfusion.

Effect of conserved intersubunit amino acid substitutions on Hfq protein structure and stability.

Hfq is a thermostable RNA-binding bacterial protein that forms a uniquely shaped homohexamer. Based on sequence and structural similarity, Hfq belongs to the like-Sm (LSm) protein family. In spite of a rather high degree of homology between archaeal and eukaryotic LSm proteins, their quaternary structure is different, usually consisting of five to eight monomers. In this work, the importance of conserved intersubunit hydrogen bonds for the Hfq spatial organization was tested. The structures and stabilities for the Gln8Ala, Asn28Ala, Asp40Ala, and Tyr55Ala Hfq mutants were determined. All these proteins have the same hexamer organization, but their stability is different. Elimination of a single intersubunit hydrogen bond due to Gln8Ala, Asp40Ala, and Tyr55Ala substitutions results in decreased stability of the Hfq hexamer. Tyr55Ala Hfq as well as the earlier studied His57Ala Hfq has reduced protein thermostability, which seems to correspond to an opening of the protein hydrophobic core.

Using coherent X-ray ptychography to probe medium-range order.

Characterization of microscopic structural order and in particular medium range order (MRO) in amorphous materials is challenging. A new technique is demonstrated that allows analysis of MRO using X-rays. Diffraction data were collected from a sample consisting of densely packed polystyrene-latex micro-spheres. Ptychography is used to reconstruct the sample transmission function and fluctuation microscopy applied to characterize structural order producing a detailed `fluctuation map' allowing analysis of the sample at two distinct length scales. Independent verification is provided via X-ray diffractometry. Simulations of dense random packing of spheres have also been used to explore the origin of the structural order measured.

[Statistical models of longevity, aging and degradation in demography, gerontology and oncology].

Several interesting papers related with the problems of mathematical modelling in connection with a study of aging and longevity have been published recently in "Advances of Gerontology"; see, for example, [1-4, 10, 11]. Following the main idea of these papers we consider here some approaches for construction of statistical models used today in survival analysis and reliability, and which can be used in demography, gerontology and carcinogenesis studies. We expose the so called dynamic regression models, which are well adapted for studies of survival in dynamic environments.

Radiation-induced melting in coherent X-ray diffractive imaging at the nanoscale.

Coherent X-ray diffraction techniques play an increasingly significant role in the imaging of nanoscale structures, ranging from metallic and semiconductor to biological objects. In material science, X-rays are usually considered to be of a low-destructive nature, but under certain conditions they can cause significant radiation damage and heat loading on the samples. The qualitative literature data concerning the tolerance of nanostructured samples to synchrotron radiation in coherent diffraction imaging experiments are scarce. In this work the experimental evidence of a complete destruction of polymer and gold nanosamples by the synchrotron beam is reported in the case of imaging at 1-10 nm spatial resolution. Numerical simulations based on a heat-transfer model demonstrate the high sensitivity of temperature distribution in samples to macroscopic experimental parameters such as the conduction properties of materials, radiation heat transfer and convection. However, for realistic experimental conditions the calculated rates of temperature rise alone cannot explain the melting transitions observed in the nanosamples. Comparison of these results with the literature data allows a specific scenario of the sample destruction in each particular case to be presented, and a strategy for damage reduction to be proposed.

Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates.

Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ- (L-1-amino-3-methylthiopropylphosphinic acid) and ß- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

Class proximity measures--dissimilarity-based classification and display of high-dimensional data.

For two-class problems, we introduce and construct mappings of high-dimensional instances into dissimilarity (distance)-based Class-Proximity Planes. The Class Proximity Projections are extensions of our earlier relative distance plane mapping, and thus provide a more general and unified approach to the simultaneous classification and visualization of many-feature datasets. The mappings display all L-dimensional instances in two-dimensional coordinate systems, whose two axes represent the two distances of the instances to various pre-defined proximity measures of the two classes. The Class Proximity mappings provide a variety of different perspectives of the dataset to be classified and visualized. We report and compare the classification and visualization results obtained with various Class Proximity Projections and their combinations on four datasets from the UCI data base, as well as on a particular high-dimensional biomedical dataset.

RNA-binding Sm-like proteins of bacteria and archaea. similarity and difference in structure and function.

RNA-binding proteins play a significant role in many processes of RNA metabolism, such as splicing and processing, regulation of DNA transcription and RNA translation, etc. Among the great number of RNA-binding proteins, so-called RNA-chaperones occupy an individual niche; they were named for their ability to assist RNA molecules to gain their accurate native spatial structure. When binding with RNAs, they possess the capability of altering (melting) their secondary structure, thus providing a possibility for formation of necessary intramolecular contacts between individual RNA sites for proper folding. These proteins also have an additional helper function in RNA-RNA and RNA-protein interactions. Members of such class of the RNA-binding protein family are Sm and Sm-like proteins (Sm-Like, LSm). The presence of these proteins in bacteria, archaea, and eukaryotes emphasizes their biological significance. These proteins are now attractive for researchers because of their implication in many processes associated with RNAs in bacterial and archaeal cells. This review is focused on a comparison of architecture of bacterial and archaeal LSm proteins and their interaction with different RNA molecules.

Second- and third-harmonic generation and hyper-Rayleigh scattering in porous-silicon-based photonic microcavities.

Optical second-harmonic (SH) generation in porous-silicon-based photonic microcavities is observed in the form of hyper-Rayleigh scattering (HRS), with the SH radiation being diffuse and depolarized. Infiltration of sodium nitrite into the pores essentially suppresses HRS and results in generation of specular and partially polarized SH. Dissimilarly, the third-harmonic (TH) radiation from both unfilled and infiltrated samples is specular and polarized. These results indicate different spatial localization of the SH and TH sources.

[Crystal structures of mutant ribosomal proteins L1].

Nine mutant forms of ribosomal proteins L1 from the bacterium Thermus thermophilus and the archaeon Methanococcus jannaschii were obtained. Their crystal structures were determined and analyzed. Earlier determined structure of S179C TthL1 was also thoroughly analyzed. Five from ten mutant proteins reveal essential changes of spatial structure caused by surface point mutation. It proves that for correct studies of biological processes by site-directed mutagenesis it is necessary to determine or at least to model spatial structures of mutant proteins. Detailed comparison of mutant L1 structures with that of corresponding wild type proteins reveals that side chain of a mutated amino acid residue tries to locate like the side chain of the original residue in the wild type protein. This observation helps to model the mutant structures.

Macrolide resistance mechanisms among Streptococcus pneumoniae isolated over 6 years of Canadian Respiratory Organism Susceptibility Study (CROSS) (1998 2004).

BACKGROUND: Resistance to macrolides in Streptococcus pneumoniae arises primarily due to Erm(B) or Mef(A). Erm(B) typically confers high-level resistance to macrolides, lincosamides and streptogramin B (MLS(B) phenotype), whereas Mef(A) confers low-level resistance to macrolides only (M phenotype). The purpose of this study was to investigate the incidence of macrolide resistance mechanisms in Canadian isolates of S. pneumoniae obtained between 1998 and 2004. Furthermore, the genetic relatedness, serotype distribution and antibiotic susceptibility profile among S. pneumoniae isolates with dual erythromycin ribosomal methylase [Erm(B)] and efflux pump [Mef(A)] were analysed. METHODS: A total of 865 macrolide-resistant (erythromycin MIC > or = 1 mg/L) S. pneumoniae isolates were collected from the Canadian Respiratory Organism Susceptibility Study (CROSS) from 1998 to 2004. The presence of erm(B) and mef(A) was determined for each isolate by PCR; mutations in the genes coding for L4 and L22 ribosomal proteins and for 23S rRNA were identified by DNA sequencing. Each isolate containing both erm(B)- and mef(A)-mediated macrolide resistance was genotyped by PFGE and serotyped using the Quellung reaction with antisera. RESULTS: Of the 865 isolates studied, 404 (46.7%) were mef(A)-positive, 371 (42.9%) were erm(B)-positive, 50 (5.8%) were positive for both mef(A) and erm(B) and 40 (4.6%) were negative for both mef(A) and erm(B). Of the macrolide-resistant isolates negative for both mef(A) and erm(B), 22 (2.5%) contained 23S rRNA A2058G, A2059G or A2059C mutations, 7 (0.8%) contained 23S rRNA A2058G or A2059G mutations along with an S20N mutation in L4 ribosomal protein, and 1 isolate contained an E30K ribosomal protein mutation alone. Of the macrolide-resistant strains positive for both mef(A) and erm(B), 36 (72%) were multidrug-resistant (macrolide-, penicillin- and trimethoprim/sulfamethoxazole-resistant), 39 (78%) isolates belonged to serotype 19A or 19F and 36 (72%) belonged to one clonal complex (> or =80% genetic relatedness) genetically related to the Taiwan 19F-14 clone. CONCLUSIONS: The prevalence of efflux-based macrolide resistance in S. pneumoniae in Canada remained steady between 1998 and 2004. Macrolide resistance due to erm(B) decreased over the same time period, with a rapid increase in isolates with both erm(B) and mef(A) macrolide resistance.