• Journal of Plant Biology
• /
• v.29 no.4
• /
• pp.285-294
• /
• 1986

The present study was attempted to investigate the effects of polyamines such as putrescine, spermidine and spermine on protein content and DNase activity in vivo and in vitro in carrot embryos. It was also investigated whether polyamines could replace role of cations required for DNase activity in vitro. The results obtained are as follows. Putrescine, spermidine and spermine increased protein content, although response to spermine reached plateau at the concentration of 0.1 mM. DNase activity was inhibited by polyamines, the inhibition being concentration-dependent and the highest att he concentration of 10 mM. The inhibition of DNase activity was the most prominent with spermine. Similar inhibitory effect to polyamines which was concentration-dependent was found in DNase activity but no change was shown on time-course in vitro. Putrescine and spermidine enhanced the DNase activity at low Mg2+ and Mn2+ concentrations, suggesting that the role of Mg2+ and Mn2+ for DNase activity could be, in part, replaced by these polyamines. These results, therefore, suggest that plyamines can modulate DNase activity through binding to DNA rather than direct effect on DNase activity.

• Journal of Veterinary Clinics
• /
• v.21 no.3
• /
• pp.248-252
• /
• 2004

DNase activity from infective larvae of the parasitic nematode Haemonchus contortus was characterized and compared to that from whole worm. DNase activity from infective larvae was detected throughout pHs 4-10, but high activity was detected under acidic conditions. The activity was not inhibited by 10 mM EDTA at pH 5.0, but was significantly inhibited at pH 7.0. The activity produced DNA, fragments with mixtures of 3'-hydroxyls (OH) and 3'-phosphates (P) at each pH with predominance of 3'-P. A unique DNase activity at 37 kDa was identified from infective larvae on zymograms. The 37 kDa DNase was detected only at pH 5.0, but not at pH 7.0, and this activity was not inhibited by EDTA at pH 5.0. These characteristics of the 37 kDa infective larval DNase resemble those of classic acidic DNases (e.g., DNase II). In contrast, 34, 36 and 38.5 kDa DNase activities were shown to be specific for whole worm. This result demonstrated that DNases in H contortus are regulated during development.

• Molecules and Cells
• /
• v.19 no.1
• /
• pp.54-59
• /
• 2005

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity $10^8$ and $10^9$ for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction ($IC_{50}=0.1-0.7{\mu}M$) and DNA degradation by DNase I ($IC_{50}=0.8-8{\mu}M$). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

• Microbiology and Biotechnology Letters
• /
• v.40 no.4
• /
• pp.348-355
• /
• 2012

In the present study, we investigated the overexpression and characterization of bovine pancreatic (bp)- DNase I in Saccharomyces cerevisiae and Pichia pastoris. The bp-DNase I gene was fused in frame with the GAL10 promoter, $MF{\alpha}$, and GAL7 terminator sequences, resulting in the plasmid, pGAL-$MF{\alpha}$-DNaseI (6.4 kb). Also, the bp-DNase I gene was fused in frame with the AOX1 promoter, $MF{\alpha}$, and AOX1 terminator sequences, resulting in the plasmid, pPEXI (8.8 kb). The recombinant plasmids, pGAL-$MF{\alpha}$-DNaseI and pPEXI were introduced into S. cerevisiae and P. pastoris host cells, respectively. When the transformed yeast cells were cultured at $30^{\circ}C$ for 48 h in galactose or methanol medium, bp-DNase I was overexpressed and the most of activity was found in the extracellular fraction. P. pastoris transformant activity showed 45.5 unit/mL in the culture medium at 48 h cultivation, whereas S. cerevisiae transformant revealed 37.7 unit/mL in the extracellular fraction at 48 h cultivation. The enzymatic characteristics, such as DNA cleavage and half life were investigated. Treatment of the recombinant DNase I from P. pastoris induced degradation of the calf thymus DNA within 1 minute, and this DNA degradation rate was higher than that of commercial bp-DNase I (SIGMA) and the recombinant DNase I from S. cerevisiae.

• Bulletin of the Korean Chemical Society
• /
• v.35 no.9
• /
• pp.2749-2752
• /
• 2014

Recent studies have shown that single-stranded DNA adsorbed onto graphene oxide is protected from DNase I cleavage. However, double-stranded DNA bound to graphene oxide and could be digested by DNase I. To elucidate whether single-stranded DNA is protect from DNase I in the presence of graphene oxide, this study conducted DNase I digestion using single-stranded DNA and single-stranded DNA containing the duplex region in the presence of graphene oxide. Addition of DNase I resulted in restoration of the fluorescence emission that had been quenched when DNA was adsorbed to graphene oxide. It indicates that DNase I cleaved the adsorbed single-stranded DNA onto graphene oxide, which was sufficient for the detection of DNase I activity.

• Korean Journal of Veterinary Research
• /
• v.44 no.3
• /
• pp.441-448
• /
• 2004

DNase activity in Haemonchus contortus reproductive tissue was characterized and compared to that in whole worm. DNase activity in reproductive tissue was detected throughout pHs 4-10 with high activity under acidic conditions. The activity was not inhibited by 10 mM EDTA at pH 5.0, but largely inhibited by pH 7.0. The activity produced DNA fragments with mixtures of 3'-hydroxyls (OH) and 3'- phosphates (P) at each pH. Three distinct DNase activities were identified and had $M_rs$ of 34, 36 and 38.5 kDa in zymograms, which were distinguished according to pH requirement and sensitivity to EDTA. Among them, the 36 kDa reproductive tissue DNase had predominant activity at pH 5.0, but very weak at pH 7.0, and this activity was not inhibited by EDTA at pH 5.0. These characteristics of the 36 kDa reproductive tissue DNase resemble those of classic acidic DNases. In contrast, 36 kDa whole worm DNase activity had high activity at both pH 5.0 and 7.0. While the 36 kDa DNase activity at pH 5.0 was similar in both reproductive tissue and whole worm samples, the activity at pH 7.0 was predominantly detected in whole worm sample. This suggests that the 36 kDa whole worm DNase at pH 5.0 differs from that at pH 7.0. Thus, results indicate that the EDTA-insensitive 36 kDa DNase at pH 5.0 is specific for H. contortus reproductive tissue.

• Journal of the Korean Chemical Society
• /
• v.29 no.3
• /
• pp.304-310
• /
• 1985

Effect of spermine on the susceptibility of calf thymus nucleohistone to DNase 1, in relation to the structural change of the nucleohistone, and cooperativity of the interaction of the nucleohistone with DNase 1 was investigated. Dansylated nucleohistone, in which the histone moiety had been derivatized by dansylation, was also used to investigate functional roles of the histone moiety on the cooperativity. The data here indicate the possibility that the nucleohistone, in contrast with the DNA, may not undergo monomolecular condensation, whereas intermolecular aggregation and enhancement of the positive cooperativity of the interaction of nucleohistone with DNase 1 may be brought about by spermine. The interaction of the DNS-nucleohistone with DNase 1 showed negative cooperativity. Based on the data here, it can be speculated that the cooperativity of the nucleohistone is influenced by the histone moiety of the nucleohistone.

• Korean Journal of Veterinary Research
• /
• v.44 no.3
• /
• pp.455-462
• /
• 2004

Change in ${\beta}$-tubulin nucleic acid and protein sequences was the only known difference between Haemonchus contortus fenbendazole (FBZ)-resistant and -susceptible isolates. This change was sufficient to determine the pathologic effect induced by FBZ treatment. This research was initiated to investigate further differences from these two isolates. Since ${\beta}$-tubulin is involved in formation of microtubule, which has functions in secretory vesicle transport, DNase activities from excretory/secretory products (ESP) of the two isolates were compared, based on pH, sensitivity to DNase inhibitors, molecular masses and production of 3'-OH. The most significant difference detected was that a 38.5 kDa DNase activity was identified from ESP of H. contortus FBZ-susceptible isolates but not from those of H. contortus FBZ-resistant isolates. However, it was shown that the 38.5 kDa DNase is expressed with similar level of activity in intestine and whole worm of H. contortus FBZ-resistant and -susceptible isolates. This result demonstrated that the secretory transport pathway of the 38.5 kDa DNase was inhibited by unknown mechanisms, which may be related with ${\beta}$-tubulin sequence change in FBZ-resistant isolates. Other DNases of 34, 36 and 37 kDa were detected from ESP of both H. contortus FBZ-resistant and -susceptible isolates. Overall DNase activities found from ESP of these two isolates were not inhibited by 10 mM EDTA at pH 5.0, but largely inhibited by pH 7.0. In addition, DNase activities in two isolates produced DNA fragments with mixtures of 3'- hydroxyls (OH) and 3'-phosphates (P) at each pH although the 3'-end labeling ratios at pH 5.0 and 7.0 were shown different. Identification of inhibition of the 38.5 kDa DNase secretion in FBZ-resistant isolates suggests existence of further differences, in addition to ${\beta}$-tubulin sequence change, in two isolates. This shows complex effect of FBZ on H. contortus biological mechanisms.

• Journal of Microbiology
• /
• v.33 no.3
• /
• pp.222-227
• /
• 1995

We have examined DNase I sensitivity of .betha.-tubulin and flagellar calmodulin genes which are transiently and coordinately activated differentiation of Naegleria gruberi amebae into flagellates. The DNase I sensitivity of .betha.-tubulin and flagellar calmodulin genes changed in parallel with the changes in transcriptional activity of the respective genes during differentiation. The two genes were resistant to DNase I inamebae stage when transcription of the two genes was inactive. Forthy minutes after initiation of differentiation, when the two genes were most actively being transcribed, the two genes showed the highest sensitsivity to DNase I. One hundred and twenty minutes after initiation, the differentiation was completed and transcriptional activity of the two genes decreased to a low level. At this stage, the two genes were resistant to DNase I treatment like the ones at the amebae stage. This change in the DNase I sensitivity of the two genes was not observed when transcription of the two genes was blocked by adding cycloheximide at the beginning of differentiation.

• The Journal of the Korean bone and joint tumor society
• /
• v.7 no.2
• /
• pp.41-50
• /
• 2001

Purpose : To investigate biochemical markers for osteosarcoma, activities of deoxyribocuclease(DNase), ribonuclease(RNase), 5'-nucleotidase, alkaline phosphatase and amylase were determined in the osteosarcoma tissue and serum of patients with osteosarcoma. Also studied were DNase, RNase in osteosarcoma tissue, isolating the enzymes from the sarcoma tissue and investigating the sarcoma specific enzymes. Materials and Methods : The experimental tissue and serum were obtained from twelve patients with osteosarcoma. The control group were obtained from the normal healthy tissue of the same patients. The tissue were centrifugalized to obtain extracts. The extracts were analized for the estimation of nucleic acid, protein contents and enzyme activities. And then each enzymes were isolated and analized by DEAE-cellulose chromatography and estimated for activities. Result : Activities of acid DNase, RNase, 5'-nucleotidase and alkaline phosphatase were significantly increased in osteosarcoma tissue. Neutral RNase in osteosarcoma tissue was shown to bo highly active, exhibiting secretory form of RNase inhibitor associated with the RNase was also increased. In the serum of patients with osteosarcoma, RNase activity was significantly increased. DEAE-cellulose column chromatographical analysis revealed that acid DNase was isolated as a single enzyme and neutral RNase as five isozymes in osteosarcoma tissue. Conclusion : The results indicated that combination of these enzymes could be used as markers for osteosarcoma. The results indicated that acid DNase and neutral RNase might play a role in genesis of sarcoma and suppression of sarcoma.