DNAase Activity Research Articles

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Conugative transfer of bacteriocin genes - new mechanismof antimicrobial action of probiotic preparations

In connection with the high rate of antibiotic resistance growth of microorganisms, the creation of alternative antimicrobial agents or methods for their delivery is a priority in biology, medicine and veterinary medicine. The results of the design and testing of the ColE7-mediated kill-anti-kill system based on the probiotic strain Nissle 1917 are presented. The genetically modified strain Escherichia coli ŽP (donor killer) has been made at University of Ljubljana (Slovenia), carrying the colicin gene СolE7 with DNA-ase activity on the conjugative plasmid, as well as the immE7 gene in the chromosome. It provides the cell with the synthesis of an immune protein that binds tightly to the corresponding colicin, inhibiting its activity within the host cell. The system has been tested with the reference E. coli strain in various experimental models: in plankton culture, in a forming and formed biofilm. It has been shown that the antimicrobial action based on the conjugative transfer of bacteriocin genes is possible. Prospects for the use of E. coli ŽP as the basis of a probiotic preparation, which, unlike analogues, will have a high antibacterial activity against enteropathogens due to a new delivery mechanism of colicin, allowing an effective treatment of strains resistant to bacteriocins are identified.

Anti-DNA antibodies in the blood of patients with schizophrenia possess DNA-hydrolyzing activity

IntroductionAutoantibodies (Abs) to different neuronal receptors and DNA were detected in the blood of patients with schizophrenia. Abs hydrolyzing DNA were detected in pool of polyclonal autoantibodies in autoimmune and infectious diseases, such catalytic Abs were named abzymes.ObjectivesTo investigate the level of anti-DNA antibodies and DNA-hydrolyzing activity of IgG from the serum of patients with schizophrenia depending on leading clinical symptoms.Aims– To measure the concentration of anti-DNA Abs in serum of patients with leading positive and negative symptoms;– to determine DNA-hydrolyzing activity of IgG.MethodsIn our study, 51 patients were included. The levels of antiDNA Abs were determined using ELISA. DNA-hydrolyzing activity was detected as the level(%) of supercoiled pBluescript DNA transition in circular and linear forms. Statistical analysis was performed in “Statistica 9.0”.ResultsAnti-DNA Abs of patients with schizophrenia not only bind DNA, but quite efficiently hydrolyze the substrate. IgG of patient with schizophrenia were shown to possess DNA hydrolyzing activity. It should be noted that DNAase activity of IgG in patients with schizophrenia with a negative symptoms was significantly higher, than in patients with positive symptoms (Table 1).ConclusionsThe data show a correlation with the level of DNase activity and leading symptoms of patients with schizophrenia.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Compounds from Lactobacillus plantarum culture supernatants with potential pro-healing and anti-pathogenic properties in skin chronic wounds

Context: It is necessary to advance the field of alternative treatments for chronic wounds that are financially accessible to the least economically developed countries. Previously we demonstrated that topical applications of Lactobacillus plantarum culture supernatants (LAPS) on human-infected chronic wounds reduce the pathogenic bioburden, the amount of necrotic tissue, and the wound area, as well as promote debridement, granulation tissue, and wound healing.Objective: To study LAPS chemically and biologically and to find potential molecules responsible for its pro-healing and anti-pathogenic properties in chronic wounds.Materials and methods: (1) Chemical analysis: extracts were subjected to a column chromatography and the fractions obtained were studied by GCMS. (2) Quantification: dl-lactic acid (commercial kit), phenolic compounds (Folin–Ciocalteu), H2O2 (micro-titration), and cations (flame photometry). (3) Biological analysis: autoinducers type 2 (AI-2) (Vibrio harveyi BB170 bioassay), DNAase activity (Agar DNAase), and Pseudomonas aeruginosa biofilm inhibition (crystal violet technique).Results: According to its biological activity, the most significant molecules found by GCMS were the following: antimicrobials (mevalonolactone, 5-methyl-hydantoine, benzoic acid, etc.); surfactants (di-palmitin, distearin, and 1,5-monolinolein); anesthetics (barbituric acid derivatives), and AI-2 precursors (4,5-dihydroxy-2,3-pentanedione and 2-methyl-2,3,3,4-tetrahydroxytetrahydrofurane). Concentrations measured (µg/mL): dl-lactic acid (11.71 ± 1.53) and H2O2 (36 ± 2.0); phenolic compounds (485.2 ± 15.20); sodium (370 ± 17); potassium 920 ± 24); calcium (20 ± 4); and magnesium (15 ± 3). DNAase from LAPS had activity on genomic DNA from PMNs and P. aeruginosa.Discussion and conclusion: The molecules and biological activities found in LAPS could explain the observed effects in human chronic wounds.

Isolation and Characterization of Multi Drug Resistant Pathogen from Soil Samples Collected From Hospital Disposal Site

Antibiotic resistant bacteria was isolated from Uttarpara State General Hospital near Kolkata. The microscopical, biochemical tests suggested it to be Neisseria sp. Our major aim was to check the effect of several aqueous plant extracts on MDR and finding out tentative MIC value, study of any physiological changes brought about by the natural extract by phase contrast microscopy and ultimately to study any DNAase activity executed by the most successful plant extract on the multidrug resistant bacteria.

Enzymatic Rate Enhancements: A Review and Perspective

The sea urchin embryo nuclei which retained their ability to maintain the DNA synthesis in an in vitro system were isolated. The DNA synthesis isolated nuclei was shown to be an ATP-dependent process which is inhibited by low concentrations of actinomycin D, a polymerase alpha araCTP inhibitor. The newly synthesized DNA is represented by short fragments of about 4S. After addition of Ca2+, Mg2+-dependent DNAase to sea urchin embryo nuclei, the synthesis of short DNA fragments is enhanced. This stimulating effect of Ca2+, Mg2+-dependent DNAase is ATP-dependent and is observed only within a narrow range of enzyme concentrations (of the order of 1-5 units of DNAase activity per ml of incubation sample). The increase in the enzyme concentration to 10 or more units of activity results in the depression of DNA synthesis. It is concluded that DNA replication in sea urchin embryo nuclei depends on the presence of active DNAases as well as on the number of accessible initiation sites of DNA replication.

Modulatory effect of vitamins A, C and E mixtures against tefluthrin pesticide genotoxicity in rats

The present experiments to be reported were declared the effect of mixtures from the three antioxidant vitamins (A, C and E) on some hematologic parameters, liver and testes nucleic acid system, blood immune-system, chromosomal aberration and sperm-shape of tefluthrin (LD 50) intoxicated rats. A total of 90 male albino rats were used divided into six groups (15 rats each). Oral ingestion of technical tefluthrin significantly decreased the count of RBCs, Hb content and stimulated plasma LDH activity, also reduced the levels of blood immuno globulins (IgG, IgA and IgM). Under the effects of tefluthrin liver and tests nucleic acids (RNA and DNA) contents were decreased and the activities of nucleases (RNAase and DNAase) were also inhibited relative to these values of normal healthy control group. Chromosomal aberrations and sperm-shape abnormalities were significantly increased after ingestion of tefluthrin, compared to the control group. Mitotic index (MI) and sperm count was decreased significantly due to the potential cytotoxicity of tefluthrin. Treatment of intoxicated rats with antioxidant vitamins (A, C and E) mixtures reduced the harmful influences of tefluthrin. Blood IgG, IgA and IgM levels, RBCs count and Hb content were improved relative to normal healthy animal group. The frequency of chromosome aberrations in bone marrow cells of intoxicated rats treated with vitamins as well as the frequency of sperm abnormalities were decreased and readjusted near to that of the healthy control animals, also MI and sperm counts were increased significantly near to the control group after vitamins ingestion as treatments. It is interesting to note that, the ingestion of vitamins mixtures readjusted and normalized the hematologic parameters around those of normal healthy control. In case of liver and nucleic acids system, the disturbed effects of the pesticide induction of RNA and DNA contents as well as RNAase and DNAase activities were alleviated by the treatments with the present antioxidative vitamins mixtures, in which the nucleic acids contents and nucleases activities of liver and testes of tefluthrin intoxicated rats were ameliorated and normalized compared with those of normal control.

Purification and biochemical characterization of a mycelial alkaline phosphatase without DNAase activity produced byAspergillus caespitosus

Biochemical properties of a termostable alkaline phosphatase obtained from the mycelium extract of A. caespitosus were described. The enzyme was purified 42-fold with 32% recovery by DEAE-cellulose and concanavalin A-Sepharose chromatography. The molar mass estimated by Sephacryl S-200 or by 7% SDS-PAGE was 138 kDa and 71 kDa, respectively, indicating a homodimer. Temperature and pH optima were 80 degrees C and pH 9.0. This enzyme was highly glycosylated (approximately 74% saccharide content). The activity was enhanced by Mg2+ (19-139%), NH4+ (64%), Na+ (51%) and Mn2+ (38%). 4-Nitrophenyl phosphate (4-NPP) was preferentially hydrolyzed, but glucose 1-phosphate (93%), UTP (67%) and O-phosphoamino acids also acted as substrates. V(lim) and K(m) were 3.78 nkat per mg protein and 270 micromol/L in the absence of Mg2+ and 7.35 nkat per mg protein and 410 micromol/L in the presence of Mg2+, using 4-NPP as substrate. The purified alkaline phosphatase removed the 5'-phosphate group of a linearized plasmid without showing DNAase activity, indicating its potential for recombinant DNA technology.

DNA-hydrolyzing IgG antibodies from the blood of patients with acquired immune deficiency syndrome

DNAase activity of 110 samples of IgG from the blood of AIDS patients was analyzed. It was shown that the relative activity of preparations varies very much from patient to patient, but 96% preparations show detectable level of DNAase activity. Several rigid criteria were applied and it was shown that DNAase activity is an intrinsic property of antibodies from AIDS patients. It was shown that catalytic activity could posses not only intact IgG, but also separated light chains of polyclonal antibodies. The abzymes catalyze DNA hydrolysis effectively in a wild range of pH (5.0-9.5). K(M) and V(MaKC) values of antibody-dependent hydrolysis of DNA was estimated.

Biologically Active Polypeptides and Hydrolytic Enzymes in Sea Anemones of Northern Temperate Waters

The content of biologically active polypeptides in aqueous and ethanol extracts of seven sea anemone species collected near Sakhalin Island (Sea of Okhotsk) and in Posyet Bay (Sea of Japan) was analyzed. Water extracts of the sea anemone Cribrinopsis similis showed the highest hemolytic activity, while ethanol extracts proved to have toxic properties. The levels of toxic and hemolytic activity of extracts of sea anemones inhabiting northern temperate waters were 2 to 3 orders of magnitude lower, compared to tropic species. The reason for this is likely to be the differences in the habitat conditions and biological traits of these animals. The water extracts of all species possessed proteolytic, phospholipase A2, and low DNAase activities, except Actinostola sp., whose aqueous extract contained a high activity alkaline DNAase. The species studied contained a wide range of proteinase inhibitors, O-glycosyl hydrolases (glycosidases and polysaccharide hydrolases). Water extracts of C. similis and Stomphia coccinea possessed the highest laminarinase activity. High activity of N-galactopyranosidase was found in water extracts of S. coccinea and Oulactis orientalis.

Purification of Neurospora crassa alkaline phosphatase without DNAse activity for use in molecular biology

Alkaline phosphatase, excreted by Neurospora crassa preg (c) and purified to apparent homogeneity by 7.5% PAGE, did not show DNAase activity and removed the terminal 5'-phosphate group from plasmid Bluescript M13(+) linearized with EcoRI. The preg (c) strain may therefore replace other sources of alkaline phosphatase for use in dephosphorylating linearized plasmidial DNA in cloning experiments.

Activation of Human T Helper 1 and DNAase Expression in CD4 +T Cells Induced by Short Immunomodulating Peptides

Activation of human T helper 1 cells took place when lymphomononuclear cells from healthy donors were incubated in the presence of short synthetic peptides encompassing sequences present in extracellular matrix proteins. Active peptides conformed to a common structural pattern ("2-6-11 motif") [N.López-Moratalla et al., Biochem. Biophys. Acta (1994) 1221, 153-158] conferring immnunomodulating properties. The release of IL-2 and IFNγ, as well as LAK and NK-dependent cytotoxicity induced by these peptides, could be blocked by anti-HLA-DR antibody. Activated CD4 + cells isolated from the mixed incubated population contained secretion granules with DNAase activity. These results suggest that these immunomodulating peptides presented by HLA-II play a key role in the differentiation of CD4 + T cells towards a Th1 functional phenotype.

Optimization of medium for extracellular nuclease formation from Rhizopus stolonifer

A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 μM Mn(2+), Co(2+) or Mg(2+), and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA.

Preparation of a nuclease-free alpha-amylase and its use in DNA purification

A thermostable alpha-amylase fromBacillus licheniformis when heated to 80°C for 20 min retains >90% of its activity but loses all DNAase activity. This allows the amylase to be used to purify yeast DNA.

Effects of nutritional status of Aspergillus nidulans on its nuclease activity

Before nutrient limitation set in, mycelium of Aspergillus nidulans contained little free nuclease activity but treatment of extracts with trypsin led to the appearance of substantial DNAase and RNAase activity as a result of activation of a cryptic nuclease. Onset of nitrogen or sulphur limitation led to the appearance of DNAase activity but not necessarily RNAase activity. Carbon or phosphate limitation did not lead to a similar increase in free DNAase activity.

DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.

Genetic and structural characterization of endA: A membrane-bound nuclease required for transformation of Streptococcus pneumoniae

The endA gene encoding the membrane nuclease of Streptococcus pneumoniae, which is necessary for DNA uptake in genetic transformation, was cloned in a streptococcal vector. This was accomplished by insertional mutagenesis of the gene, cloning of the mutant allele, and substitution of the wild-type allele by chromosomal facilitation of plasmid establishment. Plasmids carrying the endA+ gene complemented cells with endA- in the chromosome to restore DNAase activity and transformability. Determination of its DNA sequence showed the gene to encode a 30 kDa protein, EndA, with a typical signal sequence for membrane transport at its amino end. In vitro synthesis of EndA showed the initial translation product to be enzymatically active without further processing. Comparison with EndA found in cell membranes indicated that the enzyme retained its signal sequence, which apparently anchored the otherwise hydrophilic protein to the membrane. From the nucleotide sequence in the vicinity of endA and the effect of various insertions and deletions, it appears that endA is the last gene in an operon containing at least two other genes. Neither of these upstream genes, nor the downstream gene, are essential for either cell viability or transformability.

Lens fiber differentiation correlated with activation of two different DNAases in lens embryonic cells

In order to identify the different DNAases present in the lens differentiating tissue, we have used an assay which reveals their activity directly on DNA-containing gels after SDS polyacrylamide gel electrophoresis. DNAase renaturation from nuclear embryonic lens extracts does not occur after separation in 0.1% SDS polyacrylamide gel electrophoresis in contrast to that observed with purified micrococcal nuclease. When the SDS concentration in the running buffer and separating gel is decreased to 0.075%, renaturation of lens DNAase and enzyme activities are observed. Isoelectrofocusing was carried out in a polyacrylamide gel which was overlaid with an agarose gel containing DNA, permitting the visualization of the pI of DNAase activity. The presence of several DNAase isoenzymes was demonstrated in 11-day embryonic lenses. In epithelial lens nuclei, high molecular weight (MW) isoenzymes with basic pI were predominant. In post-mitotic fiber lens nuclei, two lower MW isoenzymes with acidic pI were detected as well as a high MW activity with a basic pI.

DNAase activity in terminal differentiating embryonic chick lens tissues : ∗B. ALLINQUANT, P. BAUSERO, E. CHAUDUN, M. SANWAL, Y. COURTOIS and M.F. COUNIS U. 118 INSERM, 29, rue Wilhem PARIS and ∗U 134 INSERM Hopital Salpåtrière, PARIS FRANCE

DNAase activity in terminal differentiating embryonic chick lens tissues : ∗B. ALLINQUANT, P. BAUSERO, E. CHAUDUN, M. SANWAL, Y. COURTOIS and M.F. COUNIS U. 118 INSERM, 29, rue Wilhem PARIS and ∗U 134 INSERM Hopital Salpåtrière, PARIS FRANCE

Two dimensional NMR studies on the solution structure of d-CTCGAGCTCGAG

Two dimensional (2D) FT-NMR investigations have been carried out on the self-complementary dodecanucleotide d-CTCGAGCTCGAG, which has cleavage sites for the restriction enzyme Xho I (between C and T). The central TCG portion is also known to show a preference for DNAase activity. Complete resonance assignments have been obtained for the non-exchangeable sugar and base protons of the oligonucleotide. Information regarding sugar geometries, glycosidic torsion angles and other structural parameters has been obtained from the relative intensities of the cross peaks in the COSY and NOESY spectra. The results indicate that deoxyribose rings of C1 and C7 adopt a conformation different from the remaining sugars in the double helical oligonucleotide. The central TCG portion also exhibits variations in the backbone structure. The base stacking in the double helix shows interesting sequence dependent effects suggesting that the sequence effects are not localised to nearest neighbours but extended over longer stretches.