OBJECTIVETo study the effect of DNase I on biofilm formation of Staphylococcus aureus.

METHODSThe growth curve of S. aureus was detected using a spectrophotometer. The adhesion of S. aureus was analyzed using flat colony counting method, and the biofilm formation was assayed using the 96-well crystal violet staining method.

RESULTSExposure to different concentrations of DNase I did not obviously affect the growth of S. aureus but significantly inhibit the formation of bacterial biofilms in a dose-dependent manner. DNase I inhibited the adhesion of S. aureus at different growth stages. When combined with antibiotics, DNase I resulted in a signi?cant decrease in the established bio?lm biomass compared to antibiotics or DNase I used alone.

CONCLUSIONDNase I can effectively inhibit biofilm formation of S. aureus and enhance the inhibitory effect of antibiotics against S. aureus biofilms.

Anti-Bacterial Agents ; Biofilms ; drug effects ; Deoxyribonuclease I ; chemistry ; Staphylococcus aureus ; growth & development

Animals ; Archaeal Proteins ; genetics ; metabolism ; Deoxyribonuclease I ; genetics ; metabolism ; Gene Editing ; methods ; Humans ; Natronobacterium ; enzymology ; genetics

T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5′-GCTCTTCT^GAAGAGC-3′) at each end. Thus, this plasmid can be easily converted into T-vectors by a nickase (quadruple nicking), which results in two double strand breaks with 3′-thymidine overhangs. DsRed indicator gene, which is inserted between the restriction sites, helps identifying the PCR recombinants. Using this pNBQ-T plasmid the insertion efficiency of a PCR product was examined. We successfully identified white colony of the recombinants with the inserted myostatin promoter gene: the cloning efficiency was 93%. Therefore, this simple method utilizing pNBQ-T plasmid will serve as a useful and efficient technique for preparation of home-made T-vectors.
Clone Cells ; Cloning, Organism ; Deoxyribonuclease I* ; Methods ; Myostatin ; Plasmids ; Polymerase Chain Reaction

The rapid involution of the rat ventral prostate after castration is an active process initiated by removal of the inhibitory effects of androgen on prostatic cell death. The degradation of genomic DNA into nucleosomal-sized fragments if an early event in this process and is catalyzed by Ca2+Mg2+-dependent endonuclease activity which is dependent upon calcium ions. The morphologic correlation of the involution process involves a series of structural changes which are collectively referred to as apoptosis. Since the castration-induced endonuclease is dependent upon calcium ions for maximal activity, a potential involvement of a intracellular calcium in the castration-induced prostatic cell death was investigated. Acute disturbance in intracellular calcium homeostasis within the ventral prostate by means of a potent calcium channel blocker, nifedipine, simultaneous with castration resulted in a significant decrease in prostatic apoptosis. This result points to a potential role intracellular calcium levels in the mechanism of activation of castration-induced death of the androgen-dependent epithelial cells in the ventral prostate.
Animals ; Apoptosis* ; Calcium Channels* ; Calcium* ; Castration ; Cell Death ; Deoxyribonuclease I ; DNA ; Epithelial Cells ; Homeostasis ; Ions ; Nifedipine* ; Prostate* ; Rats*

Structural and functional changes in the major apurinic/apyrimidinic DNA endonuclease (APEX) gene in human colorectal cancers were investigated. DNAs were prepared from surgically removed 25 human colorectal tissues and direct sequencing of PCR-amplified APEX gene covering the entire protein coding region was performed. Point mutations in 3 and silent mutations in 3 out of 25 colorectal cancer patients were found. Base substitutions in intron II were also found in 2 patients. T<-> C or some A<-> G transitions were the most typical pattern of the mutations. AP DNA endonuclease (APE) activities in normal and tumor tissues were 65.7 EU/mg and 21.7 EU/mg, respectively. APEX protein was detected in both normal and tumor tissues and no remarkable difference in the amount of APEX protein between colorectal cancer tissues and their normal counterparts was observed. The incidence of APEX gene mutation in colorectal cancer was 12% which is relatively lower than that of other genes associated with colorectal tumor, but a significant reduction of APE enzyme activities in tumor tissues, especially in those with APEX mutations, was observed. These results indicate that the decreased APE enzyme activity might be closely related to the colorectal tumorigenesis, although no quantitative correlation between APE enzyme activity and APEX content exists.
Carcinogenesis ; Colorectal Neoplasms* ; Deoxyribonuclease I ; DNA Repair* ; DNA* ; Hominidae ; Humans* ; Incidence ; Introns ; Open Reading Frames ; Point Mutation

Biologic resurfacing of the damaged joints is an area of great interest and clinical promise because of the limited potential ofdamaged articular cartilage healing. Several methods such as spongiolization. joint dehridement and ahrasion of suhchondral hone. perichondral grafts, and osteochondral grafts have heen used to repair cartilage defects, but the results were not satisfactory. Rccently autologous chondrocyle transplantation with a pcrioslcal patch was paid an altention for its advantage , the regeneration with hyalin cartilage. But it have many disadvantages such as too expensive cost. second staged operation, and technically difficult to isolatc chondrocytes from a small volume of donor site, so we performed that a definecl cartilaee delect in the ribbit patella was treated with transplanta1ion of in virto expanded allogenic chondrocvtes and then compared with an autologous chondrocytes transplantation. Adult rabbits were used to transplant autogenously and allogenously and allogenically harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously 3x 3mmin size , extending down to the calcified zone. Chondrocytes were isolated in the femoral condyle of the opposite knee or othe rabbit knee. And then enzymatic digestion ( collagenase A and DNase I ) was performed for 5 hours room temperature in a spinner bottle and cells were seeded in a 25cm2 culture flask in Dulheccos modified essential medium (DMEM), supplemented with l0% fetal hovine serum (FBS). The culture medium was changed twice weekly. After 14 days of culture, the cells were isolated hy irypsinization and transplanted into previously made chondral defects with an autogenous periosteal patch taken from the medial aspect of tibia. Healing ol' the defects was assessed by gross examination, immunohistochemical stain, and light microscope with hematoxylin-eosin stain at 8, 16, and 24 weeks. Allogenic and autologous chondrocytes transplantation significantly increased the amount of newly tormed repair tissue compared to that found in control knees in which the Jesion was solely covered hy a periosteal patch. The repair tissue, however, had a tendency of incomplete bonding to adjacent cartilage. This study shows that allogenic and autologous articular chondrocytes that have heen expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesion. There is no signilicant diffcrence hetween allogenic and autologous chondrocytes transplantation.
Adult ; Cartilage ; Cartilage, Articular ; Chondrocytes* ; Collagenases ; Deoxyribonuclease I ; Digestion ; Humans ; Hyalin ; Joints ; Knee ; Patella* ; Rabbits ; Regeneration ; Tibia ; Tissue Donors ; Transplants

BACKGROUND: Recently, we have reported that biodegradable poly [-(4-aminobutyl)-L-glycolic acid] (PAGA) can condense and protect plasmid DNA from DNase I. In this study, we investigated whether the systemic administration of pCAGGS mouse IL-10 (mIL-10) expression plasmid complexed with PAGA can reduce the development of insulitis in non-obese diabetic (NOD) mice. METHODS: PAGA/mIL-10 plasmid complexes were stable for more than 60 minutes, but the naked DNA was destroyed within 10 minutes by DNase I. The PAGA/DNA complexes were injected into the tail vein of 3 week-old NOD mice. RESULTS: Serum mIL-10 level peaked at 5 days after injection, could be detected for more than 7 weeks. The prevalence of severe insulitis at 12 week-old NOD mice was markedly reduced by the intravenous injection of PAGA/DNA complex (15.7%) compared to that of naked DNA injection (34.5%) and non-treated controls (90.9%). In conclusion, systemic administration of pCAGGS mIL-10 plasmid/PAGA complexes can reduce the severity of insulitis in NOD mice. CONCLUSION: The study presents the PAGA/DNA complex has the potential for the application of the prevention of autoimmune diabetes mellitus.
Animals ; Deoxyribonuclease I ; Diabetes Mellitus, Type 1 ; DNA* ; Genetic Therapy ; Injections, Intravenous ; Insulin-Secreting Cells* ; Interleukin-10* ; Mice ; Mice, Inbred NOD* ; Plasmids* ; Prevalence ; Veins

PURPOSE: Aggregatibacter actinomycetemcomitans is associated with localized aggressive periodontitis. It produces cytolethal distending toxin (CDT), which induces cell cycle arrest in the G2/M phase. The CDT holotoxin is composed of CdtA, CdtB, and CdtC. CdtB has structural homology to human DNase I and is an active component of the CDT complex acting as a DNase. In particular, the pattern homology seen in the CdtB subunit has been associated with specific DNase I residues involved in enzyme catalysis, DNA binding, and metal ion binding. So, to study the functions and regulation of recombinant CdtB, we made up a quantity of functional recombinant CdtB and tested it in relation to the metal ion effect. MATERIALS AND METHODS: We constructed the pET28a-cdtB plasmid from A. actinomycetemcomitans Y4 by genomic DNA PCR and expressed it in the BL21 (DE3) Escherichia coli system. We obtained the functional recombinant CdtB by the refolding system using the dialysis method and then analyzed the DNase activity and investigated the metal ion effect from plasmid digestion. RESULTS: The recombinant CdtB subunit was expressed as the inclusion bodies. We were able to obtain functional recombinant CdtB subunit using refolding system. We confirmed that our refolded recombinant CdtB had DNase activity and was influenced by the metal ions Mg2+ and Ca2+. CONCLUSION: We suggest that the factors influencing recombinant CdtB may contribute to CDT associated diseases, such as periodontitis, endocarditic, meningitis, and osteomyelitis.
Aggressive Periodontitis ; Bacterial Toxins ; Catalysis ; Cell Cycle Checkpoints ; Deoxyribonuclease I ; Deoxyribonucleases ; Dialysis ; DNA ; Edetic Acid ; Escherichia coli ; Humans ; Inclusion Bodies ; Ions ; Meningitis ; Osteomyelitis ; Periodontitis ; Plasmids ; Polymerase Chain Reaction

Gene therapy is a potential therapeutic strategy for treating hereditary movement disorders, including hereditary ataxia, dystonia, Huntington's disease, and Parkinson's disease. Genome editing is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome using modified nucleases. Recently, clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 (CRISPR/Cas9) has been used as an essential tool in biotechnology. Cas9 is an RNA-guided DNA endonuclease enzyme that was originally associated with the adaptive immune system of Streptococcus pyogenes and is now being utilized as a genome editing tool to induce double strand breaks in DNA. CRISPR/Cas9 has advantages in terms of clinical applicability over other genome editing technologies such as zinc-finger nucleases and transcription activator-like effector nucleases because of easy in vivo delivery. Here, we review and discuss the applicability of CRISPR/Cas9 to preclinical studies or gene therapy in hereditary movement disorders.
Biotechnology ; Deoxyribonuclease I ; DNA ; Dystonia ; Genetic Engineering ; Genetic Therapy ; Genome ; Huntington Disease ; Immune System ; Movement Disorders* ; Parkinson Disease ; Spinocerebellar Degenerations ; Streptococcus pyogenes

OBJECTIVENephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage.

METHODSSixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria.

RESULTSThe result indicated that mean levels of sera NSMCE2 have a significantly increase (P<0.01) in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase I and II were significantly elevated in nephrolithiasis patients (P$lt;0.01).

CONCLUSIONThis study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.

Adult ; Blood Proteins ; analysis ; Case-Control Studies ; Creatinine ; blood ; Deoxyribonuclease I ; blood ; Endodeoxyribonucleases ; blood ; Hemoglobins ; analysis ; Humans ; Ligases ; blood ; Malaysia ; Middle Aged ; Nephrolithiasis ; blood ; enzymology ; Urea ; blood