Substrate-induced modulation of protein-protein interactions within human mitochondrial cytochrome P450-dependent system.

Steroidogenesis is strictly regulated at multiple levels, as produced steroid hormones are crucial to maintain physiological functions. Cytochrome P450 enzymes are key players in adrenal steroid hormone biosynthesis and function within short redox-chains in mitochondria and endoplasmic reticulum. However, mechanisms regulating supply of reducing equivalents in the mitochondrial CYP-dependent system are not fully understood. In the present work, we aimed to estimate how the specific steroids, substrates, intermediates and products of multistep reactions modulate protein-protein interactions between adrenodoxin (Adx) and mitochondrial CYP11 s. Using the SPR technology we determined that steroid substrates affect affinity and stability of CYP11s-Adx complexes in an isoform-specific mode. In particular, cholesterol induces a 4-fold increase in the rate of CYP11A1 - Adx complex formation without significant effect on dissociation (koff decreased ∼1.5-fold), overall increasing complex affinity. At the same time steroid substrates decrease the affinity of both CYP11B1 - Adx and CYP11B2 - Adx complexes, predominantly reducing their stability (4-7 fold). This finding reveals differentiation of protein-protein interactions within the mitochondrial pool of CYPs, which have the same electron donor. The regulation of electron supply by the substrates might affect the overall steroid hormones production. Our experimental data provide further insight into protein-protein interactions within CYP-dependent redox chains involved in steroidogenesis.

SPR-Based study of affinity of cytochrome P450s / redox partners interactions modulated by steroidal substrates.

The goal of this work was to test the hypothesis that the affinity of protein-protein interactions in the cytochrome P450-dependent monooxygenase system is modulated by the low-molecular-weight compounds (substrates or inhibitors). The surface plasmon resonance (SPR) based study was carried out using the recombinant protein preparations of three microsomal cytochromes P450 (CYP17A1, CYP21A2, and CYP2C19) and their redox partners: cytochrome b5 (CYB5A), NADPH - cytochrome P450 reductase (CPR), and also iron-sulfur protein adrenodoxin (Adx). As a result, we have revealed some specificity of the influence of the steroid substrates on the binding affinity of CYPs with their redox partners, namely: the lack of effect on CPR/CYPs and Adx/CYP complex formation, and a significant effect on interactions between CYB5A and steroidogenic CYPs. The equilibrium dissociation constant (Kd) value of the CYB5A/CYP17A1 complex decreased by 5 times in the presence of progesterone (P4), which was due to a 10 times increase in the association rate constant (kon). In this case, a twofold increase in the dissociation rate constant (koff) value of CYB5A/CYP17A1 complex formation was observed. It was also demonstrated that the affinity of CYB5A/CYP17A1 interaction increased in the presence of two other steroidal substrates 17α-hydroxyprogesterone and pregnenolone and that effect was comparable with P4. In contrast, only the twofold decrease in the affinity of CYB5A/CYP21A2 interaction in the presence of P4 was caused by a slight increase in the koff value (the kon value of the complex did not change). This indicates a different format of the steroidal substrates effects expressed in a change in the stability of the CYB5A/CYPs complexes. Thus, it was found that P4 modulated the both kinetic and equilibrium constants of CYB5A/CYP17A1 and CYB5/CYP21A2 complex formation and complexes, while not affecting the CYB5A/CYP2C19 interaction (2C19 is the cytochrome P450 isoenzyme possessing broad substrate specificity), thereby indicating a specific influence of steroidal substrates on interactions involving steroidogenic CYPs. Our results are consistent with current understanding of the role of CYB5A as a regulator of cytochrome P450 activity in P450-dependent monooxygenase system.

[A key role of the internal region of the 5'-untranslated region in the human L1 retrotransposon transcription activity].

The long 5-untranslated region (5'-UTR) of the human retrotransposon L1 harbors a unique internal promoter which ensures new copies of this mobile element to be much less dependent on an integration site at the level of transcription. The mechanism of this promoter's action still remains unclear, but due to some early studies the opinion has been -formed that the most important part for its function ("minimal promoter") is the first 100-150 nts of the 5'-UTR. In this paper we show that activity of the "minimal promoter" is rather poor in comparison with the entire 5'-UTR. The absolutely crucial part which is indispensable for the effective transcription is the internal region of the 5'-UTR (+390...+662) containing multiple binding sites for various transcription factors. This region may be considered as a transcriptional enhancer. Deletion of this segment leads to a dramatic lost of transcription level irrespectively of cell type, while deletion of the first 100 nt decreases the transcription efficiency no more than 1.5 to 2-fold. Thus, the organization of the L1 regulatory region may be much more similar to that of well-studied invertebrate LINE elements than it was thought before. Also we suggest a possible existence of an alternative sense promoter within the internal part of the L1 5'-UTR driving the synthesis of a 5'-truncated mRNA of the retrotransposon.

Heart rate variability is related to disease activity and smoking in rheumatoid arthritis patients.

We investigated the heart rate variability (HRV) parameters in patients with rheumatoid arthritis (RA) and assessed their relationship with disease characteristics. Twenty-three female patients with RA [age 48+/-7 (mean+/-SD) years] free of cardiovascular diseases and 23 age- and gender-matched healthy controls were evaluated. After careful clinical examination, the following parameters were obtained after 24-h Holter recordings: average of all normal-to normal (NN) intervals over the entire 24-h ECG recording (meanNN, ms); the standard deviation for the time between NN complexes (SDNN, ms); the standard deviation of the average NN intervals for each 5-min period (SDANN, ms) and the square root of the mean-squared differences of successive NN intervals (rMSSD, ms). We also assessed quantitative parameters of the Poincaré plot: the standard deviation of the points perpendicular to the line-of-identity (SD1, ms); the standard deviation along the line-of-identity (SD2, ms) and their ratio (SD12). HRV parameters excluding SD2 were significantly lower in patients with RA, than in control group (p<0.05). Significant correlations of SDNN and SDANN with swollen joints count, Ritchie articular index, disease activity score (DAS) and disease duration were found. SDNN also correlated with leucocyte count and smoking. SD1 significantly correlated only with disease duration. Relationships between SDNN and smoking, swollen joints count and DAS were confirmed using multivariate analysis. Our data indicate that in patients with RA reduced HRV is independently associated with high disease activity and smoking. HRV assessment may be useful as a part of cardiovascular risk stratification in RA patients.

[Different effect of the ''recombinant'' bradykinin on the contractile activity of cultured atrial and ventricular cardiomyocytes]. / Razlichnoe vliianie "rekombinantnogo" bradikinina na sokrashchenie kul'tiviruemykh kardiomiotsitov predserdii i zheludochkov.

The physiological activity of the "recombinant" bradykinin expressed by retrovirus recombinant pPS-3-neo (brd) was tested on cultural atrial (aCMC) and ventricular (vCMC) cardiomyocytes in newborn rats. The "recombinant" bradykinin was shown to have a chronotropic effect on aCMC and an inotropic effect on vCMC. The effects are in line with the action of the synthetic bradykinin preparation at a concentration of around 10(-15) M. A pretreatment of CMC by parmidine, i.e. a bradykinin antagonist, blocked the effect of bradykinin. The contractive CMC activity in the cultural cell medium, transferred by pPS-3-neo without the bradykinin gene, was not different from the control value.

[Identification of genes induced to chondrocytes by nitric oxide]. / Poisk i identifikatsiia genov, indutsiruemykh v khondrotsitakh pod deistviem okisi azota.

Nitric oxide (NO) acts as a short-lived paracrine factor and selectively activates transcription of certain genes. The spectrum of inducible genes was studied in primary chondrocytes. A cDNA library was obtained by subtraction hybridization with RNAs isolated from rabbit chondrocytes before and after treatment with nitrosoglutathione, an NO-generating agent. Some of the cloned cDNAs were homologous to known mammalian genes and human EST. NO-dependent transcriptional activation was demonstrated for the stromelysin 1 and cyclooxygenase 2 genes and, for the first time, for mcl1 coding for an apoptosis suppressor.

[Retroviral vectors in gene therapy]. / Retrovirusnye vektory v gennoi terapii.

This review describes the systems of retroviral transfer and expression of the genes that are widely applied in basic biological research and in gene therapy. Unique features of retroviruses providing a background for construction of retroviral vectors and the methods to use these vectors are discussed.

Synthesis and tumor inhibiting activity of lanthanum (III) complexes with some 1-aminocycloalkancarboxylic acids.

Complexes of La(III) with 1-aminocyclopentane-, -hexane, -heptane and -4-ethylcyclohexanecarboxylic acids were obtained. The compounds were characterized by elemental analyses, IR spectroscopy and conductivity measurements. The following general formula was derived: LaL3Cl3 x 5 H2O, where L is the corresponding 1-aminocycloalkanecarboxylic acid. The pharmacological studies showed that all complexes manifested higher cytostatic and cytotoxic effects in comparison with lanthanum chloride. Much higher cytotoxic (anti-P388/D1) and cytostatic (anti-L-1210 and anti-melanoma-B16) activity was found for the lanthanum complex with 1-aminocyclopentanecarboxylic acid.

Synthesis, toxicity and immunomodulating activity of Co(II), Ni(II), Cu(II) and Zn(II) complexes of L(-)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole (levamisole).

Co(II), Ni(II), Cu(II) and Zn(II) complexes of levamisole (LMS) were prepared and characterized by elemental analyses, IR spectroscopy, 1H and 13C NMR and mass spectrometry. The following general formula was derived: M(LMS)2Cl2, where M = Co, Ni, Cu, Zn. It was established that LMS behaved as a monodentate ligand and the coordination was accomplished through the N-7 atom. The toxicity and the immunomodulating activity of the complexes on mice and rats in comparison with uncomplexed LMS was assayed. The metals in the complexes exerted different changes in the toxicity of LMS. The complex containing Zn(II) was less toxic and manifested higher immunomodulating activity than LMS.

[Standardization of environmental electromagnetic fields generated by meteorological radars]. / Reglamentatsiia élektromagnitnykh polei, sozdavaemykh meteoro- logicheskimi radiolokatorami v okruzhaiushchei srede.

An analysis of some traditional techniques for the determination of the non-working level of electromagnetic radiation of meteorological radars is provided. New approaches with higher strictness of definition and formalism are proposed.