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).

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.

[Study of the spectrum of rabbit myocardium nuclear DNAases using models of ischemia and diabetes mellitus].

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].

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].

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).

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.

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.

[Importance of studying the level of platelet adenine nucleotide levels in various phases of chronic myeloleukoses].

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.

[Platelet activating factor inhibits irritant-induced leukocyte migration into mouse peritoneal cavity].

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.

[Physico-chemical and catalytic properties of hypoxanthine guanine phosphoribosyltransferase in thrombocytes from blood donors and patients with hemophilia 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.

[Activity of topoisomerase I and endonucleases in cells transfected by a ras oncogene].

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.

[DNA recombination in vitro initiated by Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes].

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.

[Cell nuclear DNases: multiplicity and heterogeneity].

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.

[The activity of nuclear endonucleases and topoisomerases in the liver of rats and in diethylnitrosamine-induced tumors].

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.

[Specificity of fragmentation of DNA from pBR322 plasmid by Ca,Mg-dependent endonuclease from cell nuclei of human lymphocytes].

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.

[Effectiveness of methods of isolating specific class-G rabbit antibodies for the immunoenzyme determination of human placental alkaline phosphatase].

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).

[Isolation and analysis of Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes].

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.

[Use of monoclonal antibodies in comparative studies of Ca, Mg-dependent endonuclease in cell nuclei].

Eight hybridoma cell lines derived from fusion between myeloma X-63 and mouse splenocytes were found to secrete monoclonal antibodies against Ca/Mg-dependent endonuclease of human spleen cell nuclei. Two of them, termed N and S, were used in comparative research of enzymes from different organs and species of animals. The data obtained show that N and S antibodies recognize different antigenic determinants of the enzyme molecule.

Immunochemical studies of human placental alkaline phosphatase in normal and neoplastic tissues.

Human placental alkaline phosphatase (hPLAP) is normally present in plasma membranes of syncytiotrophoblast microvilli. hPLAP is expressed by many malignant tumors and is considered to be useful as a tumor marker. The objective of the work was to develop immunochemical methods for detection of this marker.

[Dynamics of the endo-DNAase activity of cell nuclei of mouse thymus and spleen lymphocytes during the immune response].

Endo-DNAse (mostly Ca/Mg-dependent endonuclease) activity was studied in extracts of lymphocyte cellular nuclei from the spleen and thymus of mice upon their immunization with sheep red blood cells. Endo-DNAses were detected by their action on super-stranded DNA pBR 322. It has been established that endo-DNAse activity considerably changes in the course of immune response.