[Apoptosis during spinal cord trauma: prospects for pharmacological correction]. / Apoptoz pri travmaticheskom povrezhdenii spinnogo mozga: perspektivy farmakologicheskoi korrektsii.

Spinal trauma is a serious problem of modern medicine. The morphological studies illustrate the presence of two alternative pathways of cell destruction in the injured spinal cord: immediate necrotic damage and delayed apoptotic destruction of cells. The apoptosis continues for about 14 days after trauma, and it involves both neurons and glia on a significant distance from the traumatic zone. In this review, the basic stages of apoptosis in spinal cord, biochemical regulation of this process, and methods for its detection are considered. The fact, that apoptosis is a normal cell death process, and that it has reversible stages allows to consider a possibility of pharmacological correction of apoptosis. The special attention is paid to anti-apoptotic therapy with the use of antisense oligodeoxynucleotides and to the perspectives of gene therapy.

[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.

[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.

[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.

[Detection of a steady increase in endonuclease activity in dog liver cell nuclei after long term arterial hypotension and in post-resuscitation period]. / Obnaruzhenie ustoichivogo povysheniia éndonukleaznoi aktivnosti v kletochnykh iadrakh pechini sobak pri dlitel'noi arterial'noi gipotenzii i v postreanimatsionnom periode.

Presence of Ca2+, Mg(2+)-dependent endonuclease activity was found in dog liver cell nuclei. Specific characteristics of chromatin autolysis were also studied in liver cell nuclei after 2-4 hrs long arterial hypotension as compared with that within the later restoration period 1-3 months. The rate of DNA acid-soluble fraction accumulation correlated directly with the arterial hypotension duration. Quantitative evaluation of the liver tissue Ca2+, Mg(2+)-dependent endonuclease activity was undertaken under conditions of hemorrhagic shock. The enzymatic activity was not normalized both after death and in the postresuscitation period. Analysis of the chromatin autolysis and of alterations in the enzymatic activity during postresuscitation period enabled to suggest that the isozyme spectrum of Ca2+, Mg(2+)-dependent endonuclease was altered.

[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.

[DNAses of the cell nuclei: Mn2+-dependent endonuclease]. / DNKazy kletochnykh iader: Mn2+-zavisimaia éndonukleaza.

Comparison of catalytic properties of a Mn2(+)-dependent and a Ca2+, Mg2+ dependent endonucleases of rat liver cell nuclei was carried out. The Mn2(+)-dependent endonuclease has Mr 31 kDa by SDS-PAAG-electrophoresis; pH optimum 5.5; calcium-magnesium synergism less than 3 in rat liver DNA, RF M13 DNA and phage M13 DNA. The rate of hydrolysis of single strand and double strand circular DNA was the same. The Mn2(+)-dependent endonuclease split DNA by double hit manner, and didn't change the manner in the presence of different divalent cations. Ca2+, Mg2(+)-dependent endonuclease has pH optimum 6.5; calcium-magnesium synergism up to 40 in rat liver DNA and 175 in RF M13 DNA. The rate of hydrolysis of single strand DNA was higher than double-strand DNA.

[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.

[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.

[Mn2+-dependent endonuclease activity of chromatin]. / Mn2+-zavisimaia éndonukleaznaia aktivnost' khromatina.

The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+-dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same.

[Dinucleosome as a product of initial chromatin cleavage by endogenous nucleases]. / Dinukleosoma kak produkt nachal'nogo rasshchepleniia khromatina éndogennymi nukleazami.

The ability of nucleic endonucleases to recognize dinucleosomal level of chromatin structure was studied. Rat liver chromatin endonucleases were shown to be capable of DNA cleavage at dinucleosome linkers. The cleavage was observed mainly at initial stages of chromatin autohydrolysis, i.e. up to 10-20th min of incubation in the medium containing 5 mmol of magnesium chloride and 2 mmol of calcium chloride. Thus, mononucleosomal DNA in dinucleosomes and other even chromatin subunits was larger and more homogeneous than in odd subunits. The cleavage of autodigested chromatin by bovine spleen and snake venom exonucleases and by nuclease S1 has shown that dinucleosomal structure is independent of differences in the length of internal and external dinucleosome linkers. The initial endonucleolysis appears to be characterized by different accessibility of the linkers for chromatin nucleases.

[Conditions for initial chromatin endonucleolysis and the characteristics of its products]. / Usloviia nachal'nogo éndonukleoliza khromatina i kharakteristika go produktov.

The restricted initial autohydrolysis of chromatin is used for obtaining its matrix-active fractions. Application of the method for isolating cell nuclei in the presence of 10 mM CaCl2 makes it possible to appreciably inhibit the cleavage of chromatin DNA in the course of isolation, namely to 0.9% according to the acid-soluble fraction. It is shown that at the initial stages of autohydrolysis the method of solubilization is by one order of magnitude more sensitive as compared to the measurement according to the acid-soluble fraction. Initial autohydrolysis is characterized by rapid increment of the amount of the solubilized fraction, delayed formation of mononucleosomes and by the capacity to a 20% activation in the presence of 10(-5) M histamine.

[Specific effect of endonucleases from Brevibacterium ammoniagenes on DNA]. / Izuchenie osobennostei deistviia endonukleaz Brevibacterium ammoniagenes na DNK.

The previously described deoxyribonucleases from Brevibacterium ammoniagenes have been characterized. It was shown that they are endonucleases with molecular weights of 60 000 (I), 10 000 (II) and 20 000 (III). The rate of endonuclease I effect on native DNA exceeded that on the denatured DNA 2-fold. The mechanism of its action is of a single hit type. The enzyme hydrolyzes two chains of DNA simultaneously in two symmetrical sites and splits the bond 5'-P to form fragments with terminal 5'-OH and 3'-P. Endonuclease I was characterized as deoxyribonucleate-3'-oligonucleotide hydrolase (EC 3.1.4.6).

[Methylation and degradation of chromatin DNA in isolated rat liver cell nuclei]. / Metilirovanie DNK zhromatina i ego rasshcheplenie v izolirovannykh kletochnykh iadrakh pecheni krys.

Methylation of chromatin DNA in rat liver cell nuclei incubated in a medium with [3H]CH3-S-adenosyl methionine was studied. It was shown that under the given experimental conditions DNA methylation and chromatin degradation by endogenous nuclear nuclease (nucleases) with a formation of chromatin structural subunits occur simultaneously. An analysis of methylated chromatin DNA degradation products based on a number of approaches demonstrated a predominant methylation of extra-nucleosomal DNA. The data obtained suggest that chromatin of isolated nuclei contain sites with supermethylated DNA fragments incorporating not less than 400 nucleotide pairs. These sites possess an increased sensitivity to endogenous nuclease.