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PubMed1989

Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs.

Binding mechanisms of ADPR-transferase to restricted double-stranded DNA fragments of SV40 and pBR322 DNA were determined by nuclease protection techniques. Top and bottom strands of double-stranded DNA were identified by specific labeling with 32P. Protection against specific exonucleases identified binding of ADPR-transferase to DNA termini, whereas binding to internal regions of linear DNAs was probed by protection against endonucleases. ADPR-transferase protein protected against exonucleolytic attack from lambda exo and exoIII in all DNA fragments tested, demonstrating that the enzyme protein binds indiscriminately to all DNA termini. Extending earlier results [Sastry, S.S., & Kun, E. (1988) J. Biol. Chem. 263, 1505-1512], the modifying effect of the binding of ADPR-transferase to DNA induced changes in DNA conformation, as evident from altered pause sites that appeared following digestion of DNA fragments by lambda exonuclease in the presence of ADPR-transferase. In contrast to the nonselective binding of ADPR-transferase to DNA termini, ADPR-transferase conferred protection endonuclease attack (DNase I and micrococcal nuclease) only to the 209-bp EcoRI-PstI SV40 DNA fragment. These results indicate that binding of ADPR-transferase to relatively rare internal regions of restricted DNA fragments exhibits some degree of specificity. Specificity of binding appears to be related to the coincidental relative A+T-rich regions in DNA, and to DNA bending, both identified in the 209-bp SV40 DNA fragment. Synthetic polydeoxyribonucleotides containing dA-dT bind ADPR-transferase stronger than polydeoxyribonucleotides containing dG-dC. It was deduced from endonuclease protection patterns that binding of the enzyme protein leaves no defined footprints on the 209-bp SV40 DNA fragment, but there is significant modification of DNA structure following binding of the enzyme protein. Methylation protection assays and the prevention of the binding of ADPR-transferase to T4 DNA by its glucosylation indicate that the enzyme binds in the major groove of DNA. The 36-kDa A peptide fragment of ADPR-transferase [Buki, K. G., & Kun, E. (1988) Biochemistry 27, 5990-5995] exhibits the same protection against endonucleolytic enzymes as the intact ADPR-transferase molecule.

Animals↗

PubMed1980

Hypoxanthine in deoxyribonucleic acid: generation by heat-induced hydrolysis of adenine residues and release in free form by a deoxyribonucleic acid glycosylase from calf thymus.

A slow conversion of adenine residues to hypoxanthine occurs in single-stranded DNA when heated in neutral aqueous buffers. The rate of this reaction at pH 7.6 and 110 degrees C is k = 4 x 10(-8) s-1, as determined by base analysis of heat-treated DNA that contains radioactively labeled adenine residues. It is proposed that adenine deamination is one of several forms of hydrolytic damage that may occur as spontaneous premutagenic lesions in DNA in vivo. Cell extracts from calf thymus and human fibroblasts contain a DNA glycosylase activity with specifically catalyzes the release of free hypoxanthine from DNA or polydeoxyribonucleotides that contain dIMP residues. Several properties of the purified enzyme from calf thymus are described: It has an approximate molecular weight of 31 000. No cofactors are required for activity. The enzymatic release of hypoxanthine occurs readily from double-stranded polydeoxyribonucleotides that have either thymine or cytosine residues in the complementary strand. Single-stranded polymers are 10-20-fold more slowly attacked, and there is no detectable cleavage of monomeric dIMP. Hypoxanthine is liberated from DNA directly as a free base. Thus, when poly(dI) x poly(dC) containing both [3H]-dIMP and [32P]dIMP residues was employed as the substrate, 3H-labeled hypoxanthine but no 32P-labeled material was released in ethanol-soluble form. The hypoxanthine-DNA glycosylase presumably acts in DNA repair by preventing deaminated adenine residues from being expressed as mu.

Adenine↗

PubMed1985

Antitemplate effect of polynucleotides and their hybrid complexes.

In continuation of efforts to correlate the antitemplate activities of modified polynucleotides with their structure, and to understand the factors governing both their potency and stability, a group of single-stranded poly(ribo- and deoxyribo-) nucleotides, and the "hybrid" double-stranded complexes were prepared and investigated. The double-stranded hybrid poly(A,hs5U).poly(dT) section was found to be more stable to murine blood nucleases than was the single-stranded poly(A,hs5U). In a comparative study as inhibitors of the DNA polymerase alpha from rat hepatoma, the results showed that the modified polynucleotides were more potent than the unmodified ones, in general, the polydeoxyribonucleotides were better antitemplates than their ribo counterparts and the poly(A70,hs5U30).poly(dT) hybrid was more active than either of the single-stranded components. Thus it is possible to increase the nuclease resistance of the modified polyribonucleotides by forming hybrid complexes with complementary polydeoxyribonucleotides, and at the same time, to augment their antitemplate activities.

Animals↗

PubMedDate not supplied

[Deoxyribonucleoprotein and nucleic acid changes in the tissues of rats after a flight on the Kosmos-1129 biosatellite].

The concentration of polydeoxyribonucleotides and nucleic acids was measured in the spleen, thymus, liver, bone marrow and blood of rats flown for 18.5 days on Cosmos-1129. The exposure led to an increase in the polydeoxyribonucleotide content in the thymus and a decrease of the DNA and RNA concentration in the spleen and thymus. These changes returned to normal at R+6.

Animals↗

PubMed1990

The pharmacology and clinical pharmacology of defibrotide: a new profibrinolytic, antithrombotic and anti-platelet substance.

Defibrotide, a deoxypolyribonuclide, has been found to modulate endothelial cell function causing increase in t-PA and decrease in PAI levels and also increase in PGI2 production. In addition, it increases platelet c-AMP levels and decreases MDA and TXB2 formation in human. Defibrotide inhibits platelet aggregate formation in vitro experiments as well as end-to-end anostomosis in rats. So, defibrotide inhibits the activation of platelets. Besides an increase of protein C and S levels a synergic action of heparin was observed in animal experiments. A strong antithrombotic effect has been observed in animal models. The drug has a beneficial effect in the cases of DVT, POVD, stroke and thromboembolism. Through its action we may say that the drug acts in a novel fashion in contrast to the other drugs used in this area. Defibrotide is a single-stranded polydeoxyribonucleotide obtained from deoxyribonucleic acid of mammalian lungs by controlled depolimerization. Since 1981 in our laboratory and in the clinical department we have been investigating a newly developed agent defibrotide in vitro experiments, animal experiments, and also its clinical pharmacology and clinical application. Some of our findings are already published and compared with literature (40, 43, 46). Because of the limited space we are not going to review the literature in detail but we are going to summarize our observations on this compound in the following order. I--in vitro experiments, II--Animal experiments, III--clinical pharmacology in human.

Animals↗

PubMed1996

The anti-ischemic drugs defibrotide and oligotide analogously inhibit leukocyte-endothelial cell adhesion in vitro.

Defibrotide (a polydeoxyribonucleotide) and oligotide (an oligodeoxyribonucleotide) obtained from mammalian single-stranded DNA, have been demonstrated to have anti-ischemic activity in some experimental models of ischemia/reperfusion of kidney in rats. We hypothesized that their anti-ischemic activity could be related to an inhibition of leukocyte-endothelial cell adhesion and also the consequent generation of oxygen free radicals by leukocytes. We studied the in vitro adhesion of neutrophils to human umbilical vein endothelial cells under basal conditions and following neutrophil or endothelial cell activation (using 10(-7) fMLP and 500 U/ml TNF-alpha, respectively). Defibrotide and oligotide significantly inhibited neutrophil adhesion to endothelial cells (after only 1 min of drug treatment). When the anti-LFA-1 70H12 F(ab)2 monoclonal antibody was used, the drugs exerted only slight additional inhibition of the adhesion of fMLP-activated neutrophils to endothelium. These results, confirmed in NIH/3T3-ICAM-1-transfected cells, demonstrate that defibrotide and oligotide interfere with leukocyte adhesion to endothelial cells by an LFA-1-dependent mechanism.

3T3 Cells↗

PubMed1992

Effect of oral defibrotide on tissue-plasminogen activator and tissue-plasminogen activator inhibitor balance.

Defibrotide, a polydeoxyribonucleotide of mammalian origin, has been shown to reduce the blood level of the plasminogen activator inhibitor, and so to increase the activity of tissue plasminogen activator without any adverse effect. A randomized, double-blind, placebo-controlled study has been done in 22 patients, 14 with peripheral vascular disease, 6 with coronary heart disease and 2 with cerebrovascular disease. Patients were given defibrotide 400 mg b.d. or identical placebo for 30 days and the parameters of fibrinolysis were evaluated before and after the treatment. A significant increase in tissue plasminogen activator activity at rest and after venostasis was observed after defibrotide; tissue plasminogen activator antigen at rest and after venostasis was not affected by either treatment. Defibrotide significantly reduced plasminogen activator inhibitor activity and antigen at rest. Only one patient complained of gastric pain after placebo treatment. The study shows that defibrotide has profibrinolytic property and that it could be used to explore the role of plasminogen activator inhibitor in venous and arterial thrombosis.

Administration, Oral↗

PubMed2000

Effects of defibrotide on aorta and brain malondialdehyde and antioxidants in cholesterol-induced atherosclerotic rabbits.

The effects of a high-cholesterol diet in the presence and absence of defibrotide, a single-stranded polydeoxyribonucleotide compound, on the lipid peroxidation product malondialdehyde, endogenous antioxidant enzymes catalase, glutathione peroxidase, and the antioxidant thiol compound GSH were investigated. Forty male New Zeland white rabbits were divided into four groups each consisting of 10 rabbits. Group I received a regular rabbit chow diet and group II 1% cholesterol plus regular chow, group III was given defibrotide (60 mg/kg per day p.o. in water) and was fed with regular chow, and group IV received defibrotide plus 1% cholesterol for 9 weeks. Blood cholesterol and malondialdehyde, catalase, glutathione peroxidase, and GSH were determined before starting the experimental diet regimen (basal). After 9 weeks, the same parameters were determined in blood, aorta, and brain tissues (end -experiment). Aortic tissue was examined under a light microscope for morphological alterations indicative of atherosclerosis. The increase in serum total cholesterol was greater in group II than group IV. Plasma malondialdehyde in group II was higher than in group III. Brain malondialdehyde in group II was higher than all other groups, and aortic malondialdehyde in this group was higher than group I and III. Serum catalase activity decreased in group II and increased in group III, compared with basal values. Brain catalase activity in group I was higher than group II, and aorta catalase in group IV was higher than in group I and III. Blood glutathione peroxidase activity in group III and IV was higher than basal. GSH concentrations decreased significantly in the cholesterol-fed groups (group II and IV). Histological alterations in the cholesterol-fed groups were more pronounced in group II. The increased levels of malondialdehyde in plasma, aorta, and brain tissue of group II suggest a role of oxygen free radicals in the pathogenesis of cholesterol-induced atherosclerosis. The higher malondialdehyde values in the brain tissues of animals in group II compared with group IV suggest a protective role of defibrotide in the brain against lipid peroxidation in the oxidant stress of cholesterol-induced atherosclerosis. Increased catalase activities in the blood and aortic tissues and increased glutathione peroxidase activities in the blood of rabbits receiving defibrotide suggest an induction of these antioxidant enzyme activities by defibrotide. These results imply that anti-atherosclerotic, anti-ischemic effects of this drug may be due to the beneficial effects on the oxidant-antioxidant balance of various tissues.

Animals↗

PubMed1985

Usefulness of defibrotide in protecting ischemic myocardium from early reperfusion damage.

Defibrotide, a partially depolymerized polydeoxyribonucleotide obtained from mammalian lungs, was found to stimulate prostacyclin (PGI2) production and to possess significant antithrombotic and fibrinolytic activities. The present study was designed to evaluate the actions of defibrotide on feline myocardial ischemia, produced by 3 hours of occlusion of the left anterior descending coronary artery (LAD) and followed by 2 hours of reperfusion. Intravenous administration of defibrotide (32 mg/kg/hour subsequent to a 32 mg/kg bolus injection), beginning 30 minutes after LAD occlusion, resulted in a 60% reduction in loss of CK specific activity from ischemic myocardium at 5 hours, while the nonischemic myocardium remained unaffected. Defibrotide largely antagonized the increase in ST segment during LAD occlusion and prevented the appearance of a Q wave during early reperfusion, which was found in all vehicle-treated cats. Although 2 of 8 vehicle-treated cats died from ventricular fibrillation and another had severe ventricular tachyarrhythmia, none of the defibrotide-treated cats had similar severe changes in the electrocardiogram and all 7 cats survived the 5-hour observation period. Defibrotide had no direct action on general hemodynamic functions. In a separate set of experiments, defibrotide (0.1 mg/ml) produced an 8- to 10-fold stimulation of PGI2 release. The data suggest a remarkable protective potential of defibrotide on reperfusion damage of the ischemic myocardium, which may be associated with a PGI2-related mechanism.

Adenosine Triphosphate↗

PubMed1978

Electrostatic complexes of mitomycin C with nucleic acids and polyanions.

Reductively activated mitomycin C exhibits strong, non-covalent electrostatic binding to polyanions such as polyvinylsulfate and polyphosphate. The protonated C-2 amino group generated by the reduction is most likely responsible for this type of interaction. At moderate drug and salt concentrations only covalent binding to nucleic acids is observable. This is shown to be guanine-specific in DNA for the first time, as well as in synthetic polyribo- and polydeoxyribonucleotides at 10--20 times higher binding levels than previously tested. At higher mitomycin C concentration, however, strong non-covalent electrostatic binding to nucleic acids also occurs, resulting in a binding ratio up to 1 mol drug bound per mol mononucleotide, although this non-specific binding is relatively inhibited compared to polyvinylsulfate. Salts also have an inhibitory effect on the non-specific binding to nucleic acids. A series of mitomycin derivatives were compared for their binding and cross-linking abilities using DNA as substrate, with the following results: (a) the presence of a basic nitrogen . funtion at C-2 promotes binding, both covalent and electrostatic, presumably by kinetically facilitating the approach between positively charged nitrogen and DNA. (b) The aziridine ring is the major covalent binding site, indispensable for crosslinking and determines the guanine-specificity of the binding.

Anions↗

PubMed1988

Preferential alkylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of guanines with guanines as neighboring bases in DNA.

The base sequence of DNA has been shown to influence the kinds and amounts of alkylation of purine bases by N-methyl-N-nitrosourea [W. T. Briscoe and L-E. Cotter, Chem. Biol. Interact. 56, 321 (1985)]. In the present study, the alkylation of DNA polymers of defined sequence by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has been investigated. The assay involved treating poly (dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), and calf thymus DNA with BCNU, followed by hydrolysis to release the modified purine bases and separation and quantitation of these by HPLC. Analysis of the results revealed that there was a 24-fold increase of 7-(beta-hydroxyethyl)guanine (HOEtG) in poly(dG).poly(dC) relative to poly(dA-dG).poly(dC-dT). There was also a 3-fold increase in HOEtG in poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and calf thymus DNA relative to poly(dA-dG).poly(dC-dT). A 2- to 4-fold increase of 7(beta-aminoethyl)guanine (AmEtG) was observed in poly(dG-dC).poly(dG-dC) relative to the other polymers tested. This study has determined that guanines in certain base sequences in polydeoxyribonucleotides are more susceptible to BCNU alkylation at the N-7 position than guanines in other sequences.

Alkylation↗

PubMed1985

DNA sequence has an effect on the extent and kinds of alkylation of DNA by a potent carcinogen.

A system has been developed to study the effects of base sequence (neighboring bases) upon the alkylation of guanine (G) and adenine (A) bases in DNA. The study was performed on the synthetic polydeoxyribonucleotides, poly(dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), as well as calf thymus DNA. Each polynucleotide was treated with N-[3H]methyl-N-nitrosourea (MNU), depurinated, and the freed alkylpurines separated by HPLC and quantitated by liquid scintillation counting. The amounts of 3-methylguanine (3-MG), 7-MG, and O6-MG relative to guanine, and 3-methyladenine (3-MA) and 1-MA plus 7-MA relative to adenine, and also the O6-MG/7-MG ratios were highly reproducible for a given polynucleotide. Significant differences were found in the amounts of each of the methylpurines formed when compared among the six synthetic polynucleotides and DNA. This evidence is interpreted as an effect upon alkylation which is ultimately dependent upon the base sequence. These findings may have significance in defining the specificity of chemical carcinogens in terms of the susceptability to modification of nucleotide sequences such as those found in certain oncogenes.

Adenine↗

PubMed1987

Defibrotide, an antithrombotic substance which prevents myocardial contracture in ischemic rabbit heart.

Defibrotide, a polydeoxyribonucleotide obtained from mammalian lungs, reduced in a dose-dependent fashion the ischemic contracture due to low perfusion (0.2 ml/min) of isovolumic left heart of rabbit and abolished the irregular rhythm of the heart, thereby restoring the cardiomechanical activity upon reperfusion (20 ml/min). Defibrotide stimulated the release of PG-like material from the heart in a dose-dependent manner without modifying the basal contractility. Both PGE2 and PGI2 (10 ng/ml) have an antiischemic activity on this preparation as shown by the partial reduction of the ischemic contracture and by the improvement of heart contractility upon reperfusion. Indomethacin infusion (1 microgram/ml) completely removed both the antiischemic activity of Defibrotide (400 micrograms/ml) and its ability to increase the generation of prostaglandins in the rabbit heart. These results suggest that Defibrotide has a beneficial influence on ischemic rabbit heart through an increase in prostaglandin synthesis. However other mechanisms not necessarily related to prostaglandin generation, such as a direct effect on membrane function deactivation and mitochondrial Ca2+ overload, should be considered in explaining the antiischemic activity of Defibrotide in the rabbit heart.

Animals↗

PubMed1992

A novel insight into the mechanism of the antithrombotic action of defibrotide.

Defibrotide is a polydeoxyribonucleotide sodium salt with antithrombotic properties. These properties have been attributed to its profibrinolytic activity [increase of tissue plasminogen activator (t-PA) activity, concomitant decrease of that of plasminogen activator inhibitor (PAI)], but there could conceivably be other factor(s). To look for these, we studied Defibrotide in a thrombosis model (pulmonary thromboembolism in mice) in which free radicals play a pivotal role. Defibrotide was found to be active after both intravenous and oral administration. Defibrotide behaved in vitro like a scavenger of H2O2 but not of O2.- in cell-free systems. Defibrotide added in vitro to cellular systems decreased the stimulated release of beta-glucuronidase from polymorphonuclear cells (PMNs), the luminol chemiluminescence induced by oxygen species generated by stimulated PMNs and the generation of O2.- from stimulated macrophages. We think that the antithrombotic activity of Defibrotide is based on other factor(s) in addition to profibrinolytic activity, i.e., some scavenger activity and desensitization of cells involved in thrombus formation must also be taken into account.

Animals↗

PubMed1995

Effects of defibrotide on leukocytosis in rabbits with diet-induced atherosclerosis.

The antithrombotic drug Defibrotide (DFT) (a polydeoxyribonucleotide with a mean MW of 20,000 Daltons) reduces the number of leukocytes and platelets in thrombi. Because leukocytes and platelets are of importance in the genesis of endothelial lesions leading to atherosclerosis, DFT was given to challenge leukocytosis in rabbits with diet-induced atherosclerosis (0.25% cholesterol for 16 weeks). After 9 weeks of cholesterol feeding and at the end of experiment, oral DFT (60 mg/Kg per day) had decreased the leukocyte count raised by the cholesterol diet. Leukocyte stickiness and leukocyte differential counts were not modified by either oral cholesterol or by oral cholesterol plus oral DFT. At the end of experiment, oral DFT had normalized the platelet count increased by cholesterol diet. The red blood cell count decreased by oral cholesterol at 9 weeks and at the end of experiment was normalized by DFT. The % of aortae endothelial surface involved in the atherosclerotic process was decreased by oral DFT. The frequencies of intimal thickening in blood vessels of kidneys and hearts and in cardiac valves were reduced by oral DFT by 47%, 29% and 17%, although these reductions were not statistically significant. It is suggested that DFT, by preventing the increase in the number of leukocytes and platelets and deactivating them, as demonstrated in papers already published, was able to counteract against the atherosclerotic process.

Animals↗

PubMed1985

Cardioprotective effects of defibrotide in acute myocardial ischemia in the cat.

We investigated the effects of Defibrotide (D), a natural polydeoxyribonucleotide, on acute myocardial ischemia (AMI) in anesthetized cats. A permanent ligature was placed around the left anterior descending coronary artery (LAD) 12-14 mm from its origin. Ventricular fibrillation and death were exceptional and when they occurred the cats were not included in the evaluation. Pretreatment of cats with D, 32 mg Kg-1 h-1, i.v. infusion, maintained throughout the 5 h occlusion period, reduced AMI-ST segment increases and increased the diminished pressure-rate index (PRI). AMI-induced changes in lactate, ATP and CPK in ischemic tissue were prevented by D. PGI2 gave the same results as D. Atenolol prevented the loss of myocardial CPK, but had no favourable effects on lactate and ATP in ischemic tissue. The beneficial effects of D in AMI reported here could be partly attributed to its ability to enhance PGI2 release from vascular walls; D might also relieve ischemia by improvement of local tissue oxygenation, energy supplies and platelet function by its ability to deaggregate platelet clumps.

Acute Disease↗

PubMed1986

Protective activity of defibrotide against lethal acute myocardial ischemia in the cat.

Defibrotide (D) is a natural polydeoxyribonucleotide from mammalian lungs with profibrinolytic and antithrombotic activities. D also has PGI2-stimulating and tissue plasminogen activator (TPA)-releasing activities, but has no anticoagulant properties. The protective effects of D were demonstrated very recently in a model for non-lethal ischemia in the cat. In the experiments reported here Defibrotide was tested in a model for acute myocardial ischemia leading to ventricular fibrillation (VF) and death of the cat. Occlusion of the coronary artery (LAD) at its origin induced VF and death in 17 of 20 control cats. When cats were treated with D (32 mg Kg-1, bolus i.v., + 32 mg Kg-1 h-1, i.v., after LAD occlusion) 19 of 20 animals survived until the end of experiments. D also prevented changes in plasma and myocardial CPK, hemodynamics and ECG. D was compared with a variety of pharmacological agents which are used clinically for specific cardiovascular diseases. The ability of D to promote considerable generation of PGI2 from vascular walls plus its ability to prevent the decreases in CPK-activity and ATP in the myocardial tissue may have roles in its beneficial effects against ischemic heart in the cat. However, the mechanism/s of the substantial protective effect of D against cardiac death has still to be clarified.

Animals↗

PubMed1992

Defibrotide has antiischemic activity in perfused rabbit hearts, preventing tissue Ca++ overloading.

Defibrotide (D), a polydeoxyribonucleotide obtained from mammalian lungs, reduced the ischemic contracture due to low perfusion (0.2 ml/min) of the isovolumic left heart of the rabbit and abolished the irregularity of the rhythm of the heart, thereby restoring the cardiomechanical activity upon reperfusion (20 ml/min). D stimulated the release of PG-like material. Indomethacin infusion completely prevented both the antiischemic activity of D and its ability to increase the generation of prostaglandins in the rabbit heart. Measurement by atomic absorption spectroscopy of calcium content in ischemic heart tissue and its mitochondrial fraction indicated that the ischemic procedure significantly increased tissue calcium content in both. D, Prostacyclin (PGI2) and Nifedipine protected the heart from ischemic ventricular contracture and prevented accumulation of calcium in the heart. The effect of D on preventing Ca++ overload was completely abolished by indomethacin infusion. The results indicate that the beneficial effects of Defibrotide in experimental ischemia are primarily due to a release of Prostaglandin E2 (PGE2) and PGI2, which in turn may inhibit the detrimental effects of calcium overload in myocytes and mitochondria.

Animals↗