SecM leader peptide as an allosteric translation inhibitor: a molecular dynamics study.

The SecM leader peptide regulates translation of the SecA protein, being a part of the Sec translocase, that reversibly arrests the ribosome. In the present study the structure of the SecM complex with the E. coli A/A,P/P-ribosome was obtained by means of docking and molecular dynamics simulation methods. It has been established that binding of the SecM leader peptide in the nascent peptide exit tunnel leads to a turn of the aminoacylating proline residue away from the C-terminal SecM glycine residue, which is adverse to the peptidyltransferase reaction. Besides, the SecM binding leads to a disturbance of the A-tRNA contacts with the tip of the H38 helix of the 23S rRNA (the A-site finger, ASF) and ribosomal protein uL16. Allosteric interrelation between these events has been proved by a construction of networks of concerted changes in non-covalent interactions throughout the whole ribosome, whereupon the A1614 and A751 residues of the 23S rRNA in the exit tunnel that formed stacking interactions with the SecM residues during the MD simulations, were found to be the principal triggers, inducing crucial alterations in the A-tRNA binding. The allosteric signal from the SecM peptide to the ASF, according to our model, is transmitted through ribosomal protein uL22, and there is reason to believe that this sensor is used not only by the SecM leader peptide, but also by other peptides that cause translation arrest.

Investigation of radezolid interaction with non-canonical chloramphenicol binding site by molecular dynamics simulations.

Radezolid is a promising antibiotic of oxazolidinone family, which is able to overcome effect of some linezolid resistance mechanisms of bacterial ribosomes. The structure of the radezolid complex with ribosomes was never published but, by analogy with linezolid, it is considered to prevent the binding of aminoacyl-tRNA to the A-site of the ribosome large subunit. However, as with linezolid, it can be assumed that radezolid binds to the alternative binding site existing in the A,A/P,P-ribosome. In the present article we have investigated this issue by molecular dynamics simulations and proposed the structure of the radezolid complex with a E. coli ribosome, which is consistent with available data of biochemical investigations of radezolid action.

Investigation of Allosteric Effect of 2,8-Dimethylation of A2503 in E. coli 23S rRNA by Molecular-Dynamics Simulations.

Ribosome is a molecular machine that synthesizes all cellular proteins. It also is a target of about half of the clinically used antibiotics. Adaptive chemical modification of ribosomal RNAs residues is one of the ways to provide resistance to certain antibiotics. A curious example of such modification is 2,8-dimethylation of A2503 in 23S rRNA, which induces resistance to phenols, linkosamides, oxazolidinones, pleuromutilins, and certain macrolides. In this article the effect of 2,8-dimethylation of A2503 on conformation and mobility of RNA residues of the 70S E. coli ribosome was investigated employing molecular dynamics simulations method. Significant alterations were detected both in the immediate environment of the 2503 23S rRNA residue and in the nucleotides located deeper in the nascent peptide exit tunnel (NPET), which are known to be involved in signal transmission from the antibiotics bound in the NPET to the peptidyl transferase center. These alterations shift the ribosome towards the A/A, P/P-state from the conformationally different state - P/P, E/E one in our case. The obtained results allow us to conclude that the effect of m2m8A2503 modification involves additional stabilization of the A/A, P/P-state favoring the peptidyl transferase reaction (PTR) contrary to antibiotics that inhibit PTR.

A noncanonical binding site of linezolid revealed via molecular dynamics simulations.

Linezolid, an antibiotic of oxazolidinone family, is a translation inhibitor. The mechanism of its action that consists in preventing the binding of aminoacyl-tRNA to the A-site of the large subunit of a ribosome was embraced on the basis of the X-ray structural analysis of the linezolid complexes with vacant bacterial ribosomes. However, the known structures of the linezolid complexes with bacterial ribosomes poorly explain the linezolid selectivity in suppression of protein biosynthesis, depending on the amino acid sequence of the nascent peptide. In the present study the most probable structure of the linezolid complex with a E. coli ribosome in the A,A/P,P-state that is in line with the results of biochemical studies of linezolid action has been obtained by molecular dynamics simulation methods.

A noncanonical binding site of chloramphenicol revealed via molecular dynamics simulations.

Chloramphenicol, an antibiotic belonging to the family of amphenicols, is an inhibitor of translation. On the basis of X-ray structural analysis of the binding of chloramphenicol to free bacterial ribosomes, the chloramphenicol action mechanism that consists in preventing the binding of aminoacyl-tRNA to the A-site of the large subunit of the ribosome was adopted. However, the known structures of chloramphenicol complexes with bacterial ribosomes poorly explain the results of the experiments on the chemical modification of 23S rRNA, the resistance to chloramphenicol caused by mutations in 23S rRNA and, which is particularly important, the selectivity of chloramphenicol in suppression of translation, depending on the amino acid sequence of the nascent peptide. In the present study the putative structure of the chloramphenicol complex with a bacterial ribosome in the A,A/P,P-state has been obtained by molecular dynamics simulations methods. The proposed structure of the complex allows us to explain the results of biochemical studies of the interaction of chloramphenicol with the bacterial ribosome.

Structural Insight into Interaction between C20 Phenylalanyl Derivative of Tylosin and Ribosomal Tunnel.

Macrolides are clinically important antibiotics that inhibit protein biosynthesis on ribosomes by binding to ribosomal tunnel. Tylosin belongs to the group of 16-membered macrolides. It is a potent inhibitor of translation whose activity is largely due to reversible covalent binding of its aldehyde group with the base of A2062 in 23S ribosomal RNA. It is known that the conversion of the aldehyde group of tylosin to methyl or carbinol groups dramatically reduces its inhibitory activity. However, earlier we obtained several derivatives of tylosin having comparable activity in spite of the fact that the aldehyde group of tylosin in these compounds was substituted with an amino acid or a peptide residue. Details of the interaction of these compounds with the ribosome that underlies their high inhibitory activity were not known. In the present work, the structure of the complex of tylosin derivative containing in position 20 the residue of ethyl ester of 2-imino(oxy)acetylphenylalanine with the tunnel of the E. coli ribosome was identified by means of molecular dynamics simulations, which could explain high biological activity of this compound.

Investigation of Ribosomes Using Molecular Dynamics Simulation Methods.

The ribosome as a complex molecular machine undergoes significant conformational changes while synthesizing a protein molecule. Molecular dynamics simulations have been used as complementary approaches to X-ray crystallography and cryoelectron microscopy, as well as biochemical methods, to answer many questions that modern structural methods leave unsolved. In this review, we demonstrate that all-atom modeling of ribosome molecular dynamics is particularly useful in describing the process of tRNA translocation, atomic details of behavior of nascent peptides, antibiotics, and other small molecules in the ribosomal tunnel, and the putative mechanism of allosteric signal transmission to functional sites of the ribosome.

Molecular Dynamics Investigation of a Mechanism of Allosteric Signal Transmission in Ribosomes.

The ribosome is a molecular machine that synthesizes all cellular proteins via translation of genetic information encoded in polynucleotide chain of messenger RNA. Transition between different stages of the ribosome working cycle is strictly coordinated by changes in structure and mutual position both of subunits of the ribosome and its ligands. Therein, information regarding structural transformations is transmitted between functional centers of the ribosome through specific signals. Usually, functional centers of ribosomes are located at a distance reaching up to several tens of angstroms, and it is believed that such signals are transduced allosterically. In our study, we attempted to answer the question of how allosteric signal can be transmitted from one of the so-called sensory elements of ribosomal tunnel (RT) to the peptidyl transferase center (PTC). A segment of RT wall from the E. coli ribosome composed of nucleotide residues A2058, A2059, m(2)A2503, G2061, A2062, and C2063 of its 23S rRNA was examined by molecular dynamics simulations. It was found that a potential signal transduction pathway A2058-C2063 acted as a dynamic ensemble of interdependent conformational states, wherein cascade-like changes can occur. It was assumed that structural rearrangement in the A2058-C2063 RT segment results in reversible inactivation of PTC due to a strong stacking contact between functionally important U2585 residue of the PTC and nucleotide residue C2063. A potential role for the observed conformational transition in the A2058-C2063 segment for regulating ribosome activity is discussed.

Modeling Interactions of Erythromycin Derivatives with Ribosomes.

Using a method of static simulation, a series of erythromycin A analogs was designed with aldehyde functions introduced instead of one of the methyl substituents in the 3'-N-position of the antibiotic that was potentially capable of forming a covalent bond with an amino group of one of the nucleotide residues of the 23S rRNA in the ribosomal exit tunnel. Similar interaction is observed for antibiotics of the tylosin series, which bind tightly to the large ribosomal subunit and demonstrate high antibacterial activity. Binding of novel erythromycin derivatives with the bacterial ribosome was investigated with the method of fluorescence polarization. It was found that the erythromycin analog containing a 1-methyl-3-oxopropyl group in the 3'-N-position demonstrates the best binding. Based on the ability to inhibit protein biosynthesis, it is on the same level as erythromycin, and it is significantly better than desmethyl-erythromycin. Molecular dynamic modeling of complexes of the derivatives with ribosomes was conducted to explain the observed effects.

[Heliogeophysical disturbances and exacerbations of cardiovascular disease].

A relationship between heliogeophysical disturbances and exacerbations of cardiovascular disease was studied. The index of exacerbations of cardiovascular disease was a number of calls for ambulance in Yakutsk city to patients with hypertension, hypertension crisis, myocardial infarction, cerebral stroke. Statistical processing of the experimental data, using superposed epoch technique, has allowed defection of "pre-storm" (2-4 days before the maximum of geophysical disturbances) and "post-storm" (2-4 days after it) maxima in the dynamics of referring of patients with cardiovascular pathology for emergency care.

[Original experience in plastic surgery of pelviureteral segment in hydronephrosis in children].

The study of postoperative urodynamics of the upper urinary tract was made in 339 patients with obstruction of the pyeloureteral segment and on experimental animals (morphological tests). This study revealed factors deteriorating functional capacity of anastomosis in early postoperative period. The findings allow the surgeons to update the technique of the pyeloureteral segment resection and uretero-pyeloanastomosis, to determine indications for applying nonsteroid anti-inflammatory drugs in the postoperative period, to reveal advantages and establish indications for drainage-free pyeloplasty. Thus, use of drainage-free modified technique of pyeloplasty in certain indications is a safe and less traumatic method of the patients' management which increases efficacy of Anderson-Hines operation.

[Characteristics of vertebral and muscular tonic syndromes in acute and remote periods of cervical whiplash injuries]. / Osobennosti vertebral'nogo i myshechno-tonicheskogo sindromov v ostrom i otdalennom periodakh khlystovoi travmy shei.

The study elicited the peculiarities of vertebral and muscular tonic syndromes in acute and remote periods of whip cervical trauma (WCT). Forty patients in acute period of WCT (2nd-3rd degree of severity) and 30 patients in remote period of WCT, who experienced pain and other symptoms 6 months after the trauma (late whip syndrome--LWS) were examined. The control group included 30 patients with neck and arm pain due to cervical osteochondrosis. In WCT, comparing to cervical osteochondrosis, more marked movement restriction in sagittal plane, more frequent blockade of the lower cervical spine segments, stronger correlation between pain syndrome and movement restriction in the cervical segments, more frequent muscular tonic syndrome in the anterior neck muscles and deeper neck flexors were found. In LWS, in contrast to the acute period of WCT, dissociation between more restricted active and more preserved passive movements in the cervical segments, weaker correlation between emerging of pain syndrome and restriction of movement volume, more frequent blockade of the upper cervical segments, more frequent occurrence of supraspinal muscles and shoulder-scapular syndromes were detected. The data obtained revealed a complex mechanism of symptoms formation in WCT that should be taken into account in treatment planning for acute and remote periods of cervical trauma.

[Antitoxic features of magnesium oxide under combined radiation and thermal injury]. / Antitoksichnye svoistva okisi magniia pri kombinirovannom radiatsionno-termicheskom porazhenii.

Male Wistar rats were exposed to 7.5 Gy total body gamma radiation followed to the additional full-thickness thermal bum. It was shown, that single administration of magnesium oxide in 1 hour after combined injury significantly corrected the early signs of endogenous intoxication. The level of bacterial endotoxemia decreased as well as serum concentration of toxic oligopeptides; general blood serum toxicity has been reduced too. Four-fold magnesium oxide's using as an enterosorbent in combination with antibiotics (doxycyclini, gentamicini or ciprofloxacin) has ensured 73-100% rats survival. All untreated animals dead within 30 days after combined injury.

Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development.

RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.

[Biological preparation based on Lactobacillus acidophilus, a new agent in the early treatment of combined radiation-thermal injuries]. / Biopreparat na osnove Lactobacillus acidophilus--novoe sredstvo rannego lecheniia kombinirovannykh radiatsionno-termicheskikh porazhenii.

Male mice F1 (CBA x C57BL6) were used for experiments. Animals were exposed to 7 Gy gamma-radiation and additionally inflicted to full-thickness thermal burn 10% body surface. As it has been revealed, single subcutaneous injection of the created biopreparation based on inactivated lactobacillus microbic biomass increased mice survival with combined injury from 23% to 73%. Therapeutic efficacy of this remedy did not correlate with postradiation damages of the hemopoietic system. Injection of killed L. acidophilus mixture strikingly averted development of the intestine autoinfection. Possible mechanisms of the benefit therapeutic action of the new preparation are discussed.

[Effects of a synthetic carbon-mineral sorbent and antibiotics on the development of combined radiation and thermal injury]. / Vliianie sinteticheskogo uglerod-mineral'nogo sorbenta (SUMS-1) i antibiotikov na razvitie kombinirovannogo radiatsionno-termicheskogo porazheniia.

Male Wistar rats exposed to whole-body irradiation, the midline absorbed dose was 7.5 Gy. Full-sickness thermal burn 15% of body surface inflicted immediately after irradiation. Experimental study of the therapeutic efficacy of enterosorption alone or in combination with antibiotics doxycycline and ciprofloxacin performed. The strong decrease of bacterial endotoxemia, toxic oligopeptides' level and general blood toxicity revealed after treatment compared with non-treated animals with combined injuries. Corrections of postirradiation intestinal dysbacteriosis revealed too. The best result observed when carbon mineral sorbent and antibiotics administered daily within the first 10-14 days after combined injury. Survival of treated animals increased up to 80% (all rats of control group died during 30 days after combined injury).

[Evaluation of the effectiveness of the use of pefloxacin in combined radiation-thermal injury]. / Otsenka éffektivnosti primeneniia pefloksatsina pri kombinirovannom radiatsionno-termicheskom porazhenii.

The efficacy of pefloxacin, a novel promising antibacterial drug of the group of fluoroquinolones, was studied on mice against doxycycline in the prevention and treatment of infectious complications of radiation/thermal injury. According to the data on the effect of the antibiotics on the resistance to exogenic infection and the development of dysbacteriosis pefloxacin was recommended for the use in the scheme of combined therapy of radiation/thermal injury.

[Experimental rationale for the feasibility of using sulacillin for the prevention of infectious complications of combined radiation and thermal injuries]. / Eksperimental'noe obosnovanie vozmozhnosti primeneniia sulatsillina dlia profilaktiki infektsionnykh ozlozhnenii kombinirovannykh radiatsionno-termicheskikh porazhenii.

Concurrent radiation and thermal injury (IRTI) was simulated in Wistar rats. For prevention of the autoinfectious complications sulacillin, a combination of ampicillin and sulbactam, was used. The use of sulacillin was started on the onset of IRTI and continued for 7 days. The drug was administered intramuscularly twice a day. It was observed that the 8-day survival of the animals increased by more than 40 per cent and the statistical levels of bacteremia and bacterial endotoxemia significantly decreased. The experiments showed that sulacillin had no side immunodepressive effect and did not aggravate the affection of the blood system. The drug was recommended for further studies to provide evidence for rational schemes of antibacterial therapy in IRTI.