The effect of 15-ketosterol analogues with a 5,6-dimethylhept-3-en-2-yl chain at C17 on cholesterol metabolism in Hep G2 hepatoma cells

The effect on cholesterol metabolism in Hep G2 hepatoma cells was studied for new analogues of 15-ketosterol [3beta-hydroxy-5alpha-cholest-8(14)-en-15-one] (I): (24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-diene-15-one (II), (24S)-3alpha-hydroxy-24-methyl-5-alpha-cholesta-8(14),22-diene-15-one (III), and (24S)-24-methyl-5alpha-cholesta-8(14),22-diene-3,15-dione (IV). Analogues (I) and (II) were found to be equally effective inhibitors of cholesterol biosynthesis after a 3-h incubation with Hep G2 cells; however, (II) produced a stronger inhibitory effect after a 24-h incubation or after an incubation of cells preliminarily treated with the inhibitor in a medium containing no ketosterol. The ability of ketosterols to inhibit cholesterol biosynthesis decreased in the order (II) > (IV) > (III). Ketosterol (II) inhibited, whereas ketosterol (III) stimulated the biosynthesis of cholesteryl esters. (IV) stimulated the biosynthesis of cholesteryl esters at a concentration of 1-10 microM and exerted no marked effect at a concentration of 30 microM. These results indicate that delta8(14)-15-ketosterols containing a modified side chain are of interest as regulators of cholesterol metabolism in liver cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.     

Changes in the Activity of Antioxidant Enzymes in Wheat Leaves and Roots as a Function of Nitrogen Source and Supply

The activity of enzymes participating in the systems of antioxidant protection was assayed in the second leaf and roots of 21-day-old wheat seedlings (Triticum aestivum L.) grown in a medium with nitrate (NO^– ₃ treatment), ammonium (NH⁺ ₄ treatment), or without nitrogen added (N-deficiency treatment). The activities of superoxide dismutase (SOD), peroxidase, ascorbate peroxidase, glutathione reductase, and catalase in the leaves and roots of the NH⁺ ₄ plants was significantly higher than in the plants grown in the nitrate medium. The activity of SOD decreased and ascorbate peroxidase markedly increased in leaves, whereas the activity of ascorbate peroxidase increased in the roots of N-deficient plants, as compared to the plants grown in nitrate and ammonium. Low-temperature incubation (5°, 12 h) differentially affected the antioxidant activity of the studied plants. Whereas leaf enzyme activities did not change in the NH⁺ ₄ plants, the activities of SOD, peroxidase, ascorbate peroxidase, and catalase markedly increased in the NO^– ₃ plants. In leaves of the N-deficient plant, the activity of SOD decreased; however, the activity of other enzymes increased. In response to temperature decrease, catalase activity increased in the roots of NO^– ₃ and NH⁺ ₄-plants, whereas in the N-deficient plants, the activity of peroxidase increased. Thus, in wheat, both nitrogen form and nitrogen deficiency changed the time-course of antioxidant enzyme activities in response to low temperature.     

Effect of salt stress on antioxidant system of plants as related to nitrogen nutrition

In plants of wheat (Triticum aestivum L.) grown in the media with nitrate (NO ₃ ^? plants), ammonium (NH ₄ ⁺ plants), and without nitrogen (N-deficient plants), the response to oxidative stress induced by the addition of 300 mM NaCl to the nutrient solution was investigated. Three-day-long salinization induced chlorophyll degradation and accumulation of malondialdehyde (MDA) in the leaves. These signs of oxidative stress were clearly expressed in NO ₃ ^? and N-deficient plants and weakly manifested in NH ₄ ⁺ plants. In none of the treatments, salinization induced the accumulation of MDA in the roots. Depending on the conditions of N nutrition, salt stress was accompanied by diverse changes in the activity of antioxidant enzymes in the leaves and roots. Resistance of leaves of NH ₄ ⁺ plants to oxidative stress correlated with a considerable increase in the activities of ascorbate peroxidase and glutathione reductase. Thus, wheat plants grown on the NH ₄ ⁺ -containing medium were more resistant to the development of oxidative stress in the leaves than those supplied with nitrate.     

Substrate specificity and biochemical properties of M3.BstF5I DNA methyltransferase from the BstF5I restriction-modification system

Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of λ phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min⁻¹) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5′-GGATC-3′ DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5′-GGATG-3′ recognition site.     

Antibodies against synthetic peptide fragments of T-cadherin inhibit binding of low density proteins with T-cadherin]

Potential antigenic determinants of the atypical lipoprotein-binding proteins T-cadherin (p105) and its precursor (p130) from cells of human smooth muscles were synthesized by the solid phase method according to the Fmoc-scheme. These corresponded to the 51-61, 140-160, 161-179, 260-271, 340-352, 350-362, and 370-385 sequences of p130 and were chosen on the basis of computer analysis of its antigenic structure. The conjugates of the peptides with horseradish peroxidase were used for the immunization of mice and rabbits. Antisera against the peptides corresponding to the 140-160, 161-179, and 260-271 sequences of p105 were shown by immunoblotting to react with p105, which we isolated from the vascular cells of smooth muscles and earlier identified as T-cadherin. These antisera inhibited the binding of low density lipoproteins with p105 in a dose-dependent manner. These results confirmed the identification of the p105 protein as T-cadherin and demonstrated the fundamental possibility of studying the interaction of this protein with low density lipoproteins by using antipeptide antibodies that inhibit binding.     

The Effect of Long-term Space Flights on Human Urine Proteins Functionally Related to Endothelium

It is known that long-term space flights lead to dysregulation of the cardiovascular system, and the endothelium is the most important functional element of such dysregulation. In order to find the signs of endothelial dysfunction in cosmonauts who have been in long-term space flights, we collected urine samples from 21 cosmonauts before the flight and on the first and seventh days after landing. The urine samples were investigated by chromatography–mass spectrometry analysis. Proteins were identified using the MaxQuant software and the SwissProt database. The software package Perseus was used for semi-quantitative analysis. The reconstruction of associative molecular networks was performed using the ANDSystem software. We identified 200 different proteins in urine samples of 21 Russian cosmonauts. The ANDSystem software made it possible to determine seven processes related to endothelium functioning. These processes had direct relations to 17 urine proteins, which were functionally associated with the endothelium. At the same time, eight proteins (such as serotransferrin, prostate-specific antigen, fibrinogen gamma chain, UFO tyrosine kinase receptor, aminopeptidase N, vascular cell adhesion molecule 1, osteopontin, and syndecan-4) were significantly changed (p < 0.01) at different points of the recovery period (the first and seventh days). Thus, we performed the first study of the urine protein composition in cosmonauts for the evaluation of signs of endothelial dysfunction after space flight using proteomics methods.     

M.BstF5I-2 and M.BstF5I-4 DNA Methyltransferases from BstF5I Restriction–Modification System of Bacillus stearothermophilus F5

The BstF5I restriction–modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction–modification systems. Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands. Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4. We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5"-CATCC-3" catalyzed by BstF5I-2 and BstF5I-4 DNA [N6-adenine]-methyl-transferases from the BstF5I restriction–modification system.     

Gene cloning, comparative analysis of the protein structures from Fsp4HI restriction-modification system and biochemical characterization of the recombinant DNA methyltransferase

Genes coding for the restriction-modification system Fsp4HI, recognizing the sequence 5'-GCNGC-3' have been cloned in Escherichia coli ER2267 cells and its primary structure has been determined. This RM system consists of two genes: the DNA-methyltransferase gene which is followed by the restriction endonuclease gene in the same direction. The analysis of amino acid sequences of the proteins showed that M.Fsp4HI belongs to C5 DNA-methyltransferases, and the restriction enzyme shares more or less significant homology to just a few restriction endonucleases with related recognition sequences. M.Fsp4HI enzyme was purified by means of column chromatography. According to the results of biochemical study it was considered that M.Fsp4HI has its optimal activity at 30 degree C and pH 7.5. M.Fsp4HI modifies the first cytosine residue in the sequence 5'-GCNGC-3'.     

Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis

T-cadherin is a unique member of the cadherin superfamily of adhesion molecules. In contrast to “classical” cadherins, T-cadherin lacks transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosilphosphoinositol moiety. T-cadherin is predominantly expressed in cardiovascular system. Clinical and biochemical studies evidence that expression of T-cadherin increases in post-angioplasty restenosis and atherosclerotic lesions—conditions associated with endothelial dysfunction and pathological expression of adhesion molecules. Here, we provide data suggesting a new signaling mechanism by which T-cadherin regulates endothelial permeability. T-cadherin overexpression leads to VE-cadherin phosphorylation on Y731 (β-catenin-binding site), VE-cadherin clathrin-dependent endocytosis and its degradation in lysosomes. Moreover, T-cadherin overexpression results in activation of Rho GTPases signaling and actin stress fiber formation. Thus, T-cadherin up-regulation is involved in degradation of a key endothelial adhesion molecule, VE-cadherin, resulting in the disruption of endothelial barrier function. Our results point to the role of T-cadherin in regulation of endothelial permeability and its possible engagement in endothelial dysfunction.