[Isolation and purification of recombinant truncated (delta2-27) cytochrome P450 2B4 expressed in E. coli cells as a fusion protein with glutathione-S-transferase]. / Vydelenie i ochistka rekombinantnogo "usechennogo" (delta2-27) tsitokhroma P450 2B4, ékspressirovannogo v kletkakh E. coli v sostave gibridnogo belka s glutation-S-transferazoi.

This paper describes the modification of the method by Coon and Pernecky (Meth. Enzymol. 1996, 272, 25-34) for purification of truncated (delta 2-27) recombinant form of cytochrome P450 2B4 expressed in E. coli as fusion protein with glutathione-S-transferase. The modifications included optimisation of conditions for proteolytic reaction of fusion protein with thrombine, removal of this protease from purified cytochrome P450 preparations using column chromatography on hydroxyapatite, introduction of the additional step for obtaining of spheroplasts using of lysozyme, and optimisation of conditions for enzyme stabilisation during of its purification and storage. The overall yield of purified cytochrome was 20% and the specific content of P450 was 14,5 nmol/mg protein was measured. This method is suitable for large-scale isolation of high purified cytochromes P450 which are necessary for study of structure-functional relationships of this hemoprotein with protein partners as well as for investigation of its structure and mechanism of action.

[Study of the spectrum of rabbit myocardium nuclear DNAases using models of ischemia and diabetes mellitus]. / Izuchenie spektra iadernykh éndoDNKaz miokarda krolika na model'iakh ishemii i sakharnogo diabeta.

There were alterations in the spectrum of nuclear endo-DNAases in the rabbit myocardium on experimental models of diabetes mellitus and other diseases where the perfused heart was involved i.e. disappearance of the enzyme with 130 kDa from the control extracts was followed by a great increase in the content of low molecular forms in the nuclear preparations obtained from the ischemic heart, as well as that with 145 kDa in all the abnormalities under study.

[Nuclear endo-DNAse from rabbit myocardium in ischemia and diabetes mellitus]. / Iadernaia éndodnkaza miokarda krolika pri ishemii i sakharnom diabete.

The activity of deoxyribonucleases was altered in the myocardium of New Zealand rabbits under normal conditions, in diabetes mellitus as well as in various experiments, where model heart perfusion was carried out using media of various ion composition and pH value. Studies of Ca2+, MG(2+)- dependent deoxyribonuclease from myocardial nuclear extracts exhibited that protective cell mechanisms appear t be induced at the first steps of tissue ischemic impairments as well as that diabetes decreased cardiac sensitivity to ischemia.

[The clinical significance of detecting the inhibition of topoisomerase I by the sera of patients with systemic scleroderma]. / Klinicheskaia znachimost' vyiavleniia ingibirovaniia topoizomerazy I syvorotkami bol'nykh sistemnoi sklerodermiei.

Topoisomerase I activity was studied by electrophoresis in agarose gel according to plasmid DNA relaxation. Sera from 62 patients with systemic scleroderma, 35 with Raynaud's syndrome, 8 with focal scleroderma, 15 with systemic lupus erythematosus, 20 with rheumatoid arthritis and 20 healthy subjects were examined. Out of 62 sera from SSD patients, anti-topoisomerase activity was found in 67.8% of cases. The test appeared positive in 79% of patients with diffuse and 63% with limited disease patterns. The mean age and disease standing were similar in the positive and negative groups. An increase of the skin count and more frequent occurrence of trophic disorders in patients with inhibition of the enzyme were recorded. 40% of the patients demonstrated the coincidence of the results with the use of the topoisomerase test and ELISA. In patients with other rheumatic diseases and in the healthy subjects, no inhibition of the enzyme was found.

Nuclear topoisomerase I and DNase activities in rat diethylnitrosamine-induced hepatoma, in regenerating and fetal liver.

DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue.

[Activity of topoisomerase I and endonucleases in cells transfected by a ras oncogene]. / Aktivnost' topoizomerazy I i éndonukleaz v kletkakh, transfitsirovannykh onkogenom ras.

Activities of nuclear endonucleases and topoisomerase I were measured in rat fibroblasts which were at the stages of tumor transformation: control embryonal fibroblasts--CEF; cells immortalised by transfection of S1A segment of SA7 adenovirus--REF-1; intermedius cells transfected once by EJras oncogene--REF-1EJ; and cells transformed after the second transfection by the same oncogene--REF-2EJ. The topoisomerase I and Ca2+, Mg2+-dependent endonuclease was most decreased at the stage of immortalised cells, and the intermedius stage (REF-1EJ) was characterized by the lower activity of Ca2+, Mg2+-dependent endonuclease. The highest activity of Mn2+-dependent endonuclease is seen in REF-2EJ cells. In model experiments the ability of Ca2+, Mg2+-dependent endonuclease to split non-stochastically the EJras oncogene inserted into pBR322 plasmid was shown. The role of the investigated enzymes in the restriction of plasmid integration, cellular immortalisation and recombination of plasmids with chromosomes during cell transformation is discussed.

[The activity of nuclear endonucleases and topoisomerases in the liver of rats and in diethylnitrosamine-induced tumors]. / Aktivnost' iadernykh éndonukleaz i topoizomeraz v pecheni krys i opukholiakh, indutsirovannykh diétilnitrozaminom.

The DNA endonuclease (Aendo) and DNA topoisomerase (Atopo) activities in liver nucleus extracts of normal rats, in DENA-induced hepatomas and in liver tissues around tumours were investigated. The profile of nuclear endonucleases measured in the presence of 2 mM CaCl2 + 5 mM MgCl2, or 5 mM MnCl2, or 5 mM MgCl2, or 2 mM CaCl2 (pH 7.4), or I mM EDTA (pH 5.0) was different in normal and tumour tissues. Mn2+-dependent endonuclease was the main endonuclease in the tumour tissue, whereas Ca2+, Mg2+-dependent endonuclease was the main one in the normal liver and in the tissue around the tumour. An increase in the Mn2+-dependent endonuclease activity correlated with a decrease in the hepatoma differentiation level. Atopo of types I and II increased in the tissue around the tumour. Aendo and Atopo of cellular nuclei decreased in animals given DENA without the liver tumour.

[New means of isolating restriction endonuclease preparations using organic solvents]. / Novyi sposob vydeleniia ochishchennykh preparatove restriktsionnykh éndonukleaz s ispol'zovaniem organicheskikh rastvoritelei.

A new procedure is developed for isolation of highly purified preparations of restrictional endonoucleases Bam HI and Eco RI by means of fractionation with isopropyl alcohol. Restrictional endonuclease Bam HI, practically free of unspecific nucleases, was isolated after ultrasonic destruction of cells, precipitation of the restrictases with isopropanol and chromatography on DEAE cellulose. Additional chromatography on hydroxyapatite enabled to obtain the homogenous preparation of Bam HI restrictase, as shown by polyacrylamide gel disc electrophoresis. Other organic solvents (acetone, ethanol) might be also used for purification of the restrictional endonucleases.