S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.     

Analysis of polymorphism at nine nuclear genome DNA loci in maris

Population genetic survey of the indigenous populations of the Marii El Republic, represented by the two major ethnographic groups of Maris, Meadow (five samples from Morkinsk, Orshansk, Semursk, Sovetsk, and Zvenigovsk districts) and Mountain (one sample from Gornomariisk district) Maris, was carried out. All Mari groups were examined at nine polymorphic DNA loci of nuclear genome, VNTR(PAH) (N = 422), STR(PAH) (N = 152), VNTR(ApoB) (N= 294), VNTR(DAT1) (N = 363), VNTR(eNOS) (N = 373), ACE (N = 412), IVS6aGATT (N = 513), D7S23(KM.19) (N = 494), and D7S8 (N = 366). Allele and genotype frequency distribution patterns were obtained for individual samples and ethnographic groups, as well as for the ethnic group overall. In each of six Mari samples examined, the deficit of heterozygotes was observed, i.e., the mean observed heterozygosity was lower than the expected one. The indices of mean heterozygosity, Hs = 0.455, and interpopulation differentiation, FST = 0.0024, for the Mari gene pool were obtained using a set of DNA markers analyzed. Analysis of the genetic distances and between population differentiation (FST) showed that the main part of genetic diversity in Maris was determined by the differentiation between the populations of Meadow Maris. The contribution of the differences between the ethnographic groups of Mountain and Meadow Maris to the ethnic gene pool was small. It is suggested that the main role in the formation of the Mari gene pool is played by the geographic factor.     

Thermodynamics of GTP and GDP Binding to Bacterial Initiation Factor 2 Suggests Two Types of Structural Transitions

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 °C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 °C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (− 868 ± 25 and − 577 ± 23 cal mol^− 1 K^− 1, respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the γ-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.     

Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization

Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.     

Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB,and its role in benzothiazinone resistance

Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl‐β‐d ‐ribose 2′‐epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro‐group to an amino‐group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB‐BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.     

Compact internal representation of dynamic situations: neural network implementing the causality principle

Animals for survival in complex, time-evolving environments can estimate in a “single parallel run” the fitness of different alternatives. Understanding of how the brain makes an effective compact internal representation (CIR) of such dynamic situations is a challenging problem. We propose an artificial neural network capable of creating CIRs of dynamic situations describing the behavior of a mobile agent in an environment with moving obstacles. The network exploits in a mental world model the principle of causality, which enables reduction of the time-dependent structure of real situations to compact static patterns. It is achieved through two concurrent processes. First, a wavefront representing the agent’s virtual present interacts with mobile and immobile obstacles forming static effective obstacles in the network space. The dynamics of the corresponding neurons in the virtual past is frozen. Then the diffusion-like process relaxes the remaining neurons to a stable steady state, i.e., a CIR is given by a single point in the multidimensional phase space. Such CIRs can be unfolded into real space for execution of motor actions, which allows a flexible task-dependent path planning in realistic time-evolving environments. Besides, the proposed network can also work as a part of “autonomous thinking”, i.e., some mental situations can be supplied for evaluation without direct motor execution. Finally we hypothesize the existence of a specific neuronal population responsible for detection of possible time-space coincidences of the animal and moving obstacles.     

Cytotoxicity of RNases is increased by cationization and counteracted by K(Ca) channels

K(Ca) channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of K(Ca) channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing K(Ca) channels was found. These results suggest that cells without the K(Ca) channel activity cannot counteract toxic effect of RNases.     

Interaction between sensory and cognitive processes in visual recognition: the role of the associative areas of the cerebral cortex

Monkeys were taught to differentiate stimuli with different types of information and to perform a spatial choice. Development of Alzheimer's disease in experimental monkeys entailed a deficit of operative memory, a considerable enhancement of entropy related to correct decisions. In control monkeys, no significant changes of these characteristics occurred. In monkeys, following a bilateral removal of the sulcus principalis, the 7th field, or both, the operative memory deficit was also determined by two components. The specifics of disorders in the operative memory due to Alzheimer's disease suggest that cholinergic mechanisms determining the sensory processing differ from those involved in decision-making. The structural-functional organization of interaction between sensory and cognitive processes controlled by the motivation and attention system, is discussed.     

Cortical neuronal connections and reconstruction of visual space

We investigated distribution of retrograde-labelled cells in cortical areas 17, 18, and the transition zone 17/18 of both hemispheres in cats after microiontophoretic horseradish peroxidase (HRP) injections into the single cortical columns of area 17, 18, 19 or 21a. On the base of clustered pattern of intrinsic labelling, asymmetric location of labelled callosal cells that was associated with the appropriate pattern of labelling in layers A and A1 of lateral geniculate nucleus, we suggest that cortical neuronal connections are eye-specific and may provide for each eye a separate binding of visual hemifields. After HRP injections into columns of area 19 or 21a, the disparate inputs from areas 17, 18 and transition zone 17/18 were revealed. Such connections may provide a local depth information and the selection of stereoscopic surfaces in central sectors of visual space.