Effects of Ukrain on the activities of DNA-nicking enzymes.

We studied the effects of Ukrain, a novel antitumor drug, on the activities of calcium, magnesium-dependent endonuclease (CME) and manganese-dependent endonuclease (MnDE) in rat liver nuclei, the activity of topoisomerase I assessed by pUC19 plasmid relaxation and CME activity in the nuclei of lymphocytes from colon cancer patients. Ukrain was found to exert a dose-dependent inhibiting effect on both CME and MnDE, similar to that exerted by erythropoietin, which was used as a reference preparation. Both Ukrain and erythropoietin also caused dose-dependent inhibition of topoisomerase I activity. The influence of Ukrain on CME activity in the nuclei of the lymphocytes of colon cancer patients was differential, depending on treatment efficacy. The results suggest that DNA-nicking enzymes may be a target of Ukrain and may mediate its antitumor effects.

Induction of apoptosis in cultured Chinese hamster ovary cells by Ukrain and its synergistic action with etoposide.

The induction of apoptosis by Ukrain, a novel antitumor drug, was studied in Chinese hamster ovary (CHO) cells bearing multiple copies of recombinant human erythropoietin gene incorporated into their genome (cell lines CHO-k38 and -k38/12). Ukrain was found to be capable of the in vitro induction of apoptosis in the cell lines studied. The effect was less expressed in cells with type I multiple drug resistance (k38/12). Ukrain acted synergistically with etoposide, i.e., the combined effect of both agents was evident at significantly reduced concentrations. This suggests that pharmacological compositions of the drugs may reduce the effective doses used in chemotherapy and thus significantly diminish its toxic side effects. Ukrain was found to exert an unusual effect, manifested as the inhibition of protein secretion by target cells. This phenomenon may be used for the express determination of cell sensitivity to colchicine-like cytostatics, including Ukrain.

[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.

[Characteristics of nuclear endo-DNAase from rat liver by molecular mass and cationic dependence]. / Kharakteristika iadernykh éndo-DNKaz pecheni krys po molekuliarnoi masse i kationnoi zavisimosti.

Study of the isoenzymatic spectrum of nuclear DNase from rat liver demonstrated that the extracts obtained in the presence of the protease inhibitor, PMSF, contained four polypeptides with molecular masses of 120, 54, 31 and 28 kDa possessing the endo-DNase activity. Long-term storage at -20 degrees C and autodigestion of the nuclei revealed the presence of additional polypeptides with a lower molecular mass and possessing an endo-DNase activity. Treatment of rat liver nuclear extracts with trypsin caused the appearance of an active polypeptide (M(r) 145 kDa). This finding and the multiplicity of endo-DNases of different molecular masses suggest the existence of a precursor possessing a much higher molecular mass. Chromatographic separation of endo-DNases on Sephacryl S-300 revealed three fractions of 400 and more kDa possessing a Ca2+/Mg(2+)-dependent activity, however, only after trypsin treatment.

In vitro proteolysis of endonucleases in rat liver nuclei extracts.

DNA endonucleases in rat liver nuclei extracts were examined by SDS-polyacrylamide gel electrophoresis followed by zymogram analysis. Four polypeptides of 120, 54, 31 and 28 kDa, which have DNA endonuclease activity, were shown to occur in the extract isolated in the presence of phenylmethanesulfonyl fluoride (PMSF), a proteinase inhibitor. Isolation without PMSF, as well as storage at -20 degrees C, or autodigestion, resulted in multiplication of active polypeptides in the extracts. Trypsin digestion led to the appearance of an active > 140 kDa polypeptide, indicating the existence of a potential endonuclease precursor in the nuclear extract.

Intermolecular homologies of human interferon-alpha.

Human interferon-alpha 2 (IFN) was analyzed by homology search computer program with the use of protein primary structures data bases. Results indicate that four domains with heightened ability to form homology pairs with different proteins exist in the IFN molecule. These domains occupy regions 35-56, 72-85, 97-110 and 124-136, mainly between the alpha-helical cylinders on the tertiary structure models. Additionally, results show in IFN structure the presence of amino-acid motifs that create the opportunity for this cytokine to influence directly the processes of DNA functioning in cell nuclei.

[Importance of studying the level of platelet adenine nucleotide levels in various phases of chronic myeloleukoses]. / Znachenie issledovaniia urovniia adeninnukleotidov trombotsitov v raznykh fazakh zabolevaniia khronicheskim mieloleikozom.

Content of adenine nucleotides was studied in various compartments of blood platelets obtained from donors and patients with chronic myeloleukosis at various steps of the disease. Dissimilar biochemical defect was found in content of ATP and ADP, which varied in thrombocyte storage granules depending on severity of the disease. The data on adenine nucleotides content may be used for diagnosis of various steps of chronic myeloleukosis, for detection of thrombocytes anomaly and for evaluation of the state of medullary hemopoiesis.

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.

[Platelet activating factor inhibits irritant-induced leukocyte migration into mouse peritoneal cavity]. / Faktor aktivatsii trombotsitov ingibiruet migratsiiu leikotsitov v briushnuiu polost' myshei, indutsirovannuiu irritantami.

The effect of platelet-activating factor (PAF) on the leucocytes accumulation into mouse peritoneal cavity, that induced by high-molecular irritants carrageenan and polyacrylic acid pentaerytritol (carbopol) was studied. It was found that injection of PAF into peritoneal cavity one hour before i.p. injection of the irritants did not change their effects. On the contrary the injection of PAF simultaneously with the irritants or 3 and 6 hours after these injections inhibited the leucocyte accumulation into the peritoneal cavity and/or these injections increased the leucocyte adhesion to the peritoneal covering. It was supposed that finding effects are mediated by the change of leucocytes adhesion under action of PAF.

[Physico-chemical and catalytic properties of hypoxanthine guanine phosphoribosyltransferase in thrombocytes from blood donors and patients with hemophilia A]. / Fiziko-khimicheskie i kataliticheskie svoistva gipoksantinguaninfosforiboziltransferazy trombotsitov donorov i bol'nykh gemofiliei A.

Studies of some physico-chemical and catalytic properties of hypoxanthine guanine phosphoribosyl transferase HGPRT) in thrombocytes of donors and patients with hemophilia A showed that kinetic parameters (Km, Vmax) of the enzyme were similar both under normal and pathological conditions. However, some differences were detected in thermolability and inhibition of HGPRT by purine metabolites: IMP, GMP and inosine. These data suggest that molecular impairment may occur in HGPRT under conditions of hemophilia A.

Early postirradiation chromatin degradation in thymocytes.

The decrease in the average DNA size in thymocytes starts soon after in vivo irradiation and at approximately 45 min reaches a plateau, thereafter showing only minor changes up to 3 h. This fall in extent of chromatin cleavage coincides with the accumulation of 1.0-1.5 kb DNA fragments. Double-strand breaks generated by endonucleases are not randomly distributed along DNA but clustered in such a way that they give rise to fragments of 1-5 nucleosomes in size. Cycloheximide treatment partially inhibits nuclease activity in nuclear extracts isolated from thymus of irradiated mice. This suggest that DNA fragmentation is an early event in programmed death of thymocytes mediated by irradiation. The data indicate that it requires protein synthesis and that it precedes release of polydeoxyribonucleotides.

[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.

[DNA recombination in vitro initiated by Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes]. / Rekombinantsiia DNK in vitro, initsiiruemaia Ca/Mg-zavisimoi éndonukleazoi iader splenotsitov cheloveka.

The system of DNA recombination in vitro was constructed. It comprises two plasmids, the derivatives of pBR322 deleted in the genes for tetracycline resistance, and the recombinogenic extract of the thymus lymphocytes nuclei of mice. The system permits to study the effect of proteins and factors on the efficiency of recombination resulting in reconstruction of the tetracycline resistance gene. Double-strand cuts in one of the deleted plasmids were necessary for recombination. Double-strand cuts by Ca/Mg-dependent endonuclease of the human spleen lymphocytes nuclei were more efficient as compared with the ones of DNAase I, restriction endonucleases PaeI and SalI in the initiation of recombination. The possible role of Ca/Mg-dependent endonuclease in recombination in vivo is discussed.

[Cell nuclear DNases: multiplicity and heterogeneity]. / DNKazy kletochnykh iader: mnozhestvennost' i geterogennost'.

In order to determine the ratio of activities of major endonucleases of rat liver chromatin, a stepwise fractionation of cell nuclear extracts by chromatography on phosphocellulose and gel filtration through Toyopearl HW60 was carried out. This procedure resulted in partially purified preparations of Ca2+,Mg2+-dependent endonuclease (55 +/- 10 kD), Ca2+,Mg2+-dependent endonuclease (30 +/- 10 kD), Mn2+-dependent endonuclease (30 +/- 5 kD) and acid cation-independent endonuclease. The Ca2+,Mg2+-dependent endonuclease with Mr of 55 +/- 10 kD made up to 57% of the nuclear extract activity in the presence of Ca2+ + Mg2+ and revealed a high calcium-magnesium synergism. Under the same experimental conditions, the 30 +/- 10 kD enzyme made up to 33% of the nuclear extract activity and revealed a low synergism. The activity of Mn2+-dependent endonuclease made up to 26% of the total nuclear extract activity in the presence of Mn2+, that of acid endonuclease--11% of the extract activity in 1 mM EDTA at pH 5.0. It was assumed that the low molecular weight Ca2+,Mg2+-dependent endonuclease represents a product of limited proteolysis of high molecular weight Ca2+,Mg2+-dependent endonuclease.

[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.

[Specificity of fragmentation of DNA from pBR322 plasmid by Ca,Mg-dependent endonuclease from cell nuclei of human lymphocytes]. / Spetsifichnost' fragmentatsii DNK plazmidy pBR322 Ca/Mg-zavisimoi éndonukleazoi kletochnykh iader limfotsitov cheloveka.

Fragmentation of the plasmid pBR322 DNA by a purified preparation of Ca/Mg-dependent endonuclease has been studied. It was shown that on the first steps of reaction the double-stranded cuts are introduced into the superhelical DNA independent of singlestranded ones. The doublestranded cuts are introduced into superhelical and linear DNA in 12 sites enriched with GC-pairs, 9 of them include pentanucleotide CGCGG(CCGCC) that is functionally significant. Relaxation of the plasmid DNA by topoisomerase I blocks the sitespecific action of the enzyme. Ca/Mg-dependent endonuclease is concluded to be topologically dependent enzyme, possibly, participating in the recombination processes.

[Effectiveness of methods of isolating specific class-G rabbit antibodies for the immunoenzyme determination of human placental alkaline phosphatase]. / Issledovanie éffektivnosti metodov vydeleniia spetsificheskikh krolich'ikh antitel klassa G dlia immunofermentnogo opredeleniia platsentarnoi shchelochnoi fosfatazy cheloveka.

Four different chromatographic methods of IgG isolation from rabbit antisera to placental alkaline phosphatase (HPAP) have been compared. The antibodies were obtained by ion-exchange chromatography and affinity chromatography on protein-A-sepharose, on the sepharose with immobilized antigen. IgG samples were characterized by the content of specific antibodies to HPAP and checked in enzyme immunoassay (EIA). IgG purified on immobilized antigen were found to be the optimal both from the point of view of the specific antibodies content and EIA sensitivity, but satisfactory results could be also obtained with ion-exchange and protein-A-chromatography purified IgG. The last two isolation methods are simpler and provide 3-10 ng/ml sensitivity of HPAP detection, which is lower, as compared with the test employing affinity antibodies (1 ng/ml), but allows the detection of HPAP in serum samples.

[Isolation and analysis of Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes]. / Vydelenie i issledovanie Ca/Mg-zavisimoi éndonukleazy iz kletochnykh iader limfotsitov selezenki cheloveka.

A scheme for the isolation of Ca,Mg-dependent endonuclease from human spleen lymphocyte nuclei has been developed. The isolation procedure resulted in protein preparations (Mr = 57 kD) possessing an enzymatic activity and stable upon storage for over a period of one year. The enzyme is an endonuclease which predominantly cleaves double-stranded DNA by a mixed single- and double-hit mechanism with the formation of 5'-phosphate and 3'-OH terminal groups. Its maximal activation is induced by Ca2+ plus Mg2+. The enzyme is also active in the presence of Mn2+, Ca2+, Mg2+ and Zn2+ and is inhibited by Co2+. NaCl and KCl (0.15-0.2 M) and p-chloromercuribenzoate (1 mM) also inhibit the enzyme. ATP has no activating effect.