Role of L1CAM in the Regulation of the Canonical Wnt Pathway and Class I MAGE Genes.

Molecule L1CAM is specific for nerve cells and tumors of various localizations. The expression of L1CAM is significantly higher in melanoma in comparison with benign nevi and correlates with the progress of melanoma and transition from radial to vertical growth. Monoclonal antibodies to L1CAM effectively and specifically attenuate melanoma growth, though stimulates the epithelial-mesenchymal transition. shRNA-mediated knock-down of L1CAM showed the involvement of L1CAM in regulation of activity of the canonical Wnt pathway and expression of genes of class I melanoma-associated antigens (MAGE).

[Exogenic proteins in the human exhaled breath condensate].

The analysis of the protein composition of exhaled breath to diagnose diseases of the respiratory system raises a problem of differentiation proteins of expressed in the tissues of the lungs and respiratory tract (endogenous) and got in the respiratory system from the ambient air in the process of respiration (exogenous). In this work an attempt was made to estimate a set of exhaled exogenic proteins by mass spectrometry coupled with nanoflow HPLC. Six-month isolation of healthy donors indoors with air cleaned of dust leads to removal from the spectrum of exhaled proteins of some keratins that are considered therefore to be exogenic. Non-keratin proteins may also circulate between the ambient air and human respiratory ways, but their concentration appears to be significantly lower the keratin concentrations (especially epidermis keratin). Among non-keratins dermcidin seems to be the most significant exogenic protein of exhaled air. The conclusion of the diagnostic value of exhaled proteins can be done only after careful comparison of the results of quantitative and qualitative analysis of their composition in norm and pathology for a statistically significant sample of donors.

[Genetic and social factors in developing of aggression].

The association of 5-HTTLPR gene polymorthism and aggression was studied in control group of males and females and in the athletes. The sport activities were found to decrease the aggression: the effect persist for the synchronized swimmers and for the wrestlers. Control group of males were characterized by higher aggression scores (Assault, Negativism, Suspicion and Verbal aggression scales of Buss-Durkee Hostility Inventory) compared to females. For all female-subjects irrespective of sport activities and age, the association between the variants of 5-HTTLPR gene and the Indirect Aggression and Negativism scores were found: carries of SS genotype has higher scores on Indirect Aggression and lower scores on Negativism. For the males the association was different: The averaged Hostility scores were higher for the carriers of LL-genotype. The brain processes, presumably underlying the association between aggression and 5-HTLPR gene, were studied in male control group. The increased MMN component of ERP, which responsible for the automatic change detection, and decreased P3a component, related to involuntary attention and cognitive control were found in LL-carries. It might be considered as a sign that SS-carries process the information with more cognitive resources. Probably they perceive the stimulus as more complicated, which lead to activation the additional resources of frontal cortex. It might be also suggested that the carries of SS-genotype tend to deeper processing of the incoming information. Probably, it is this more "serious" analysis of external information, which underlies the rejection of impulsive aggressive actions.

[Human temperature portrait and its relations with aerobic working capacity and the level of blood lactate].

In study with participation of 53 healthy men volunteers and infra-red thermograph application we obtained data confirming thermal portrait (i.e. skin temperature distribution in muscle rest conditions with minimal thermoregulatory activation) interrelations with maximal aerobic capacity (r = +0.6) and lactate level after critical muscle load (r = -0.7). Acute regional cooling (by 1 minute feet placing in ice water) led to temperature rise in certain breast and back skin local areas and increase in oxygen uptake, pulmonary ventilation and respiratory coefficient. Moreover lactate level in peripheral blood reduced. Summarizing obtained results we assume brown adipose tissue (BAT) activity influencing on thermal portrait formation in conditions with environmental temperature below thermoneutral. This hypothesis permits to explain negative correlation between skin temperature and body mass index and 2-fold increase in oxygen uptake during acute cold exposure. Nevertheless further investigations are needed to clarify physiological mechanisms providing significant correlation between skin temperatures in rest thermoneutral conditions on the one hand and maximal aerobic capacity, anaerobic threshold and lactate content after critical muscle load on the other hand.

[Linear B-cell epitope prediction].

Combination of sliding window method with physical properties scale of amino acids is a classical approach for linear B-cell epitope prediction. But it was shown that accuracy of these methods is poor. We reviewed classical and new algorithms of epitope prediction and present own implementation of one of them. The AAPPred software is available online at http://www.bioinf.ru/aappred/.

Short-term highly intense physiological stress causes an increase in the expression of heat shock protein in human leukocytes.

Extracellular heat shock protein with molecular weight of 70 kDa is a signal molecule of the immune system. It is secreted by the peripheral blood, liver and muscle cells in response to physiological, thermal, and mental stresses. The main goal of our study was to compare the levels of expression of heat shock protein (70 kDa) matrix ribonucleic acid in leukocytes and serum concentrations of the protein before and after physiological stress. In order to solve this problem, we developed enzyme immunoassay of serum heat shock (70 kDa) protein concentration and a method for evaluating the expression of matrix ribonucleic acid of this protein in leukocytes by the real time PCR. The concentration of 70 kDa heat shock protein in the serum increased 1.7 times as a result of even a short-term highly intense physiological stress, while the expression of its matrix ribonucleic acid in leukocytes increased 1.5 times. The individual features determine the response to physiological stress. Probable sources of 70 kDa heat shock protein are discussed.

Comparative study of stabilometric parameters in sportsmen of various disciplines.

Stabilometric parameters were compared in sportsmen of various disciplines (biathlon, boat racing, judo, and water polo). A decrease in the role of gravitational factor in sport activity was accompanied by the impairment of balance characteristics, which remained within normal limits of the mean population level.

[Modulation of cardiac rhythms in humans exposed to extremely weak alternating magnetic fields].

The influence of extremely weak alternating magnetic fields (EW AMF) with amplitudes of < or = 2 microT on the heart rate variability in humans has been studied. The volunteers were placed in a large- volume square coils system (2x2x2 m), which provided the exposure of the whole body to extremely weak alternating magnetic fields homogeneous in amplitude. It was shown that the exposure of volunteers to different types of extremely weak alternating magnetic fields can both increase and decrease the magnitude of stress. In particular, the field tuned to the nuclear spins of hydrogen atoms (amplitude 1.6 microT, frequency 76 Hz) induces a decrease in the Baevsky's stress index, while the field tuned to the magnetic moments formed by the orbiting electrons in some atoms (amplitude 0.192 microT, frequency 3000 Hz) increases the stress index. The results obtained provide a possible explanation for the mechanism of adverse effects of some particular types of technogenic and natural extremely weak alternating magnetic fields on the human cardiovascular system.

[Genetic basis of time perception in athletes].

The association between the subjective time perception and polymorphism of some genes, regulating activity of serotonin and dopamine, was studied in 89 synchronized swimmers. COMT gene, responsible for dopamine destruction, influences on reproduction of short time intervals (1-2 s). 5-HT2A and MAOA genes, regulating activity of serotonin, influence on subjective time flow. 5-HTT and COMT genes, regulating activity of serotonin and dopamine respectively, are related with accuracy of orientation in time. Association of time perception with different genes and mediators suggests different perception mechanisms, in different time ranges, in concordance with the previous physiological studies. The current study reveals that these physiological mechanisms have different molecular-neurochemical basis that helps to overcome the gap between the investigation on systemic and molecular levels.

[Investigation of ribosomes of E. coli and T. maritima by atomic force microscopy].

Subunits 70S, 50S, and 30S of ribosomes of E. coli and T. maritima have been studied by atomic force microscopy. A considerable heterogeneity of structures was visualized when 70S and 30S subunits were sorbed on mica. The linear size and the height of molecules were estimated. It was found that the heights of ribosomes of E. coli and T. maritima substantially differ. The average height of 70S ribosomes of E. coli was 9.4 + 0.01 nm and that of T. maritima was 10.35 +/- 0.02 nm. The differences in the dimensions were probably determined by special organization of the mobile ribosomal element the L7/L12-stalk.

[C-reactive protein level and rate of detection of autoantibodies to beta(1)-adrenoreceptors in patients with supraventricular tachyarrhythmias].

In order to assess concentration of C-reactive protein (CRP) and prevalence of autoantibodies against beta(1)-adrenoreceptors (abeta(1)-AR) in patients with cardiac supraventricular arrhythmias we studied 53 patients with arrhythmias and 20 healthy control subjects. Patients with idiopathic arrhythmias (atrial fibrillation or flutter and atrial tachycardia, n=35) comprised group I. Group II was formed of 15 patients with supraventricular arrhythmias and dilated cardiomyopathy (DCM) or chronic myocarditis. Patients of group III (n=23) had supraventricular arrhythmias and arterial hypertension (AH). CRP concentration was determined by recently developed well standardized high sensitivity method. abeta(1)-AR were detected in blood serum by direct immunoassay. Synthetic fragment containing 26 amino acids of abeta(1)-AR second loop was used as antigen. Patients with supraventricular arrhythmias and DCM or chronic myocarditis had higher median CRP (8.0 mg/1) than patients with idiopathic arrhythmias (0.78 mg/l), with supraventricular arrhythmias and AH (1.57 mg/l), or control group (0.6 mg/l). Groups I, II and III showed similar prevalence of ab1-AR (51.4, 40.0, 52.2%, respectively), that was significantly higher than in control subjects (10%) (p<0.005). These results provide evidence of the possible presence of autoimmune and/or inflammatory processes that may be involved in the genesis of supraventricular arrhythmias.

[Spectral parameters of heart rate variability and frequency of detection of autoantibodies to beta(1)-adrenoreceptors in patients with tachyarrhythmias: idiopathic and at the background of primary myocardial diseases].

In order to assess parameters of heart rate variability (HRV) and prevalence of autoantibodies against the beta(1)-adrenoreceptors in patients with cardiac arrhythmias we studied 42 patients with arrhythmias and 20 healthy control subjects. Thirty one patients with idiopathic arrhythmias were included in group I: with paroxysmal atrial fibrillation or flutter (n=13), paroxysmal atrial tachycardia (n=2) and paroxysmal ventricular tachycardia (n=16). Group II was formed of 11 patients with paroxysmal ventricular tachycardia and dilated cardiomyopathy or chronic myocarditis. ab1-AR were determined in blood serum by direct immunoassay. Synthetic fragment containing 26 amino acids of ab1-AR second loop was used as antigen. Groups I (54.8%) and II (63.6%) showed similar prevalence of ab1-AR, which was significantly higher than in control subjects (10%) (p<0.005). HRV parameters in I group were lower in ab1-AR-positive compared with ab1-AR-negative patients. At the same time HRV parameters in ab1-AR-positive patients were significantly different from those in controls (p<0.05). In group II HRV parameters of ab1-AR-positive and ab1-AR-negative patients were significantly lower than in control subjects (p<0.05). We suppose, that ab1-AR could participate in dysfunction of chronotropic heart regulation and contribute to development of arrhythmias in patients with structurally normal hearts.

[Selective cytotoxicity of an antibody-ricin A chain conjugate for human tumor cells]. / Izbiratel'naia tsitotoksichnost' kon''iugata antitelo-A-tsep' ritsina dlia opukholevykh kletok cheloveka.

The toxic subunit of a plant ricin has been conjugate by a disulfide bond to a polyclonal rabbit antibody specific for the L-chain of human IgG. Both the antibody and ricin A-chain retained their original biological activity after conjugation. This conjugate proved to be a potent cytotoxin for surface Ig positive Burkitt lymphoma EB-3 cells, growing in vitro and produced 50% inhibition of protein synthesis at level of 1.4 x 10(-9) M. When tested for cytotoxic action on target cells, the composite conjugate molecule was at least 100 times more effective than antibodies alone, ricin A-chain alone or a conjugate ricin A-chain--normal rabbit IgG.

[Effect of pH on the conformation and stability of the plant toxin ricin]. / Vliianie pH na konformatsiiu i stabil'nost' struktury rastitel'nogo toksina ritsina.

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.

[Ricin structure: the study by the fluorescence quenching method]. / Struktura ritsina: issledovanie metodom tusheniia fluorestsentsii.

To elucidate the details of pH-induced conformational transformation of ricin [I] in the region surrounding tryptophan residues, we studied parameters of fluorescence of the native toxin and its isolated A- and B-subunits at pH 4.0, 5.0 and 7.4. The studies were carried out using resolution of fluorescence spectra according to different degree of tryptophan accessibility to ionic (iodide) and non-ionic organic (acrylamide) quenchers. Application of the new method allowed to reveal three classes of tryptophan residues differing in their accessibility to quenchers alpha-residues are accessible neither to ions nor to organic molecules; beta-residues are accessible only to organic molecules; while surface gamma-residues are accessible to both types of quenchers. The fluorescence spectra were assessed for each class of tryptophan residues. The major part of them was shown to be localized in apolar rigid microenvironment. Fluorescence of ricin and especially of its isolated subunits proved to be strongly dependent on the pH value. At pH less than 5 the structure of B-chain loosens, this process being reflected by an increase in accessibility of tryptophan residues to quenchers. In acidic solution at least one out of seven tryptophan residues in the ricin molecule undergoes conformational transformation. Positive charge prevails in the regions surrounding quencher-accessible tryptophan residues. Binding of lactose leads to a slight compactization of the toxin structure that causes, in its turn, short-wave shifts of the fluorescence spectra and reduction of Stern-Volmer constants for intraglobular tryptophan residues.

[Selective cytotoxic effect of an immunotoxin on human erythroid tumor cells]. / Izbiratel'noe tsitotoksitchesoe deistvie immunotoksina na opukholevye éritroidnye kletki cheloveka.

The possibility of efficient directed elimination of human erythroblastoid cells by the conjugate of IgM-monoclonal antibody HAE9 directed against the erythroblast antigen and the A-chain of a plant toxin ricin has been demonstrated. The conjugate contained 2 molecules of A-chain per one antibody molecule. The efficiencies of the cytotoxic effect of native ricin and the conjugate were compared according to the number of binding sites on the surface of K562 cells as well as to the internalization rate of these molecules. As was shown, that the number of binding sites for the antibody approaches 2.7.10(4) molecules/cell, K a being equal to 1.7.10(8) M-1 while for ricin these indices constitute 2.4.10(5) and 4.6.10(8) M-1. Almost 100% of antibodies and 36% of ricin are internalized within 10 min at 37 degrees C. At a concentration 10(-11) of native ricin and 10(-10) of immunotoxin the 50% inhibition of growth of K562 cells carrying the erythroblast antigen on their surface is observed.

[The structure of toxic protein, the mistletoe lectin, at different pH: the study using intrinsic fluorescence method]. / Struktura toksichnogo belka-lektina iz omely-pri razlichnykh pH: issledovanie metodom sobstvennoi fluorestsentsii.

The effects of pH on the conformation of mistletoe lectin I and its isolated A- and B-subunits has been investigated by using the methods of intrinsic fluorescence. By the denaturating action of guanidine hydrochloride and the influence of the quenchers (I-, Cs+, acrylamide) the structural stability of the native protein and its isolated subunits was estimated. Treatment of the protein with the denaturant and quenchers revealed its different structure at pH 7.0 and 4.0. At pH 4.0 tryptophan residues become more accessible to quenchers, positive charge of the surrounding area increases and the protein becomes more stable to the action of denaturant. The structure of the isolated A- and B-chains of mistletoe lectin I differs considerably from that of the whole protein: a) its stability to the action of guanidine hydrochloride is lower; b) it depends on the ionic strength of the solvent; c) it is characterized by increased accessibility of tryptophan residues to quenchers (for B-chain). Differences between the conformations of the isolated chains at pH 7.0 and 4.0 are marked more strongly; moreover, at pH 4.5 the B-chain undergoes structural transition. The possible relationship between structural peculiarities of mistletoe lectin I and the mechanism of its transmembrane transfer is discussed.

[Interaction of the toxic plant protein--ricin, with model membranes. A fluorescence method of study]. / Vzaimodeistvie rastitel'nogo toksichnogo belka--ritsina s model'nymi membranami. Issledovanie metodom fluorestsentsii.

The fluorescence method has been used to investigate ricin and its isolated subunits interaction with some model membranes. Three liposome types were used as a model of biological membrane: 1) liposomes constructed from lecithin and cholesterol (9:1, M:M) 2) from ganglioside receptors GM1 and 3) from the mixture of GM1, lecithin and cholesterol (1:9:1). Interaction of the protein with liposome evokes changes in the parameters of both intrinsic protein fluorescence and fluorescence of the covalently bound dansyl. Binding constants were calculated from a decrease of the intrinsic fluorescence intensity as well as from the changes in the dansyl rotation anisotropy. Measurements were carried out at neutral and acidic pH. There was good correlation of the results obtained by different methods. It was shown that association constants were different for intact ricin and its subunits. The constants also depend on liposome composition and pH of the solution. The present study has demonstrated that interaction of ricin with liposome is accounted for not only by receptor centers but also by other hydrophobic regions of ricin that are inaccessible in the native toxin and may represent the region of the subunits interaction.

[Features of the structure of catalytic subunits of toxins, inhibiting protein synthesis. I. The effect of pH and interaction with the B-chain of ricin]. / Osobennosti struktury kataliticheskikh sub''edinits toksinov, ingibiruiushchikh belkovyi sintez. I. Vliianie pH, vzaimodeistvie s B-tsep'iu ritsina.

A comparative study of gelonin and A-chains of ricin, mistletoe lectin I and diphtheria toxin was undertaken. The effect of pH was studied on: a) the conformation of the proteins under study using intrinsic fluorescence; b) interaction of these proteins with ricin B-chain using gel-filtration. Structural stability of the proteins was assessed according to denaturing action of guanidine hydrochloride and temperature, and localization of tryptophan residues was determined using fluorescence quenching by I-, Cs+ and acrylamide. All investigated proteins were shown to undergo the conformational changes when a environment became acidic. In comparison with an intact protein--gelonin, the A-chains of ricin, a mistletoe lectin and a diphtheria toxin are less stable. At pH less than 5.0 tryptophan residues became more accessible to quencher and a positive charge of the surrounding area increases (in the case of gelonin it is negatively charged). No reliable interaction of a ricin B-chain with both gelonin and A-chain of diphtheria toxin was observed. The interaction of a ricin B-chain with a A-chain of mistletoe lectin I is weaker than that with ricin A-chain and is practically pH-independent.

[Role of the interchain interaction domain of chain a in viscumin cytotoxicity]. / Znachenie oblasti mezhsub''edinichnogo vzaimodeistviia v A-sub''edinitse viskumina dlia aktivnosti toksina.

The sequence coding for the viscumin (mistletoe lectin I, MLI) A-chain (MLA) was cloned from Viscum album genomic DNA with the use of synthetic primers. This yielded three recombinant (r) MLA variants differing in number of amino acid substitutions. The rMLA structure and properties were probed using monoclonal antibodies against native MLA. Native MLI B-chain (MLB) was shown to facilitate the rMLA folding. Native MLI and chimeric proteins consisting of rMLA and native MLB did not differ in cytotoxic effect on 3T3 fibroblastoid cells. Residues were identified that are located in the MLB-contacting region and have a considerable effect on the immunochemical and cytotoxic properties of rMLA.