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. hPLAP was isolated from placenta using a procedure with preparative isoelectric focusing as a final purification step, and was used to obtain rabbit antisera. Anti-hPLAP antibodies were obtained by affinity chromatography on the immobilized hPLAP antigen and were used in immunoenzyme tests for detection of hPLAP in sera and tissues of cancer patients with different malignancies. Anti-hPLAP antibodies were also used for localization of hPLAP on the histological level. The described methods may be of use in cancer diagnostics and therapy.

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.

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.

[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.

[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.

[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.

[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.

[Ca/Mg-dependent endonuclease as a probe for detecting chromatin changes in lymphocytes in chronic lymphoid leukemia]. / Ca/Mg-zavisimaia éndonukleaza kak zond dlia vyiavleniia izmenenii khromatina limfotsitov pri khronicheskom limfoleikoze.

Chromatin fragmentation of bovine peripheral blood lymphocytes from normal animals and the ones suffering from chronic lympholeucosis (CLL) by DNase I, micrococcal nuclease and purified Ca/Mg-dependent endonuclease from nuclei of human splenocytes was studied. The lymphocytes chromatin from CLL animals was shown to be more resistant to nucleases, than the one from normal animals. It was found that difference between fragmentation of chromatin samples from normal and CLL bovines was more dramatic when Ca/Mg- dependent endonuclease was used versus traditionally exploited DNase I and micrococcal nuclease. The data suggest that purified Ca/Mg-dependent endonuclease can be a useful enzymatic probe for detection of lymphocytes chromatin changes during CLL.

[Comparative study of 21S RNP particles and ribosomes from the cytoplasm of the cells of sarcoma M-1 in vitro]. / Sravnitel'noe izuchenie 21S RNP-chastits i ribosom tsitoplazmy kletok sarkomy M-1 in vitro

Preparations of RNP-particles from rat sarcoma M-1 were obtained by means of treatment with different detergents: anione - sodium deoxycholate (1%) and non-ionic - Triton X-100 (1%). Triton X-100 caused some increase in content of the small ribosomal subunit and also in the content of 21 S and 7.5 S particles in the preparation. The latter particles as well as ribosomes were resistant to different detergents. 21 S RNP-particles were stable in 1 M KCl. In a cell-free system 21 S and 7.5 S RNP-particles lost a significant amount of labelled protein, which appeared partially in the ribosomes.

[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.

[The role of adenine nucleotides in the functional activity of platelets in hemophilia A]. / Znachenie issledovaniia adeninnukleotidov v funktsional'noi aktivnosti trombotsitov pri gemofilii A.

Content of adenine nucleotides was studied in various pools of thrombocytes from donors and from patients with hemophilia A. Biochemical deficiency of adenine nucleotides appears to occur in the pool of thrombocytes obtained from patients with hemophilia A; estimation of ADP and ATP in the storage granules as well as of the nucleotides specific radioactivity may serve as a criterion of thrombocyte disfunction.

[Inhibition of chromatin autolysis in the process of isolating and incubating rat liver cell nuclei]. / Ob ingibirovanii autoliza khromatina v protsesse vydeleniia i inkubatsii kletochnykh iader pecheni krys.

Dependence of endogenous nuclear nuclease/nucleases/ activity on pH value and ion composition of the isolation and incubation media was studied. Change in buffer pH value up to pH 9.0 and addition of Ca2+ instead of Mg2+ significantly inhibited the endogenous nuclease activity in rat liver nuclei. On the basis of these data the procedure for isolation of nuclei developed by Shaveau was modified. Almost complete inhibition of the endogenous nuclease activity was achieved during destruction of chromatin DNA from the isolated nuclei in presence of exogenous nuclease/micrococcal/.

[Nonpathogenic Neisseria studied for the content of histidine decarboxylase]. / Issledovanie nepatogennykh neisserii na soderzhanie gistidindekarboksilazy.

Histidine decarboxylase activity was not found in cells of nonpathogenic neisseria, obtained from bronchi of patients with infectious asthma. The absence of the histidine decarboxylase activity in nonpathogenic neisseria cells suggests that the sensitizing activity of these cells was not caused by a direct effect of histamine of bacterial origin on human branchopulmonary tissues.

[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.

[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.

[Thrombocyte purine nucleoside phosphorylase in various blood diseases]. / Purinnukleozidfosforilaza trombotsitov pri razlichnykh zabolevaniiakh krovi.

Activity of purine nucleoside phosphorylase (EC 2.4.2.1) was estimated in thrombocytes of donors and of patients with various hematological diseases. The enzymatic activity was decreased in chronic lymphoid leukosis chronic myeloleukemia, chronic myeloleukemia and blast transformation, acute lymphoblast and myeloblast leukemia, myeloma. Dissimilar level of purine nucleoside phosphorylase activity was found in non-Hodgkin disease. The activity was only slightly increased in Waldenström's macroglobulinemia; it was similar to normal level in hypoplastic and autoimmune hemolytic anemias. A decrease in the enzymatic activity was observed in thrombocytes of the patients with hereditary forms of hemorrhagic diatheses (hemophilia A, Willebrand disease). Normal level of the enzymatic activity was noted in Glanzmann thrombasthenia.

[Adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase of blood platelets in hereditary coagulopathies]. / Adeninfosforiboziltransferaza i gipoksantin-guaninfosforiboziltransferaza trombotsitov krovi pri nasledstvennykh koagulopatiiakh.

Activities of adenine phosphoribosyltransferase (EC 2.4.2.7 APRT) and hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8 HGPRT) were studied in thrombocytes of healthy donors, patients with hemophilia A and B and of women--heterozygote carriers of the pathologic gene. The data obtained suggest that HGPRT test may be used as a genetic marker of hemophilia as well as to detect the heterozygote carriers; estimation of APRT activity is suitable test for differentiation of hemophilia forms.

[Comparative characteristics of the catalytic properties of platelet adenosine deaminase in normal states and in chronic myeloid leukemia]. / Sravnitel'naia kharakteristika kataliticheskikh svoistv adenozindezaminazy trombotsitov v norme i pri khronicheskom mieloleikoze.

Properties of adenosine deaminase from thrombocytes of healthy persons and of patients with chronic myeloleukosis were studied in presence of various structural analogues of purines and metal ions as well as at various temperature. Catalytic properties of the enzyme in thrombocytes of patients with chronic myeloleukosis were distinct from those of thrombocytes of healthy donors. These differences in the enzymatic properties (contrary to standard estimation of the enzyme activity) might be used both as a criterion of adenosine deaminase anomalies and for detection of the initial steps of the disease.

[Isolation and properties of cytoplasmic 21S and 7.5S RNP-particles from rat M-1 sarcoma cells]. / Vydelenie i svoistva tsitoplazmaticheskikh 21 S i 7,5 S RNP-chastits iz kletok sarkomy M-1 krys

RNP-particles, containing components with coefficient of sedimentation 119 S, 78 S, 60 S, 40 S, 21 S and 7.5 S were prepared from cells of sarcoma M-1. After separation in sucrose gradient, physico-chemical properties of 21 S and 7.5 S particles were studied. RNA of the particles was shown to belong to the GC-type. More than 10 protein fractions were found in polyacrylamide gel electrophoresis of these particles in presence of sodium dodecyl sulphate. Some of the proteins possessed electrophoretic mobility similar to that ribosomal proteins. In CsCl2 floating density of 21 S and 7.5 S particles was equal to 1.355 g/cm-3. By means of immunodiffusion method it was shown that light RNP-particles were analogous to the ribosomes of rat liver tissue and sarcoma M-1. Incorporation of 14-C-leucine into protein and 32-P-ortophosphate into RNA within 4, 7 and 12 hrs after administration of the isotopes demonstrated metabolic stability of RNA and high activity of the protein in the RNP-particles studied.