A Novel Dipeptide Ligand of TSPO.

On the basis of the first dipeptide ligand of TSPO, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), which was obtained by us earlier, we synthesized a new dipeptide, N-phenylpropionyl-L-tryptophanyl-L-leucine amide (GD-102). GD-102 exhibited anxiolytic activity in the open field test in BALB/c mice and in the elevated plus maze test in ICR mice. The minimum effective dose of GD-102 was one order of magnitude lower than that of GD-23. Compound PK11195, a selective antagonist of TSPO, completely blocked the anxiolytic activity of GD-102, which testified to the involvement of TSPO in the realization of the anxiolytic effect of GD-102. The results were confirmed by molecular docking data.

[A chamber for evaluating the free choice by laboratory rats and mice of a space containing tobacco smoke]. / Kamera dlia otsenki svobodnogo vybora laboratornymi krysami i myshami prostranstva, soderzhashchego tabachnyi dym.

An original experimental setup has been designed that allows evaluation of the free preference of space containing tobacco smoke by small laboratory animals. For the laboratory mice (C57B1/6 and BALB/c) and rats (MNRA and MR) placed every day into this box, no emotional-stress reaction (ESR) caused by the environment novelty was observed on the 19th day of experiment. A difference in the free preference of space containing tobacco smoke was observed between inbred animals with active and passive ESR phenotypes.

Alteration of human leukotriene A4 hydrolase activity after site-directed mutagenesis: serine-415 is a regulatory residue.

Leukotriene A4 hydrolase (LTA-H) is a bifunctional protein that has aminopeptidase activity, but also contains an epoxide hydrolase activity that converts leukotriene (LT)A4 to LTB4. The lipid metabolic activity of this enzyme plays a central role in the control of polymorphonuclear leukocyte function and in the development of inflammation. LTA-H is widely spread in many mammalian tissues, although it appears to be inactive in many cases. Regulation of this enzyme's activity by phosphorylation of a serine at residue 415 has recently been described. Since the activation of LTA-H in the presence of activated PMNL would likely lead to a substantial increase in the production of inflammatory lipids, regulation of LTA-H presents a novel potential target for anti-inflammatory therapy. We have now made a series of site-directed mutants at this site to test the importance of this residue to the activity of LTA-H. Replacement of the critical serine with threonine or glutamine has little effect on either the epoxide hydrolase or aminopeptidase activities. However, replacing serine with a negatively charged amino acid (either aspartate or glutamate), intended to mimic phosphorylation at that site, causes significant reduction in epoxide hydrolase activity (50-70%). These mutations have little effect on the aminopeptidase activity of the LTA-H, suggesting that the mutation models the regulatory event and is not simply due to improper folding of the protein.

Regulation of leukotriene A4 hydrolase activity in endothelial cells by phosphorylation.

Endothelial cells contain leukotriene (LT) A4 hydrolase (LTA-H) as detected by Northern and Western blotting, but several studies have been unable to detect the activity of this enzyme. Since LTA-H could play a key role in determining what biologically active lipids are generated by activated endothelium during the inflammatory process, we studied possible mechanisms by which this enzyme may be regulated. We find that LTA-H is phosphorylated under basal conditions in human endothelial cells and in this state does not exhibit epoxide hydrolase activity (i.e. conversion of LTA4 to LTB4). LTA-H purified from endothelial cells is efficiently dephosphorylated by incubation with protein phosphatase-1 in the presence of an LTA-H peptide substrate and not at all in the absence of substrate. Under conditions that lead to dephosphorylation, protein phosphatase-1 activates the epoxide hydrolase activity of LTA-H. Using peptide mapping and site-directed mutagenesis, we have identified serine 415 as the site of phosphorylation of LTA-H by a kinase found in endothelial cell cytosol. In parallel, we have studied a human lung carcinoma cell line that expresses active LTA-H. Although these cells have cytosolic kinases that phosphorylate recombinant LTA-H, they do not target serine 415 and thus do not inhibit LTA-H activity. We believe that LTA-H is regulated in intact cells by a kinase/phosphatase cycle and further that the kinase in endothelial cells specifically recognizes and phosphorylates a regulatory site in the LTA-H.

[The determination of the oxidation phenotype in inbred C57Bl/6 and BALB/c mice]. / Opredelenie fenotipa okisleniia u inbrednykh myshei linii C57Bl/6 i BALB/c.

Antipyrine oxidation was studied in C57BL/6 and BALB/c inbred mice. It was found that C57BL/6 are weak oxidant but BALB/c are strong oxidants of antipyrine. Animals F1 hybrids inherited the high capacity of antipyrine oxidation.

[Properties of creatine kinase from skeletal muscle mitochondria]. / Svoistva kreatinkinazy iz mitokhondrii skeletnykh myshts.

Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.

[Radiochromatographic study of the distribution of sidnocarb-14C and its metabolites in inbred mice]. / Radiokhromatograficheskoe issledovanie raspredeleniia sidnokarba-14C i ego metabolitov u inbrednykh myshei.

The title study permitted detecting genetic differences in sydnocarb metabolism. It was established that the rate of sydnocarb oxidation in BALB/c mice was higher than in C57B1/6 mice.

Studies of the interaction of mitochondrial creatine kinase with the mitochondrial membrane.

In this study we have investigated the binding characteristics of creatine kinase (CK) with mitochondrial membrane. Creatine kinase was found to bind membranes by electrostatic forces. At physiological pH, the interaction seems to be between positively charged groups on the enzyme protein and negatively charged groups on the membrane.

[Genetic differences in the excretion of phenazepam-14C]. / Izuchenie geneticheskikh razlichii v vyvedenii fenezepama-14C.

Content of 14C-phenazepame and its metabolites was studied in feces and urine of mice strains C57BL/6 and BALB/ c after intraperitoneal administration of the preparation at a dose of 14 mg/kg. Concentration of phenazepame and of its trihydroderivative was higher in urine of the BALB/c strain mice than in C57BL/6 strain that corresponded to genetic differences of the tranquilizer metabolism in the animal strains studied.

[Genetic differences in the anticonvulsant effect and rate of metabolism of phenazepam]. / Izuchenie geneticheskikh razlichii v protivosudorozhnom éffekte i skorosti metabolizma fenazepama.

The metabolic rate of 14C-phenazepam (14 mg/kg) in male C57BL/6 and BALB/c mice and F1 hybrids (BALB/c X C57BL/6) and the anticonvulsant effect of the tranquilizer in hybrid mice were studied. It was demonstrated that oxidation of the phenazepam molecule in the third position in BALB/c mice and in F1 hybrids proceeded at a higher rate than in C57BL/6 mice. The sensitivity to the anticonvulsant effect of phenazepam in C57BL/6 animals was greater than in BALB/c mice and F1 hybrids.

[Interaction between mitochondrial creatine kinase and mitochondrial membranes]. / Issledovanie vzaimodeistviia mitokhondrial'noi kreatinkinazy s membranami mitokhondrii.

The outer mitochondrial membrane of beef heart was disrupted by hypotonic treatment and the effects of concentration and ionic strength of creatine kinase substrate solutions and some other electrolytes and non-electrolytes on mitochondrial membrane creatine kinase were studied. It was shown that electrostatic forces play an important role in the binding of creatine kinase to the mitochondrial membrane. It was assumed that under physiological conditions mitochondrial creatine kinase may undergo a reversible association - dissociation reaction with the membrane. The changes of pH within the range of 6.0 to 9.5 had no effect on the creatine kinase binding to mitochondrial membrane. Creatine kinase from beef heart mitochondria binds likewise and at the same rate to the rat liver no creatine kinase. The number of binding sites in mg of heart mitochondrial protein is n1 = 0.54 +/- 0.11 nmole, Kd1 = 0.16 +/- 0.04 microM. The number of binding sites in mg of liver mitochondrial protein is n2 = 0.65 +/- +/- 0.03 nmole, Kd2 = 0.29 +/- 0.09 microM. Excessive cytochrome c inhibits the binding of creatine kinase to the beef heart mitochondrial membrane. The results obtained suggest that mitochondrial creatine kinase is apparently bound to the membrane phospholipids.