Microembolic Signals in Arteries of the Base of the Brain after Ischemic Stroke.

Embolic strokes make up a significant proportion of acute cerebrovascular accidents. Doppler blood flow monitoring with microembolodetection allows suggesting the embolic nature of cerebral infarction. The aim of this study was to identify factors associated with the presence of microembolic signals in patients with ischemic stroke. The study included 515 patients, microembolic signals were detected in 48 (9.3%) of them. According to multispiral CT angiography, significant differences in patients with and without microembolic signals were found for wall thickness of both common carotid arteries and for left internal carotid artery (p<0.05). Factor analysis revealed a variable that reflects the severity of left carotid arteries atherosclerosis, which was a significant predictor of registration of the microembolic signals (p=0.016).

[The Activities of Representatives of Companies-Manufacturers of Pharmaceuticals Related to Informing Specialists with Purpose of Efficient Application of Medications].

The article presents the role of representative of company-manufacturer of pharmaceuticals in informing health care specialists about medications according WHO documentation. The analysis of regulation of procedure and permitted modes of their interaction with specialists in the Russian Federation. Also, an opinion of medical specialists is presented concerning the role of representatives of pharmaceutical companies in the portion of informing about medications. The results of questionnaire survey of medical representatives from the position of their role and tasks are presented concerning information activity during interaction with specialists. The proposals are formulated concerning necessity of legislative regulation of activities of representatives of the companies-manufacturers of pharmaceuticals.

Apoptotic Endonuclease EndoG Inhibits Telomerase Activity and Induces Malignant Transformation of Human CD4+ T Cells.

Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active full-length hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.

Intracellular Localization of Apoptotic Endonuclease EndoG and Splice-Variants of Telomerase Catalytic Subunit hTERT.

The activity of telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) can be regulated by alternative splicing of its mRNA. The mechanism of hTERT splicing is not understood in detail. Apoptotic endonuclease EndoG is known to participate in this process. In the present work, the intracellular colocalization and mRNA levels of EndoG and hTERT splice-variants in normal and apoptotic cancer cells were studied. We found that the development of apoptosis increased the expression of EndoG and changed the ratio of hTERT splice-variants, which decreased the telomerase activity in the cells. The development of apoptosis was accompanied by changes in the amount of mRNA and in the localization of EndoG and hTERT splice-variants in the cytoplasm, nuclei, and mitochondria of the cells. The suppression of EndoG expression using RNA interference prevented induction of the α+ß- splice-variant of hTERT and inhibition of the telomerase activity. A high degree of the intracellular colocalization of EndoG and hTERT was shown. The changes in the expression and localization of EndoG corresponded with changes in the amount and localization of hTERT splice-variants. These data confirm the participation of EndoG in the alternative splicing of mRNA of the telomerase catalytic subunit and in regulation of the telomerase activity.

[LABORATORY AND EXPERIMENTAL STUDY OF THE COMPLEX PROBIOTIC PREPARATION "BIFILACT-BILS" IN CAPSULATED FORM].

The composition and technology of complex probiotic in hard gelatin capsules was developed in Perm Branch "Biomed" of "Microgen" State Company. The preparation contains three production strains: Lactobacillus plantarum 8P-A3, L. acidophilus K3W24 and Bifidobacterium bifidum 1. Laboratory and experimental (preclinical) study of the probiotic included investigation of the antagonistic activity, "acute" and "chronic" toxicity, the effect of the preparation on histology and hematology of laboratory animals. The result of these studies suggested of the probiotic had high inhibitory activity against pathogenic microflora when compared with probiotic monopreparations and had no toxic effects on laboratory animals.

[The comparative effectiveness of high-intensity dynamic training with the use of exercise machines and therapeutic gymnastics for the joints in the patients presenting with early rheumatoid arthritis].

The objective of the present work was to compare the effectiveness of two therapeutic exercise programs for the patients presenting with early rheumatoid arthritis (RA). The study included 51 patients. Fifteen of them (group 1) were given conventional medicamental therapy in combination with high-intensity dynamic physical exercises with the use of the Enraf-Nonius training devices (45-60 min). Eighteen patients of group 2 were offered 10 sessions of remedial gymnastics for the joints (45 min each) under the guidance of an instructor that were continued under the domestic conditions (45 min each session thrice weekly for 3 months). Eighteen patients of group 3 were given medicamental therapy alone (control). The parameters estimated in the study included the mean strength of knee joint extension and ankle joint flexion measured with the use of En-TreeM devices, articular pain (100 mm BAHI), DAS28, HAQ, and RAPID3 indices. It was shown that both programs of therapeutic exercises reduced the severity of the disease, improved the functional and motor activity of the patients and their quality of life. The majority of these characteristics were significantly different from those documented in the control group (p<0.05). The clinical effectiveness of high-intensity training with the use of exercise machines was higher than without them (articular pain was reduced by 57.9% (p<0.01), DAS28 by 24.7% (p<0.05), HAQ by 60.7% (p<0.01). RAPID3 by 47.5% (p<0.01), mean strength of extension of the weak and strong knee joints increased by 87.9% (p<0.01) and 70.5% (p<0.01) respectively, the strength of flexion of the severely and less severely affected ankle joints increased by 84.6 (p<0.01) and 68.8% (p<0.01) respectively. Compliance with regular performance of therapeutic joint exercises during 3 months was higher (83.3%) than with high-intensity dynamic training with the use of exercise machines (60%). It is concluded that the latter modality should be recommended to the younger patients with RA (below 40 years), a short history of the disease, and its low activity.

[Combined step-by-step rehabilitation of the patients presenting with early-onset rheumatoid arthritis].

The objective of the present study was to develop the program for combined step-by-step rehabilitation of the patients presenting with early-onset rheumatoid arthritis (RA); the secondary objective was to estimate the effectiveness of this program. A total of 34 patients were recruited for the participation in the study. They received medicamental therapy in combination with the rehabilitative treatment during 6 months. The hospital-based treatment included therapeutic exercises for large joints under the supervision of a specialist (45 min), occupational therapy (45 min), local aerial cryotherapy of wrist, knee, and ankle joints (10 sessions 15 min each at a temperature of -60 degrees C), ortheses, and the educational program (4 daily studies 90 min each). The outpatient and home-based treatment included therapeutic exercises for large joints (45 min), wrist exercises (45 min) three times every week, ortheses. 26 patients received only medicamental therapy (control group). The following characteristics were measured: the average power of extension of knee joints and of flexion of ankle joints (by means of En-TreeM analysis of movements), wrist grip strength, articular pain (100 mm VAS, DAS28, HAQ, RAPID3 indices). The rehabilitative program ensured excellent compliance with basal therapy, reduced requirements for symptomatic medicines, and improved adherence to the methods for the formation of the correct movement patterns, orthesis wearing, and regular therapeutic exercises. The rehabilitative treatment resulted in the relief of articular pain by 70.4% (p < 0.01), decrease of DAS28 by 31.9% (p < 0.05), HAQ by 75.8% (p < 0.01), and RAPID3 by 60.1% (p < 0.01). The grip strength of the more seriously injured wrist increased by 44.9% (p < 0.05) and that of the less damaged one by 31.3% (p < 0.05). The average extension power of the weaker knee joint increased by 88.7% (p < 0.01) and that of the stronger joint by 67.7% (p < 0.01). The average flexion power of the more seriously injured ankle joint increased by 81.6% (p < 0.01) and that of the less damaged one by 70.2% (p < 0.01). The two groups were significantly different in terms of the majority of characteristics evaluated. It is concluded that the combined rehabilitative treatment helps to control the activity of the disease, enhances the functional abilities, improves the locomotor activity and quality of life of the patients with early-onset rheumatoid arthritis.

Restoration of the Indicator Properties of Whole-cell Luminescent Biosensors.

Whole-cell biosensors are widely used to produce medical diagnostic tests, but in the long term, they tend to lose their indicator properties. Consequently, it is crucial to find ways to restore these properties and prolong the shelf life of the tests. Here, we propose to use electromagnetic radiation with optimally selected parameters of frequency, power, and exposure time. The impact of radiation parameters on biosensor luminescence was studied as well as the effects of different types of radiation coming from laser sources (λ = 875 nm), a LED source (λ = 850 ÷ 890 nm), and microwave units (at frequencies 42.22, 53.53, 61.18 и 34 ÷ 38 GHz). IR treatment resulted in dose-dependent suppression of biosensor luminescence. The luminescence level when exposed to microwave radiation depends on the radiation time and frequency. Also, it has been found that optimal selection of the main radiation parameters enables to restore indicator properties partially lost by biosensors during storage. We explain the mechanism responsible for the sensitizing effect of radiation, which implies the polarization of solvent dipoles and changes in mobility of acceptor molecules. This, in turn, leads to a shift in the chemical equilibrium states and triggers a cascade of biochemical reactions that lead to restoration of the lost indicator properties of biosensors. The study of antagonistic activity has revealed that restored biosensors provide reliable test results after the expiration of their warranty period.

Structural Study of Heterogeneous Biological Samples by Cryoelectron Microscopy and Image Processing.

In living organisms, biological macromolecules are intrinsically flexible and naturally exist in multiple conformations. Modern electron microscopy, especially at liquid nitrogen temperatures (cryo-EM), is able to visualise biocomplexes in nearly native conditions and in multiple conformational states. The advances made during the last decade in electronic technology and software development have led to the revelation of structural variations in complexes and also improved the resolution of EM structures. Nowadays, structural studies based on single particle analysis (SPA) suggests several approaches for the separation of different conformational states and therefore disclosure of the mechanisms for functioning of complexes. The task of resolving different states requires the examination of large datasets, sophisticated programs, and significant computing power. Some methods are based on analysis of two-dimensional images, while others are based on three-dimensional studies. In this review, we describe the basic principles implemented in the various techniques that are currently used in the analysis of structural conformations and provide some examples of successful applications of these methods in structural studies of biologically significant complexes.

[Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes]. / Indutsirovannaia tsisplatinom ékspressiia apoptoticheskoi éndonukleazy EndoG vyzyvaet ingibirovanie aktivnosti telomerazy i zlokachestvennuiu transformatsiiu CD4+ T-limfotsitov cheloveka.

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

[Induction of apoptotic endonuclease EndoG with DNA-damaging agents initiates alternative splicing of telomerase catalytic subunit hTERT and inhibition of telomerase activity hTERT in human CD4+ and CD8+ T-lymphocytes]. / Induktsiia apoptoticheskoi éndonukleazy EndoG povrezhdaiushchimi DNK agentami vyzyvaet al'ternativnyi splaising kataliticheskoi sub"edinitsy telomerazy hTERT i ingibirovanie aktivnosti telomerazy v CD4+ I CD8+ T-limfotsitakh cheloveka.

Activity of telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At present time exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ T-lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ T-cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.

The 70S Escherichia coli ribosome at 23 A resolution: fitting the ribosomal RNA.

BACKGROUND: The ribosome--essential for protein synthesis in all organisms--has been an evasive target for structural studies. The best available structures for the 70S Escherichia coli ribosome or its 30S and 50S subunits are based on electron microscopical tilt experiments and are limited in resolution to 28-55 A. The angular reconstitution approach, which exploits the random orientations of particles within a vitreous ice matrix, can be used in conjunction with cryo-electron microscopy to yield a higher-resolution structure. RESULTS: Our 23 A resolution map of the 70S ribosome elucidates many structural details, such as an extensive system of channels within the 50S subunit and an intersubunit gap ideally shaped to accommodate two transfer RNA molecules. The resolution achieved is sufficient to allow the preliminary fitting of double-helical regions of an earlier three-dimensional ribosomal RNA model. CONCLUSIONS: Although we are still a long way from attaining an atomic-resolution structure of the ribosome, cryo-electron microscopy, in combination with angular reconstitution, is likely to yield three-dimensional maps with gradually increasing resolution. As exemplified by our current 23 A reconstruction, these maps will lead to progressive refinement of models of the ribosomal RNA.

The Escherichia coli large ribosomal subunit at 7.5 A resolution.

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.

[Reproductive function in chronic abacterial prostatitis patients].

The results of examination of 325 patients with chronic abacterial prostatitis, varicocele showed marked changes in ejaculate parameters of these patients. Prostatitis of different duration in the same degree causes pathozoospermia, reduces ejaculate fertility. Such condition of spermatogenesis indicates involvement of other sex organs suggested by the literature data and previous experience. This necessitates extended diagnostic examination of patients with chronic prostatitis.

[Clinical implications of E. coli pathogenic factors in patients with urinary infection].

The authors show perspectives of using primers detecting nucleotide gene sequences in isolated E. coli strains; pathogenic factors of E. coli, fimbrial adhesive (type I and type P); alpha-hemolysin (hly); cytotoxic necrotising factor I (cnf-1); iron-regulated proteins for predicting the course of the inflammatory process in urological patients with bacterial infection.

[Dimethylfumarate in the treatment of relapsing-remitting multiple sclerosis]. / Dimetilfumarat v terapii remittiruyushchego rasseyannogo skleroza.

This review includes results of experimental and clinical studies of dimethyl fumarate, a new oral drug for pathogenetic treatment of multiple sclerosis (MS). The mechanism of action, data from clinical trials, including MRI-results related to tolerability and safety of the drug are reviewed. The risk management plan for possible adverse events and a place of dimethyl fumarate in the current pathogenetic treatment of MS are discussed.

[Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT]. / Reguliatsiia al'ternativnogo splaisinga kataliticheskoi sub"edinitsy telomerazy hTERT apoptoticheskoi éndonukleazoi EndoG.

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of b-deletion splice variant was determined during EndoG over-expression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.

Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 A resolution by electron cryomicroscopy and angular reconstitution.

A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to approximately 45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the "functional units" within the dimeric "morphological units" of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.

Structural organisation of the head-to-tail interface of a bacterial virus.

In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-naïve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.