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.

Allosteric regulation of the ribosomal A site revealed by molecular dynamics simulations.

The A site of the ribosome, which determines binding and orientation of a new amino acid residue for the peptidyl transferase reaction, was found to occupy two different conformational states upon the molecular dynamics (MD) simulations study of the 70S E. coli ribosome. One of the states, defined as "inactive", appeared in trajectories with E-tRNA, mutations A2531U and UU2492-3C, which are known to decrease the A site affinity to the tRNA. This conformational transition was found to be allosterically connected with conformational alterations in different sites of the macromolecular complex, including the E site of the ribosome and intersubunit bridge B7a located near the E site. The MD simulations of the ribosomes with the A2531U and UU2492-3C mutations known to decrease the A-tRNA retention in the ribosome, demonstrated partial switching of the 16S and 23S rRNA conformation towards its characteristic one in the P/P, E/E state.

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.

The Ribosome as an Allosterically Regulated Molecular Machine.

The ribosome as a complex molecular machine undergoes significant conformational rearrangements during the synthesis of polypeptide chains of proteins. In this review, information obtained using various experimental methods on the internal consistency of such rearrangements is discussed. It is demonstrated that allosteric regulation involves all the main stages of the operation of the ribosome and connects functional elements remote by tens and even hundreds of angstroms. Data obtained using Förster resonance energy transfer (FRET) show that translocation is controlled in general by internal mechanisms of the ribosome, and not by the position of the ligands. Chemical probing data revealed the relationship of such remote sites as the decoding, peptidyl transferase, and GTPase centers of the ribosome. Nevertheless, despite the large amount of experimental data accumulated to date, many details and mechanisms of these phenomena are still not understood. Analysis of these data demonstrates that the development of new approaches is necessary for deciphering the mechanisms of allosteric regulation of the operation of the ribosome.

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.

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.

[Functional distinction of secreting pools of the pancreas and participation of the pancreatic ductal system in the development of the pancreatic secret properties]. / O funktsional'nykh razlichiiakh sekretornykh basseinov podzheludochnoi zhelezy i uchastii ee protokovoi sistemy v formirovanii svoistv pankreaticheskogo sekreta.

Acute experiments on dogs with separate secretion of the pancreatic enzymes, either stimulated or inhibited, from two lobes of pancreas, and investigation into the character of kinetics of the secretion from 5-7 ductal pools of the pancreas showed the functional specifics, with the device of ductal valves and microdepot of a secret of the pancreas ductal system playing an important role. The conclusion is made that the final secret of pancreas removed to the duodenum is a product of a secret-motor activation of non-equipotentional microregions of the gland, being the components of an intervisceral dynamical mosaic.

[Hygienic assessment of trace element levels in drinking water and food products within the system of socio-hygienic monitoring system]. / Gigienicheskaia otsenka soderzhaniia mikroélementov v pit'evoi vode i produktakh pitaniia v sisteme sotsial'no-gigienicheskogo monitoringa.

The paper provides a comprehensive hygienic assessment of the levels of the essential and toxic trace elements chosen in terms of regional features in drinking water and foodstuffs of locally manufactured products. Priority pollutants of drinking water and foodstuffs are defined in the eastern, western, and central lands of the Orenburg Region. A significant imbalance of essential trace elements was revealed. Daily oral intake of priority and toxic trace elements were calculated in foodstuffs and drinking water.

[Characteristics of the rat behavior following single and multiple exposures of the hypoglycemic insulin doses]. / Osobennosti povedeniia krys posle odnokratnogo i mnogokratnykh vozdeistvii gipoglikemicheskikh doz insulina.

Rats behaviour in the "open field" changed in 2 hrs after a single episode of hypoglycaemia was abolished with glucose whereas no changes occurred in their stereotyped behaviour and intraspecies interaction. In 24 hrs quantitative parameters of the "open field" behaviour normalised although the behaviour still had an altered structure. At the same time, amphetamine-induced stereotyped behaviour's indices became reduced. These and other findings suggest some deeper behavioural changes in rats during their recovery from repeated hypoglycaemic episodes and absence of synchronisation of the insulin effects.

[Effect of a 50 Hz magnetic field on rat behavior depends on the direct current component]. / Vliianie magnitnogo polia chastotoi 50 Gts na povedenie krys zavisit ot znacheniia postoiannogo magnitnogo polia.

The influence of extremely low frequency magnetic field on rats behavior (working memory, learning, social activity) was examined. Rats were exposed to magnetic fields at three different conditions: exposure 1-50 Hz, 46 mT; DC 65 mTl; exposure II-50 Hz, 46 mTl; DC 14-17 mTl; Exposure III-sham. Field related changes were observed after exposure I: the increase of error rate in Y-maze performance, shorter adaptation time in instrumental performance, the decrease of aggression and sociability, enhancement of the defence reactions. Data obtained show that influence of 50-Hz magnetic field depends on the level of DC field. The combination presumably considered as resonant to Ca++ was more effective.

[The sequelae of the intraspecific isolation of adult rats (right-handed, left-handed and ambidextrous animals)]. / Posledstviia vnutrividovoi izoliatsii vo vzroslom vozraste krys (pravshei, levshei i ambidekstrov).

We studied the influence of social isolation at the age of 2 months on zoosocial behaviour of mixed-bred male rats with different paw preference. Paw preference was determined in the test of reaching for food in the horizontal tube. The level of aggression but not sociability was found to increase significantly in dextral and ambidextrous late isolants. In sinistrals the increase of sociability but not aggression was observed. Analysis of probabilistic ethological structure of aggressive behaviour showed its validity and revealed the appearance of pathological aggression as a result of isolation in all groups of animals. Being connected with a disfunction of the regulating role of the right brain hemisphere this feature of aggressive behaviour is most pronounced in dextrals and ambidextrals.

[The modification of the intraspecific behavior of male rats raised in isolation with different variants of interaction with related animals]. / Modifikatsiia vnutrividovogo povedeniia krys-samtsov, vyrashchennykh v izoliatsii, pri razlichnykh variantakh vzaimodeistviia s sorodichami.

It was found that intraspecific behaviour of male rats having been transformed in consequence of breeding under the conditions of social isolation could be modified afterwards by an experience of social contacts. Long-term keeping the isolatedly bred rats with their mates in adulthood decreases the level of intraspecific sociability but does not affect the relatively high level of aggression of the animals under study. On the contrary, the short-term periodic social contacts with the mates in a situation of competition for water decrease only the level of aggression but not that of intraspecific sociability. However, in any case the transformations of the species-typical ritual of aggressive behaviour in consequence of early isolation are steady and are not corrected by later social contacts.

[The capacity for competition in rats raised in isolation]. / Sposobnost' k konkurentsii u krys, vyrashchennykh v izoliatsii.

It is shown, that rats, bred in conditions of intraspecies isolation are able to competition for water in the kin group, having usual experience of intraspecies intercourse. At the same level of drinking motivation in conditions of competition for water the intraspecies activity is significantly higher in rats, bred in isolation in comparison with grouped animals. Rats-isolants significantly more often than the grouped animals use extraordinary tactics of intraspecies behaviour, which increase their competitive ability in conditions of limited access to water.

[The role of the frontal cortex of the left and right hemispheres in regulating the intraspecific interaction of rats with differing zoosocial experience]. / Izuchenie roli frontal'noi kory levogo i pravogo polusharii mozga v reguliatsii vnutrividovogo vzaimodeistviia u krys s razlichnym zoosotsial'nym opytom.

By means of the analysis of probabilistic ethological structures of the intraspecies rats behaviour in the test "stranger-resident" and by the method of isolated destruction of the frontal cortex or the left and right hemispheres,--it is established that the frontal cortex of the left hemisphere regulates the intraspecies sociability and keeps it on a certain level, because the change (increase) of the intraspecies sociability is observed only at the unilateral destruction of the left frontal cortex. Isolation from the relatives in early ontogenesis leads to the loss of regulating influence of the left frontal cortex on the intraspecies sociability. In isolated animals the frontal cortex of the left hemisphere regulates the level and probability of the appearance of aggression. The right hemisphere ensures the adequate inclusion of the aggression in the probabilistic ethological structure of the total intraspecies behaviour; at its damage the forms appear of an adequate pathological aggression, and connection is lost between the elements of aggressive behaviour and the acts of intraspecies sociability.

[Characteristics of the intraspecies interaction of rats with differing zoosocial experience]. / Osobennosti vnutrividovogo vzaimodeistviia u krys s razlichnym zoosotsial'nym opytom.

It has been found that among white outbred rats with different experience of intraspecies contact four types of reaction to the partner are singled out in the "stranger-resident" test: active, neutral passive, highly-aggressive. Belonging to one or another type is determined by the level and correlation of aggression and intraspecies sociability. Among animals isolated in an adult age, all four types of reaction to the partner are represented while in rats kept in isolation since 13--14th day of life only one type is presented--the active one. For animals of the control group the first three types of reaction are typical. It is revealed that only "late" isolation leads to a pathological increase of aggressivity in a part of the animals; an increase of aggression in "early" isolants may be considered as a consequence of sharp rise of intraspecies sociability. Conclusion is made about principal difference in mechanisms of aggression increase in "early" and "late" isolants.