Inhibitor Of Deoxyribonuclease Research Articles

Thiazole derivatives as dual inhibitors of deoxyribonuclease I and 5-lipoxygenase: A promising scaffold for the development of neuroprotective drugs

A library of 43 thiazole derivatives, including 31 previously and 12 newly synthesized in the present study, was evaluated in vitro for their inhibitory properties against bovine pancreatic DNase I. Nine compounds (including three newly synthesized) inhibited the enzyme showing improved inhibitory properties compared to that of the reference crystal violet (IC50 = 346.39 μM). Two compounds (5 and 29) stood out as the most potent DNase I inhibitors, with IC50 values below 100 μM. The 5-LO inhibitory properties of the investigated derivatives were also analyzed due to the importance of this enzyme in the development of neurodegenerative diseases. Compounds (12 and 29) proved to be the most prominent new 5-LO inhibitors, with IC50 values of 60 nM and 56 nM, respectively, in cell-free assay. Four compounds, including one previously (41) and three newly (12, 29 and 30) synthesized, have the ability to inhibit DNase I with IC50 values below 200 μM and 5-LO with IC50 values below 150 nM in cell-free assay. Molecular docking and molecular dynamics simulations were used to clarify DNase I and 5-LO inhibitory properties of the most potent representatives at the molecular level. The newly synthesized compound 29 (4-((4-(3-bromo-4-morpholinophenyl)thiazol-2-yl)amino)phenol) represents the most promising dual DNase I and 5-LO inhibitor, as it inhibited 5-LO in the nanomolar and DNase I in the double-digit micromolar concentration ranges. The results obtained in the present study, together with our recently published results for 4-(4-chlorophenyl)thiazol-2-amines, represent a good basis for the development of new neuroprotective therapeutics based on dual inhibition of DNase I and 5-LO.

Synthesis and analysis of 4-oxothiazolidines as potential dual inhibitors of deoxyribonuclease I and xanthine oxidase

In this study, seven new 4-oxothiazolidine derivatives were synthesized and assayed, along 7 known derivatives, for inhibitory properties against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. Among tested compounds, (5Z)-Ethyl-2-(2-(cyanomethylene)-4-oxothiazolidin-5-yliden)acetate (6) exhibited inhibitory activity against both enzymes (DNase I IC50 = 67.94 ± 5.99 μM; XO IC50 = 98.98 ± 13.47 μM), therefore being the first reported dual inhibitor of DNase I and XO. Observed DNase I inhibition qualifies compound 6 as the most potent small organic DNase I inhibitor reported so far. Derivatives of 2-alkyliden-4-oxothiazolidinone (1) inhibited DNase I below 200 μM, while the other tested 4-oxothiazolidine derivatives remained inactive against both enzymes. The molecular docking and molecular dynamics simulations into the binding sites of DNase I and XO enzyme allowed us to clarify the binding modes of this 4-oxothiazolidine derivative, which might aid future development of dual DNase I and XO.

New mechanism of γ-H2AX generation: Surfactant-induced actin disruption causes deoxyribonuclease I translocation to the nucleus and forms DNA double-strand breaks

We previously showed that nonionic surfactants, nonylphenol polyethoxylates (NPEOs), induced phosphorylation of histone H2AX, forming γ-H2AX. In this study, we analyzed the mechanism of γ-H2AX generation by an NPEO with 15 ethylene oxide units (NPEO(15)). In MCF-7 breast carcinoma cells, NPEO(15) treatment induced γ-H2AX in a dose-dependent manner. EDTA and ZnCl2, two inhibitors of deoxyribonuclease I (DNase I), inhibited both the γ-H2AX and DNA double-strand breaks induced by NPEO(15). NPEO(15) disrupted filamentous actin and released free DNase I as detected by cell fractionation analysis. Based on immunofluorescence staining of DNase I and monitoring DNase I-GFP localization, DNase I was translocated from the cytosol to the nucleus of cells after treatment with NPEO(15). This translocation did not occur with the common DNA damage inducers ultraviolet B irradiation and hydrogen peroxide. Other surfactants, Tween 20, Triton X-100 and Nonidet P-40, also generated γ-H2AX. These results show that γ-H2AX induction by surfactants including NPEOs, occurs via a new mechanism involving release of free DNase I with actin disruption. This mechanism is distinct from the process of γ-H2AX generation caused by direct chemically induced DNA damage.

Ca(2+)-dependent and caspase-3-independent apoptosis caused by damage in Golgi apparatus due to 2,4,5,7-tetrabromorhodamine 123 bromide-induced photodynamic effects.

To clarify the role of the Golgi apparatus in photodynamic therapy-induced apoptosis, its signaling pathway was studied after photodynamic treatment of human cervix carcinoma cell line HeLa, in which a photosensitizer, 2,4,5,7-tetrabromorhodamine 123 bromide (TBR), was incorporated into the Golgi apparatus. Laser scanning microscopic analysis of TBR-loaded HeLa cells confirmed that TBR was exclusively located in the Golgi apparatus. HeLa cells incubated with TBR for 1 h were then exposed to visible light using an Xe lamp. Light of wavelength below 670 nm was eliminated with a filter. Morphological observation of nuclei stained with Hoechst 33342 revealed that apoptosis of cells was induced by exposure to light. Electron spin resonance spectrometry showed that light-exposed TBR produced both singlet oxygen (1O2) and superoxide anion (O2-). Apoptosis induction by TBR was inhibited by pyrrolidine dithiocarbamate, an O2- scavenger, but not by NaN3, a quencher of 1O2. Furthermore, TBR-induced apoptosis was inhibited by aurintricarboxylic acid and ZnCl2, which are known as inhibitors of deoxyribonuclease (DNase) gamma, and (acetoxymethyl)-1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, a chelator of Ca2+, but not by acetyl Asp-Glu-Val-Asp-aldehyde, an inhibitor of caspase-3. These results suggested that O2- was responsible for TBR-induced apoptosis, and Ca(2+)-dependent and caspase-3-independent nuclease such as DNase gamma played an important role in apoptotic signaling triggered by Golgi dysfunction.

Changes in susceptibility of bovine sperm to in situ DNA denaturation during prolonged incubation at ambient temperature under conditions of exposure to reactive oxygen species and nuclease inhibitor.

Sperm were incubated for up to 9 days in the presence or absence of exogenous hydrogen peroxide, phenylalanine, catalase and aurintricarboxylic acid to assess the influence of reactive oxygen species and inhibition of deoxyribonucleases on sperm chromatin stability. The assessment of sperm DNA susceptibility to in situ acid denaturation by the sperm chromatin structure assay did not detect any difference in chromatin stability between sperm incubated for 9 days under aerobic and anaerobic conditions in a diluent called 14G. Exposure to exogenous hydrogen peroxide under both aerobic and anaerobic conditions and to phenylalanine under aerobic conditions (which produces hydrogen peroxide by a reaction catalysed by the aromatic amino acid oxidase present in sperm) was detrimental to sperm chromatin stability, increasing its DNA susceptibility to in situ acid denaturation over the incubation time. This effect was eliminated if catalase was present in the diluent. Inclusion of the general deoxyribonuclease inhibitor aurintricarboxylic acid in the diluent severely decreased sperm chromatin stability under both aerobic and anaerobic conditions. Aurintricarboxylic acid was mildly cytotoxic, as revealed by viability assessment, under aerobic, but not under anaerobic, incubation conditions. Exogenous hydrogen peroxide, either directly added to the diluent or generated through the enzymatic oxidation of phenylalanine, was detrimental to sperm motility and the integrity of the plasma membrane.

DNase I interaction on muscle Z-line

The effect of deoxyribonuclease I on muscle Z-line structures was re-examined. Under conditions of deoxyribonuclease I activation (presence of the divalent cation Ca2+ and Mg2+), a deoxyribonuclease I preparation did not affect Z-line structure if phenylmethylsulfonylfluoride, an inhibitor of serine proteases, was also present. In the absence of protease inhibitor, both Z-lines and M-lines were digested, even in the presence of EDTA and EGTA as inhibitors of deoxyribonuclease I. These electron microscopic observations were consistent with the following results from sodium dodecyl sulphate gel electrophoresis: when the protease was inhibited but deoxyribonuclease I was activated, myofibrillar proteins remained essentially intact. However, degradation of proteins in both rabbit psoas and chicken pectoralis myofibrils was observed in the presence of deoxyribonuclease I inhibitors when the protease inhibitor was absent. Our data strongly suggest that the interaction of deoxyribonuclease I with Z-line proteins previously reported is most likely due to contamination of the deoxyribonuclease I fraction by the serine-type proteases.

5838-DNI, a deoxyribonuclease inhibitor produced by Streptomyces sp. strain no. A-5838.

5838-DNI, an inhibitor of deoxyribonuclease (DNase) II from porcine spleen was produced by Streptomyces sp. strain No. A-5838. The structure of 5838-DNI was shown to be 1,4,4a,5,12,12a-hexahydro-4,4a,11,12a-tetrahydroxy-3,8-dimethoxy-9- methoxycarbonyl-10-methyl-1,5,12-trioxo naphthacene. Although similar in structure to tetracenomycin C, which is an antibiotic against Gram-positive bacteria, 5838-DNI has different antibacterial activity. 5838-DNI was distinguished from 5923-DNI, a previously reported DNase II inhibitor, in inhibitory activity against each enzyme. 5838-DNI showed dependency of inhibition on pH and temperature, and inhibited phosphodiesterase I in a competitive manner. These data suggest that 5838-DNI is the first reported example of an inhibitor of microbial origin which is able to inhibit DNase II and phosphodiesterase I.

A novel method for determination of deoxyribonuclease and deoxyribonuclease inhibitor.

A novel method for determination of deoxyribonuclease and deoxyribonuclease inhibitor.

A novel deoxyribonuclease inhibitor from Micromonospora.

A novel deoxyribonuclease inhibitor from Micromonospora.

A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA.

A deoxyribonuclease inhibitor has been purified from KB cells by chromatography on single-stranded DNA-cellulose. Polyacrylamide gel electrophoresis showed the purified preparation to contain two major polypeptides in sodium dodecyl sulfate, with molecular weights of 72,000 and 65,000, but only one major band (with a molecular weight of approximately 140,000) after electrophoresis under nondenaturing conditions. The protein inhibits the hydrolysis of single-stranded DNA by KB DNase, DNase I, DNase II, and nuclease S1, but has no effect on the hydrolysis of double-stranded DNA by these enzymes. The inhibitor causes a reduction in the rate of hydrolysis of DNA by the deoxyribonuclease, probably by reducing the effective concentration of substrate.

Actin is the naturally occurring inhibitor of deoxyribonuclease I.

Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3.1.4.5). In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle cells. (a) The inhibitor is a major cellular component constituting 5-10% of the soluble protein. (b) It migrates with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a characteristic molecular weight of 42,000. (c) It has an amino-acid composition closely similar to that of actin. (d) The peptide maps of the two proteins are nearly identical. (e) Skeletal muscle actin inhibits the enzymatic activity of DNase I. (f) DNase I-agarose affinity chromatography quantitatively retains purified skeletal muscle actin, and actin, specifically, from high-speed supernatants of whole cell extracts. (g) An antibody to purified inhibitor protein from calf thymus, used in indirect immunofluorescence on cells grown in culture, stains a two-dimensional network of fibers similar to that seen with an actin-specific antibody.The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions.

Studies on the function of deoxyribonuclease inhibitor after partial hepatectomy

Partial hepatectomy and acute stress (intraperitoneal injection of celite) caused an increase in the activity of serum deoxyribonuclease inhibitor. The increased activity after partial hepatectomy differed kinetically from that after acute stress. Deoxyribonuclease inhibitor was purified from rat liver about 30-fold and its properties were studied and found to be those of a protein. The activity of the inhibitor in rat liver increased after partial hepatectomy. The activity of the inhibitor may not be related to the activity of DNA synthesis as assayed by the incorporation of [ 3H]thymidine.

Purification from crude extract by affinity chromatography of the inhibitor of deoxyribonucleae I.

A one step procedure, consisting of affinity chromatgography, was worked out for the purification from crude extracts of calf thymus of the protein inhibitor specific for pancreatic deoxyribonuclease (DNAase I).For this purpose DNAase I was immobilized on agaros. This solid phase enzyme conjugate was shown to have the ability to adsorb from crude extracts one major protein, which could be eluted again with 3 M guanidine‐HCl in 1.0 M soudium acetate and 30% glycerol. This protein preparation had only a little DNAase inhibiting activity, but could be idnetified by gel electrophoresis and peptide mapping as the inhibitor protein described recently. The low recovery of inhibitor activity was due to poor stability of the inhibitor protein in the solvent system found necessary to effect its elution from the column material.

Purification from calf thymus of an inhibitor of deoxyribonuclease I.

A protein inhibitor specific for pancreatic deoxyribonuclease (DNAase I) was purified 10‐ to 20‐fold from calf thymus. This purification resulted in a nearly homogenous protein demonstrating the relative abundance of the inhibitor among the soluble proteins of the crude extract.Like the DNAase inhibitors from calf spleen described earlier the thymus inhibitor showed a high tendency to form high molecular weight aggregates of low specific activity. Conditions which decreased the rate of this self‐aggregation were found as were conditions for the dissociation of already formed inhibitor aggregates.The molecular weight of the purified inhibitor was determined under both stabilizing and disaggregating conditions and values between 45000 and 50000 were obtained.The interaction between the enzyme and the inhibitor was shown to give rise to a stable complex containing one molecule each of the involved protein species.

Increased activity of deoxyribonuclease inhibitor in rat serum after partial hepatectomy

Increased activity of deoxyribonuclease inhibitor in rat serum after partial hepatectomy

Purification from calf spleen of two inhibitors of deoxyribonuclease. I. Physical and chemical characterization of the inhibitor II.

Purification from calf spleen of two inhibitors of deoxyribonuclease. I. Physical and chemical characterization of the inhibitor II.

Crystallization from Calf Spleen of Two Inhibitors of Deoxyribonuclease I

The crystallization from calf spleen of two inhibitors of deoxyribonuclease I is reported in this paper. The inhibitors, protein in nature and designated I and II, were shown to act through the formation of stable complexes with DNase I and a method has been devised for the isolation of both complexes. The stoichiometry of complex formation was studied in detail in the case of Inhibitor II. It was found that the addition compound formed (mol wt 88,200) consisted of 1 mole of DNase I (mol wt 33,200) and 1 mole of Inhibitor II (mol wt 57,400).

An assay for and some properties of deoxyribonuclease inhibitor in rat liver

A simple, convenient assay for DNase I protein inhibitor has been devised. Some of the conditions affecting the assay such as Mg ++ concentration, pH and ionic strength, have been investigated. Some properties of DNase inhibitor have been studied. The inhibitor is relatively labile to storage, is not affected by sulfhydryl reagents, and is destroyed by heating at 60 °C. Evidence is presented that suggests the mechanism of inhibitor action is through combination with a Mg ++-DNA complex. The inhibitor-DNA-Mg ++ combination is not wholly resistant to the action of DNase I.

OPTIMAL CONDITIONS FOR THE TRANSFORMATION OF STREPTOCOCCI.

Perry, Dennis (Northwestern University Medical School, Chicago, Ill.) and Hutton D. Slade. Optimal conditions for the transformation of streptococci. J. Bacteriol. 85:636-642. 1963.-A study of the properties of the streptococcal transforming system, employing streptomycin resistance as a marker, resulted in a 1,000- to 10,000-fold increase in the rate of transformation. In some cases, as high as 0.5% transformants have been obtained. Certain aspects of the system differed markedly from those of other bacterial transforming systems, particularly with regard to the time for the appearance of competence. When a group H strain was exposed to deoxyribonucleic acid after various periods of incubation, the time at which cells were most competent was about 2 hr. Similar experiments with a different group H strain and an ungroupable strain showed that competence for both occurred after 1 hr of growth. It is significant that the period of optimal competence, though attained at different times, took place at the beginning of the logarithmic phase of growth. The temperature optimum for transformation was about 37 C. Cells preincubated in sheepblood broth exhibited a higher level of transformation than cells preincubated in plain or human serum broth. That some correlation exists between the attainment of competence and growth is evident. In contrast, human serum was most effective in the transforming media. The few preparations of bovine albumin (fraction V) employed did not appreciably support transformation. Data are also presented which show that some relationship exists between transforming efficiency and serological classification, in that homologous transformation showed a 100- to 10,000-fold greater efficiency over heterologous transformation. Antibodies to deoxyribonuclease, as well as other inhibitors of deoxyribonuclease, failed to bring about transformation in group A streptococci.

Characteristics of the DNA nucleotidyltransferase activity in non-aqueous type calf-thymus nuclei

Abstract Extracts of non-aqueous type calf-thymus nuclei have been fractionated with acid and with ammonium sulphate. The fractions so obtained have been assayed for DNA nucleotidyltransferase (EC 2.7.7.7) activity in the presence and absence of cysteine, and in the presence of one or of four deoxyribonucleoside-5′-triphosphates. The patterns of incorporation of [32P]TMP indicate that the nuclei contain both DNA nucleotidyltransferase and a physically distinct nucleotidyltransferase originally described by Krakow et al. Overall incorporation of deoxyribonucleotide catalysed by the latter enzyme is about 1 27 of that catalysed by the DNA nucleotidyltransferase, as calculated from data obtained from the acid and ammonium sulphate fractions. The total number of units of deoxyribonuclease I (EC 3.1.4.5) activity in each fraction derived from the initial nuclear soluble extract was calculated. The sum of the totals so obtained exceeded the total units found in the soluble extract by a factor of sixteen. It was inferred from that observation that the soluble extract contained an inhibitor of deoxyribonuclease I. Evidence is presented for the existence in certain control reaction mixtures, of a non-enzymic incorporation of 32P from [32P]TTP into thermally denatured DNA.