DNase Research Articles

Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli.

The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli. The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag. The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength. The self-assembly of VP1 capsoids can be induced from purified VP1 pentamers by increasing the ionic strength with (NH4)2SO4. These VP1 capsoid particles were packed in vitro with anti-sense oligonucleotides and plasmid DNA. The loading with DNA was pH-dependent. We observed the highest efficiency at pH 5. DNase I treatment of particles with encapsidated material showed that 37-55% of the bound oligonucleotides and fragments of 1.5-1.8 kb double-stranded DNA were protected against degradation.

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase.

The 36K protein attached at the 5' end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0 center dot 8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.

Isolation and Characterization of NUC70, a Cytoplasmic, Hematopoietic Apoptotic Endonuclease

Endonucleolytic DNA fragmentation is the common end point and the prevailing indicator of apoptosis. We have identified a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. We purified NUC70 to homogeneity and generated a rabbit polyclonal antibody to distinguish it from previously identified nucleases. Biochemical characterization of isolated NUC70 demonstrates that it is Ca2+/Mg2+-dependent and active over a pH range of 6-8. When incubated with isolated HeLa nuclei, NUC70 was capable of generating internucleosomal DNA fragmentation. This endonucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-ethylmaleimide, spermine, and iodoacetamide. Western immunoblots using the anti-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indicate that NUC70 expression and activity is restricted to human hematopoietic cells. No such activity was detected in human epithelial cell lines or murine hematopoietic cells. We also observed no difference in levels of NUC70 expression between apoptotic and nonapoptotic cells, suggesting that activation of NUC70 may be by posttranslational modification. We demonstrate that NUC70 activity is diminished in cells pretreated with the caspase inhibitors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytoplasmic and nuclear endonuclease activities during apoptosis show that NUC70 is a cytoplasmic endonuclease that is translocated to the nucleus after the initiation of apoptosis. We confirmed this with immunostaining studies using anti-NUC70 antibody. These results demonstrate that NUC70 is an endogenous cytoplasmic endonuclease that is activated during apoptosis in a caspase-dependent mechanism.

The safety and efficacy of a proteolytic ointment in the treatment of chronic ulcers of the lower extremity

Background: Elase is a widely used ointment consisting of a combination of 2 proteolytic enzymes, fibrinolysin and desoxyribonuclease (DNAse). It is said to promote debridement of necrotic and purulent debris from skin ulcers. Objective: Our purpose was to assess the efficacy and safety of this ointment and its components in the treatment of chronic ulcers of the lower extremity. Methods: This was a double-blind, randomized, prospective study of 84 patients with leg ulcers exhibiting necrotic and purulent debris, who were treated for 21 days with twice-daily applications of the ointment, fibrinolysin, DNAse, or who received the ointment vehicle (placebo). We assessed 6 efficacy features: ulcer size, purulent exudate, necrotic tissue, erythema, pain, and overall condition of the lesion at days 8, 15, and 21 after initiation of treatment. We also assessed the frequency of adverse effects. Results: All treatments produced some improvement in the efficacy parameters and overall condition of the ulcers by week 3, but no statistically significant difference was found when compared with placebo. No serious adverse effects were noted. A later retrospective reanalysis of the data found a statistically significant reduction of purulent exudate only at days 3 and 7 of treatment in the group treated with the complete ointment, but not in the other features. Conclusion: The proteolytic ointment provides no long-term clinical benefit in reducing purulent exudate, pain, erythema, necrotic tissue, or overall condition of chronic leg ulcers when compared with either of its two components or placebo. (J Am Acad Dermatol 1998;39:737-40.)

L-DNase II, a molecule that links proteases and endonucleases in apoptosis, derives from the ubiquitous serpin leukocyte elastase inhibitor.

The most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cation-independent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.

On the use of Zn 2+ to discriminate endonucleases activated during apoptosis

One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn 2+ is known to inhibit Ca 2+- and Mg 2+-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn 2+. In this paper, we analyse the effects of Zn 2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn 2+ is able to inhibit also this acidic endonuclease at a concentration of 1–6 mM. At a higher concentration of Zn 2+. DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn 2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn 2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.

Role of sialic acid and alkaline DNase in breast cancer.

Serum levels of sialic acid and alkaline DNase (ADA) were analysed in 495 blood samples collected from 170 breast cancer patients before and during/after anticancer treatment. Fifty-six healthy females were included in the study to define the cutoff values. The markers were analysed by highly sensitive spectrophotometric methods. Statistical evaluation of the data was done using Student's 't' test, paired 't' test and ROC curve analysis. The total sialic acid (TSA), lipid bound sialic acid (LSA) and ADA in sera of untreated breast cancer patients were significantly higher than in controls. ROC curve analysis revealed TSA and LSA to be useful markers for diagnosis of breast cancer. Serum levels of TSA and LSA were significantly decreased in complete responders as compared to their pretreatment values. The pretreatment ADA values showed much individual variation. However, responders showed higher levels of ADA than untreated patients. In nonresponders the values of the biomarkers were comparable with pretreatment levels. The study suggested that TSA and LSA can be helpful in the diagnosis of breast cancer. All three markers can be used for assessment of response to anticancer treatment in breast cancer patients.

Functional characterization of a human DNase-like protein encoded by a gene positioned in Xq28

Xib, a gene recently reported to reside on the q28 region of the human X chromosome [Pergolizzi et al. (1996) Gene 168, 267–270], contains an open reading frame homologous to those of the DNase I family enzymes. The full open reading frame of this gene has been fused to the E. coli gene of the maltose binding protein and expressed in bacteria as a chimeric protein. The partially purified chimeric protein is enzymatically active. It introduces single and double stranded breaks into supercoiled DNA, at 30°C in the absence of divalent cations and at a pH optimum of 5.2. To our knowledge this enzyme represents the first cloned human endonuclease with characteristics similar to those of acidic DNase II.

Requirement of distal and proximal promoter sequences for chromatin organization of the osteocalcin gene in bone-derived cells.

The osteocalcin (OC) gene encodes a 10 Kda bone-specific protein which is expressed with the onset of mineralization during the differentiation of normal diploid osteoblasts. We have previously reported that transcriptional activation of this gene is accompanied by the presence of two DNase I hypersensitive sites, both located in the promoter region spanning key basal (proximal site, -170 to -70) and steroid-dependent enhancer (distal site, -600 to -400) elements. Here, we have examined stably transfected ROS 17/2.8 cell lines carrying OC promoter-reporter transgenes which contain a series of 5'-deletions and determined the effects of these truncations on the chromatin organization. It has been found that: (1) DNase I hypersensitivity at -600 is not a requirement for vitamin D-dependent transcriptional upregulation; (2) basal transcriptional activity and proximal nuclease hypersensitivity depend exclusively on protein-DNA interactions occurring within the proximal promoter region, and (3) within the chromatin context, the proximal 100 bp promoter fragment, containing essential elements such as the OC box (-99 to -76) and TATA box (-44 to -31), is insufficient to support formation of the proximal nuclease hypersensitive site and transcriptional activity.

DNaseI treatment is a prerequisite for the amplification of cDNA from episomal-based genes.

DNaseI treatment is a prerequisite for the amplification of cDNA from episomal-based genes.

Homogeneous Enzyme-Based Binding Assay for Studying Glycosaminoglycan Interactions with Macromolecules and Peptides

A simple and rapid homogeneous enzyme-based binding assay is described to study the degree of interaction between glycosaminoglycans and various macromolecules/peptides. The method is based on the homogeneous inhibition of a highly positively charged enzyme, acid deoxyribonuclease II (EC 3.1.22.1), by glycosaminoglycan polyanions, such as heparin, chondroitin 4-sulfate, and dermatan sulfate. Catalytic activity of DNase II is inhibited to nearly 100% by relatively small amounts of these glycosaminoglycan molecules. In the presence of species that bind these polyanions, the activity of the enzyme is regained in an amount proportional to the concentration of the species present. Thus, the relative binding affinities of various species with a given GAG can be assessed rapidly by comparing the concentration of the compound required to reverse the enzyme inhibition to 50% of the maximum value (ED50values). The feasibility of this binding assay principle is demonstrated by measuring the ED50values of five macromolecules: polylysine, polyarginine, protamine, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), using heparins of different size, as well as chondroitin 4-sulfate and dermatan sulfate as the GAG polyanions. The applicability of the assay method is further extended to study GAG–peptide interactions. A variety of small synthetic peptides (8–13 amino acid residues) derived from the heparin-binding domains of protamine and type IV collagen are used as model peptide species. Relative GAG-binding affinities of these macromolecules/peptides are compared to previous literature values, and data obtained via a new electrode-based titration method.

The Acid Deoxyribonuclease of Neutrophils: A Possible Participant in Apoptosis-Associated Genome Destruction

Human neutrophils are terminally differentiated cells that spontaneously undergo apoptosis in tissue culture. Apoptosis in these cells can be delayed by culture in the presence of granulocyte colony-stimulating factor or other inflammatory mediators. Neutrophils were found to contain an acid endonuclease that appeared to be responsible for the internucleosomal DNA cleavage that accompanies apoptosis. As measured by a plasmid nicking assay, this endonuclease had a molecular weight (M(r)) of 35,000, a pH optimum of 5.5, and a threshold for activity of pH 6.6 to 6.8. It was weakly inhibited by divalent cations (Ca2+, Mg2+, and Zn2+) and more strongly inhibited by aurintricarboxylic acid and N-bromosuccinimide. DNA from neutrophils treated with nigericin in buffers of defined pH displayed nucleosomal ladders whose prominence varied with pH in a manner that paralleled the pH dependence of the plasmid cleavage assays, consistent with internucleosomal DNA cleavage by the acid endonuclease. We have previously shown that neutrophils undergo acidification to a pH value as low as 6.0 during apoptosis; we suggest that this endonuclease may be responsible for the DNA cleavage seen in apoptotic neutrophils.

Secretion of Neutral and Acid DNases in Cultivated Human Lymphocytes after Incubation with DNA; Possible Consequences for Inhalation Anesthesia

After incubation with DNA human lymphocytes release neutral and acid DNase activities into the culture medium; the release depends on DNA concentration and time of cultivation. The electrophoretic mobility of the released neutral DNase activity is in accordance with DNase I and the electrophoretic mobility of the released acid DNase activity with DNase II. The released DNase activities do not originate from dead cells and are not influenced by blast cell formation. The anesthetic halothane can inhibit the released neutral and acid DNase activities. Inhalation anesthesia can possibly disturb the correlation between DNA and DNases in human blood.

Activities and androgenic regulation of lysosomal enzymes in the epididymis of rhesus monkey.

The activities and androgenic regulation of seven lysosomal enzymes viz. acid phosphatase, N-acetyl hexosaminidase, alpha-mannosidase, beta-glucuronidase, DNase II, RNase II and phospholipase A was established in caput, corpus and cauda segments of monkey epididymis. Estimation of enzyme activities in the the epididymis of control, castrated and castrated-androgen replaced monkeys revealed that all the enzymes except RNase II showed higher activity in caput and corpus as compared to cauda. The enzymes were reduced markedly after castration and on subsequent androgen replacement there was a significant stimulation of the repressed activities, but the control levels were not restored. RNase II showed highest activity in cauda which was further elevated after castration. The possible role of these enzymes in sperm maturation and disposal is discussed.

Multiple proteins bind the insulin response element in the human IGFBP-1 promoter

An insulin response element (IRE) has been identified ∼100 base pairs (bp) 5′ to the transcription start site of the human insulin-like growth factor binding protein-1 (hIGFBP-1) gene. This cis element appears crucial to the multihormonal regulation of hIGFBP-1 expression in liver, since (i) an intact IRE is required for maximal stimulation of hIGFBP-1 promoter activity by dexamethasone, and (ii) the IRE confers activity. Further progress in understanding how the IRE confers insulin and glucocorticoid effects requires identification of transcription factors confering effects of these hormones. D-site binding protein (DBP), and members of the hepatic nuclear factor 3 (HNF 3) and high mobility group I/Y (HMG I/Y) protein families, each known to bind DNA elements similar in sequence to the IRE, were tested for IRE binding. DBP, HMGI and HNF 3β each protected the hIGFBP-1 IRE from DN AseI digestion. Additional studies are required to establish whether binding of any of these proteins to the IRE is important to the regulation of hIGFBP-1 expression by insulin and/or glucocorticoids.

Lipid peroxidation parameters and activities of lysosomal enzymes in diabetics

The processes of lipid peroxidation and activities of lysosomal enzymes were studied in 56 patients with type I and II diabetes mellitus. The rate of lipid peroxidation of red cell membranes was assessed from the activities of enzymatic (NADPH-dependent) and nonenzymatic (ascorbate-dependent) . lipid peroxidation, from accumulation of acylhydroperoxides, in- termolecular joints, and from spontaneous red cell hemolysis. Activities of lysosomal enzymes (cathepsines, acid DNAse, and P-galactosidase) were measured in leukoconcentrate. The activity of enzymatic system of lipid peroxidation and acylhydroperoxide content in red cell membranes were found increased. In parallel with this, a deficiency in leukocytic lysosomes of P-galactosidase and DNAse was revealed. The detected metabolic disturbances may be regarded as one of the pathogenetic mechanisms of development of diabetic angiopathies. A relationship was revealed between changes in lipid peroxidation parameters and activities of lysosomal enzymes, on the one hand, and diabetes mellitus type and duration, on the other.

Nucleic Acid Sequence and DNase Hypersensitive Sites of the 5′ Region of the Mouse Progesterone Receptor Gene

To allow study of the control of the promoter of the mouse progesterone receptor gene and its specific elements, we have isolated nine individual clones and selectively sequenced the 5′ region, which includes 5′ promoter sequence and the non-coding region. Sequence comparison was performed versus the progesterone receptor genes of other species. Moreover, potential estrogen response elements, progesterone response elements, and other transcription factor recognition sites were determined by sequence analysis. In addition, since it has been confined that chromatin structures are important for regulation of gene expression, we also examined them in different estrogen target organs using the DNase hypersensitivity assay. A different pattern of the DNase hypersensitivity sites was detected between uterus and mediobasal hypothalamus and these sites were close to specific recognition elements.

Multiple Forms of Nuclear Deoxyribonuclease in Rat Thymocytes

Three distinct endonucleases (DNase α, β and γ) were isolated from rat thymocyte nuclei. These three DNases differed in chromatographic behaviors and in apparent molecular masses. The cognate form of acidic DNases α and β did not require divalent cations for their activities, whereas DNase γ was a neutral endonuclease that required both Ca2+ and Mg2+ for full activity and was inhibited by Zn2+. These enzymes digested HeLa S3 cell nuclear chromatin to the nucleosomal fragments characteristic of apoptosis. Interestingly, apoptotic rat thymocyte nuclei induced by γ-ray irradiation or glucocorticoid retained considerable activity of DNase γ, whereas their activities of DNases α and β were markedly reduced. These results indicate that in rat thymocytes DNase γ is involved in DNA fragmentation during apoptosis.

Evidence for the Formation of a New Variant Structure of λ-DNA in vitro

In 1990, Bai et al.first directly observed an unusual triple-stranded braid-likeconformation of denatured λ-DNA Hind Ⅲ in vitro with scanning tunneling microscopy(STM). The triple-stranded braid-like conformation presented in this work was dif-ferent from the model of the right-handed triple helix DNA proposed by otherauthors.In the triple helix model, the third strand winds round the major groove ofthe double helix DNA. Several studies indicated that the triple-stranded DNA, whichwas prepared by the reaction of homopurine and homopyrimidine or single-strandedDNA and doube-stranded DNA, had a low melting temperatue (T_m), a highabsorbance at 280 run in the unwinding process and was resistant to DNase I

Bovine pancreatic deoxyribonuclease F: isoelectric focusing, peptide mapping and primary structure.

DNAase F is a minor isoform of bovine pancreatic DNAase which can be separated from the other isoforms (DNAases A, B, C and D) present in a commercial preparation by a preparative isoelectric-focusing cell (Rotofor; Bio-Rad). The ampholytes and other contaminating proteins present in DNAase F preparations can be removed by chromatography on an affinity column (Cibacron Blue 3GA-agarose) and a hydrophobic-interaction column (phenyl-Sepharose CL-4B). The complete separation of DNAase F from the other isoforms is demonstrated on a thin-layer isoelectric-focusing gel, DNAase F being the most basic (pI 5.68). A procedure is described for tryptic peptide mapping by h.p.l.c. requiring only picomolar amounts of DNAase F protein. The DNAase F map shows two peptide peaks not present in the DNAase A map, and the DNAase F map does not have a peak at the position where a C-terminal peptide of DNAase A is normally eluted. The amino acid compositions and sequences for the two new peptides suggest that Gly240 in DNAase A is changed to Arg240 in DNAase F.