• Biomolecules & Therapeutics
• /
• v.28 no.6
• /
• pp.549-560
• /
• 2020

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5'-serially deleted eNOS promoters revealed that Tax-responsive element site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser¹³³ level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser¹⁹⁸¹ and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser¹⁹⁸¹, p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

• Proceedings of the Zoological Society Korea Conference
• /
• 2000.04a
• /
• pp.78.1-78
• /
• 2000

No Abstract, See Full Text

• Environmental Mutagens and Carcinogens
• /
• v.16 no.2
• /
• pp.83-87
• /
• 1996

This study was performed by the sister chromatid exchanges (SCEs) to investigate the effects of Aphidicolin (APC) or 2, 4-dinitrophenoi (DNP) on cross-adaptive response to ultraviolet radiation (UV) or ethyl methanesulfonate (EMS) in Chinese hamster ovary (CHO) cells. The pretreatment with 1 J/m$^2$ UV decreased the yield of SCEs induced by subsequent treatment with 8 mM EMS in CHO cells. And the treatment with 10 $\mu$g/ml APC or 50 $\mu$M DNP during incubation after pretreatment with 1 J/m$^2$ UV increased the yield of SCEs induced by 8 mM EMS. The pretreatment with 2 mM EMS decreased the yield of SCEs induced by subsequent treatment with 5 J/m$^2$ UV. The treatment with 10 $\mu$g/ml APC during incubation after 2 mM EMS increased the yield of SCEs induced by 5 J/m$^2$ UV. These results suggest that APC and DNP inhibit cross-adaptive response to pretreatment with UV and subsequent treatment with EMS, and also cross-adaptive response to pretreatment with EMS and subsequent treatment with UV is inhibited by APC in CHO cells.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.20 no.2
• /
• pp.136-145
• /
• 1987

From the sea-urchin, Hemicentrotus pulcherrismus, we have purified by four column chromatographic steps for DNA polymerase $\alpha$ activity. The molecular weight of DNA polymerase u was determined to be around 137,000-138,000 by Sephadex G-200 gel filtration and SDS-polyacrylamide gel electrophoresis. The purified enzyme had the optimal activity at pH 7.4. This enzyme showed to be a function of the metal ion $K^+,\;Na^+$\;and\;Mg^{2+}$ employed as activators, the optimum $K^+$\;or\;Na^+ concentration were 20 mM or 25mM and the optimum $Mg^{2+}$ concentration was 10 mM. The enzyme activity was inhibited by N-ethyl-maleimide, aphidicolin, cytosine $\beta-D-arabinofuranoside$ 5'-triphoshate (ara CTP) and phosphonoacetic acid.

• Korean Journal of Animal Reproduction
• /
• v.22 no.2
• /
• pp.153-161
• /
• 1998

This study was conducted to examine the viability of nuclear transfer bovine embryos following embryo transfer. Donor embryos were treated with nocodazole to arrest their cell-cycle-stage at mitotic(M) phase. After releasing from nocodazole blastomeres were separated and transferred into the enucleated oocytes(BC), or cultured in medium with aphidicolin. Freshly cleaved blastomeres within 1.5h after cleavage(AC) and non-cleaved ones up to 3h after releasing from nocodazole(NC) were transferred into the enucleated oocytes. Blastocysts derived from nuclear transfer were transferred to Day 7~8 recipient cows. Some blastocysts were vitrified and thawed before embryo transfer. Developmental rates to the blastocyst stage were higher in AC(18.1%, P<0.05) than BC(8.6%) and NC(5.1%). Blastocyst development slightly enhanced with aphidicolin(1~2$\mu\textrm{g}$/ml) treatment(16.9~22.6%) compared to non treated control(11.1%). Survival rate fo vitrified nuclear transfer embryos after thawing was 75%(24/32). Twnety-three vitrified nuclear transfer embryos and 3 fresh ones were transferred to 23 recipients, 6 heads were pregnant and 1 male calf(24 kg) was born from a recipient cow recevied one vitrifiedthawed nuclear transfer embryo at 277 days after embryo transfer. This result suggests that the nuclear transfer embryos can developed to term after vitrification andembryo transfer.

• Korean Journal of Animal Reproduction
• /
• v.20 no.2
• /
• pp.143-153
• /
• 1996

To improve the efficiency of nuclear transplantation in the rabbit, this study were evaluated the influence of celly cycle of donor nuclei on the in vitro developmental potential in the nuclear transplant embryos. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G1 phase of 32-cell stage. Synchronization of the cell cylce of blastomeres were induced, first, using an microtubules polymerization inhibitor, 0.5$\mu\textrm{g}$/ml colcemid for 10h to arrest blastomeres in metaphase, and secondly, using a DNA synthesis inhibitor, 0.1$\mu\textrm{g}$/ml aphidicolin for 1.5 to 2h to cleave to 32-cell stage and arrest them in G1 phase. The separated G1 phase blastomeres of 32-cell stage were injectied into enucleated recipient cytoplasms by micromanipulation. After culture until 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The nuclear transplant embryos were co-cultured for 120h. In vitro cultured embryos were monitored every 24h to assess for development rate. After in vitro cultue for 120h, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye for counting the number of blastomeres under a fluorescence microscopy. The cleavage rate of blastomeres from 16-cell stage stage rabbit embryos treated with colcemid for 10h or aphidicolin for 6h following colcemid for 10h were not significantly different. The electrofusion rate was similar by high in S and G1 phase donor nuclei as 80.6 and 79.1%, respectively. However, the nuclear transplant embryos using G1 phase donor nuclei were developed to blastocyst at high rate(60.3%) than those using S phase donor nuclei(26.0%). Moreover, the mean blastocyst stage were increased significantly(P<0.05) with the G1 phase donor nuclei(176.6 cells and 1.50 cycles), as compared with the S phase donor nuclei(136.6 cells and 1.42 cycles). These results show that the blastomeres of G1 phase were more successful as donor nuclei in the nuclear transplant procedure, compared with S phase.

• Parasites, Hosts and Diseases
• /
• v.57 no.2
• /
• pp.185-189
• /
• 2019

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.

• Environmental Mutagens and Carcinogens
• /
• v.8 no.1
• /
• pp.1-12
• /
• 1988

The effects of aphidicolin (APC), an inhibitor of DNA polymerase alpha, or 2', 3'-dideoxythymidine 5'-triphosphate (ddTTP), an inhibitor of DNA polymerase beta, on the repair of DNA damage induced by ethyl methanesulfonate (EMS) or bleomycin (BLM) were investigated in Chinese hamster ovary (CHO)-K1 cells. Three assays were employed in this study: unscheduled DNA synthesis, alkaline elution and alkaline sucrose gradient sedimentation. It was shown that APC or ddTTP inhibited DNA induced by EMS, and thus, the post-treatment with APC or ddTTP following EMS treatment was resulted in the more amount of unscheduled DNA synthesis, and the more accumulation of DNA single-stand breaks than the cells post-incubated without APC or ddTTP. While, in the BLM induced DNA repair, only ddTTP inhibited DNA repair induced by BLM. And thus, the groups post-incubated with or without APC after BLM treatment had the same value in the amount of unscheduled DNA synthesis and of DNA single-strand breaks, while post-treatment with ddTTP was resulted in the increased amount of unscheduled DNA synthesis and the increased DNA sin -strand breaks than the group without ddTTP. These results suggested that both of DNA polymerase $\alpha$ and $\beta$ participated in the repair of DNA damage induced by EMS, but in BLM-induced DNA repair, polymerase $\beta$ participated.ipated.

• Environmental Mutagens and Carcinogens
• /
• v.10 no.2
• /
• pp.85-92
• /
• 1990

The effects of aphidicolin (APC), 2`,3`-dideoxythymidine 5`-triphosphate (ddTTP), and novobiocin (NOV) on the frequencies of chromosome aberrations induced by ethyl methanesulfonate (EMS) or bleomycin (BLM) were examined in synchronized Chinese hamster ovary (CHO)-K$_1$ cells. The cells were synchronized by the thymidine double block method. APC, ddTTP and NOV alone did not affect the frequencies of chromosome aberrations. The cells in late G$_1$ and early S phases were sensitive to the induction of chromosome aberrations by EMS, wherase cells in G$_2$ phase were most sensitive to chromosme aberration by BLM.

• Environmental Mutagens and Carcinogens
• /
• v.9 no.1
• /
• pp.23-32
• /
• 1989

Chinese hamster ovary (CHO)-K1 cells echibited a differential sensitivity in the process of DNA repair synthesis induced by ethyl methanesulfonate (EMS) or bleomycin (BLM) in relation to cell cycle. Two assays were employed in this study: alkaline elution and unscheduled DNA synthesis. The post-treat-ment with aphidicolin (APC), an inhibitor of DNA polymerase alpha, inhibited DNA repair synthesis induced by EMS in G2 phase, while APC did not show any effect on BLM-induced DNA repair synthesis in all phases. On the other hands, the 2', 3'-dideoxythymidine (ddTTP), an inhibitor of DNA polymerase beta, inhibited DNA repair synthesis induced by EMS or BLM in both of G1 and G2 phases. These results suggested that the involvement of DNA polymerase alpha and beta in DNA repair was dependent on cell stage or used chemical agent.