• 동의생리병리학회지
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• 제18권3호
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• pp.774-782
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• 2004

The water extract of Dancheonhwan (DCH) has been used to treat ischemic brain and heart damage in oriental medicine. However, little is known about the mechanism by which the water extract of DCH rescues cells from ischemic damage. Therefore, this study was designed to investigate the protective mechanisms of DCH on the H₂O₂-induced toxicity in H9c2 cardiomyoblast cells. Treatment of H₂O₂ markedly decreased the viability of H9c2 cardiomyoblast in a dose-dependent and time-dependent manner. The nature of H₂O₂-induced toxicity of H9c2 cells resulted from apoptotic death confirmed with genomic DNA fragmentation. DCH increased the viability of H₂O₂-treated H9c2 cells by about 23%, and partially suppressed the genomic DNA fragmentation and PARP cleavage. H₂O₂ also activated caspase-3 protease and -9 protease, but not both caspase-6 protease and -8 protease. H₂O₂ induced the mitochondria dysfunction, including mitochondria membrane permeability transition (MPT) and cytosolic release of cytochrome c from mitochondria, which was prevented in part by pretreatment of DCH. N-acetylcystein (NAC), a free-radical scavenger, alone increased the viability of H₂O₂-treated H9c2 cells in a dose-dependent manner. Furthermore, the combination of NAC with DCH significantly increased the viability of the H₂O₂-treated H9c2 cells in a dose-dependent manner. These data indicate that DCH has the protective effect on ROS-induced apoptosis of cadiomyoblast H9c2 cells.

• Bulletin of the Korean Chemical Society
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• 제11권1호
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• pp.49-54
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• 1990

The gas-phase thermolysis reactions of ${\alpha}$- and ${\beta}$-methylated ethyl formates, Y = $CH-X-CHR_1CH_2R_2$ where X = Y = O or S and $R_1\;=\;R_2$ = H or $CH_3$, are investigated theoretically using the AM1 method. The experimental reactivity order is reproduced correctly by AM1 in all cases. The thermolysis proceeds through a six-membered cyclic transition state conforming to a retro-ene reaction, which can be conveniently interpreted using the frontier orbital theory of three-species interactions. The methyl group substituted at $C_{\alpha}\;or\;C_{\beta}$ is shown to elevate the ${\pi}$-HOMO of the donor fragment (Y = C) and depress the ${\sigma}^{\ast}$-LUMO of the acceptor fragment ($C_{\beta}$-H), increasing the nucleophilicity of Y toward ${\beta}$-hydrogen which in turn increases the reactivity. The two bond breaking processes of the $C_{\alpha}$-X and $C_{\beta}$-H bonds are concerted but not synchronous so that the reaction takes place in two stages as Taylor suggested. The initial cleavage of $C_{\alpha}$-X is of little importance but the subsequent scission of $C_{\beta}$-H occurs in a rate determining stage.

• 대한한방소아과학회지
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• 제20권1호
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• pp.257-272
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• 2006

Objective : In this study, the ability of Oriental medicine Samultanggamibang(SMTG) to induce apoptosis was investigated in B16F10 melanoma cells. Method : Tetrazolium-based colorimetric assay was performed for cytotoxicity test. Several new assays for the basis of biochemical events associated with apoptosis such as DNA fragmentation by a flow cytometry, caspase-3 activation and PARP cleavage by Western blotting should be carried out potentially useful for the basis of biochemical events associated with apoptosis such as a flow cytometry and caspase-3 activation. Results : (1) The number of B16F10 melanoma cells was less than 30 % after exposure to 1 mg/ml SMTG for 48 h. SMTG increased cytotoxicity of B16F10 melanoma cells in a dose- and time-dependent manner. (2) The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 21 % at 24 h and 25 % at 48 h after treatment with 1 mg/ml SMTG. (3) SMTG-induced apoptosis was accompained by the activation of caspase-3 and the specific proteolytic cleavage of poly-ADP-ribose polymerase. (4) SMTG induces the activation of caspase-3 and the specific proteolytic cleavage of poly-ADP-ribose polymearse and eventually leads to apoptosis through c-Jun NH2-terminal protein kinase (JNK)-dependent manner in B16F10 melanoma cells. Conclusion : SMTG had a strong cytotoxic effect of B16F10 melanoma cells.

• Preventive Nutrition and Food Science
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• 제5권4호
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• pp.234-238
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• 2000

The cleavage products formed by autoxidation of phytofluene were evaluated in order to elucidate possible oxidation products of phytofluene under oxidative conditions. Phytofluene solubilized at 50$\mu$M in liposomal suspension was oxidized by incubating at 37$^{\circ}C$ for 72 h. Among a number of oxidation products formed, five products in the carbonyl compound fraction were identified as 6, 10, 14-trimethylpentadeca-3,5,9,13-tetraen-2-one, phytapentaenal, 5,9,13,17-tetramethyloctadeca-2,4,6,8,12,16-hexaenal, 5,9,13,17-tetramethyloctadeca-2,4,8,12,16-pentaenal, 2,7,11,15,19-pentamethylicosa-2,4,6,10,14,18-hexaenal and 4,9,13,17,21-pentamethyldocosa-2,4,6,8,12,16,20-heptaenal. These correspond to a series of products formed by cleavage in the respective eight conjugated double bonds of phytofluene. Also, 4,5-didehydorgeranyl geranoic acid was formed by autoxidation of phytofluene in liposomal suspension. The pig liver homogenate had the ability to convert phytapentaenal to 4,5-didehydrogeranyl geranoic acid, comparable to the conversion of all-trans-retinal to all-trans-retinoic acid. These results suggest that phytofluene is cleaved to a series of long-chain and short-chain carbonyl compounds under the oxidative condition in vitro and that phytapentaenal is further enzymatically converted to 4,5-didehydrogeranyl geranoic acid.

• Archives of Pharmacal Research
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• 제19권3호
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• pp.191-196
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• 1996

Bizelesin is a promising novel anticancer agent which is known to alkylate N3 of adenine to induce DNA interstrand cross-links (ISC) with in $5^I-TAATTA\; and\; 5^I-TAAAAAA$. We have investigated the base specificity for DNA ISC induced by bizelesin using oligomers containing the cross-linkable sequence $5^I-TAATTA\; and\; 5^I-TAAAAAA$. in which "N" was either A, C, G, or T. An analysis of denaturing polyacrylamide gel showed that bizelesin is able to induce DNA ISC in the duplex oligomer containing sequences $5^I-TAATTA\; and\; 5^I-TAAAAAA$. The formation of interstrand crosslinking did not occur in the sequences $5^I-TAATTA\; and\; 5^I-TAAAAAA$. DNA strand cleavage assay to determine the cross-linking site within $5^I-TAATTA$sequence showed that bizelesin alkylates guanine. These results demonstrate that bizelesin is able to induce DNA ISC at guanine but not at cytosine or thymine. In addition, guanine adducts have been found to be susceptible to DNA strand cleavage by exposure to hot piperidine. The extent of DNA strand cleavage, however, was not 100% efficient in either neutral pH buffer or hot piperidine.

• 한국수정란이식학회지
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• 제13권1호
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• BMB Reports
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• 제39권2호
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• pp.140-144
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• 2006

A deoxyriboendonuclease has been purified to near homogeneity from a fast growing mycobacterium species, M. smegmatis and characterized to some extent. The size of enzyme is about 43 kDa as determined by a denaturing gel analysis. It shows optimum activity at $32^{\circ}C$ in Tris-HCl buffer (pH 7.2) containing 2.5 mM of $MgCl_2$. Both EDTA and $K^+$ but not $Na^+$ inhibit its activity. Evidences show that the enzyme is not a restriction endonuclease but catalyzes the endonucleolytic cleavage of both the double- as well as the single-strand DNA non-specifically. It has been shown that the cleavage by this enzyme generates DNA fragments carrying phosphate groups at 5' ends and hydroxyl group at the 3' ends, respectively. Analysis reveals that no endonuclease having size and property identical to our deoxyriboendonuclease had been purified from M. smegmatis before. The property of our enzymes closely matches with the deoxyriboendonucleases purified from diverse sources including bacteria.

• 한국미생물·생명공학회지
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• 제25권5호
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• pp.459-463
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• 1997

A bacterium DGUM 2011 has been selected from various samples of industrial wastewater and soil. Based on the morphological and physiological characteristics, the isolate DGUM 2011 was identified as Rhodococcus sp. and named as Rhodococcus sp. DGUM 2011. The optimal temperature and pH for the cell growth of Rhodococcus sp. DGUM 2011 were 37$\circ$C and 7.6, respectively. When phenol was added to the minimal media as a sole source of carbon and energy, the concentrations of maximum and optimum for cell growth was 0.10% and 0.08%, respectively. When 0.05% phenol was given in the minimal media, Rhodococcus sp. DGUM 2011 completely utilize it within 24 hrs. The isolate could utilize benzoic acid, p-hydroxybenzoate, p-cresol, tyrosine and phloroglucinol. The isolate possessed both catechol 1,2-dioxygenase and 2,3-dioxygenase activity. However, the activity of catechol 1,2-dioxygenase was much higher than that of 2,3-dioxygenase, which suggests that the isolate might degrade phenol via both ortho- and meta-cleavage, mainly via ortho-cleavage.

• Asian-Australasian Journal of Animal Sciences
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• 제8권6호
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• pp.641-645
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• 1995

This study was conducted to develop a method for production of nuclear transplant bovine embryos using in vitro-matured (IVM) oocytes and to examine the effect of different conditions of electrofusion on fusion rate and developmental capacity of donor nucleus transplanted to enucleated oocytes. Eight- to sixteen-cell embryos derived from oocytes matured and fertilized in vitro used as donor blastomeres and IVM oocytes were used as recipient oocytes. Oocytes were enucleated immediately after 23-24 h IVM and then reconstituted with a donor blastomere in two different micromanipulation media. Fusion rate and subsequent development of the reconstituted oocytes was compared under the different electric stimuli and recipient oocyte ages. Success rate of enucleation was significantly higher in TCM-199 medium containing FCS than in DPBS. The high fusion rate(75-94%) and development (6.4-14.8%) to morulae and blastocyst (M + B) were obtained from 0.6-0.75 kV/cm DC voltage, although total cleavage was not different among the electric pulses. Most optimal condition of electric stimulation for fusion and development was 1 DC voltage of 0.75 kV/cm, in which 80.5% of oocytes were fused, 80.0% and 31.7% of which was cleaved and developed to M + B, respectively. No M + B was obtained from 1.2 kV/cm DC voltage regardless of pulse frequency. Recipint oocyte age at electrofusion greatly affected the cleavage and subsequent development to M + B, showing high rate at 40-41 h oocyte maturation. These results suggest that a suitable condition of electrofusion for donor nuclei derived from IVF may be 1-2 DC pulses of 0.7 kV/cm for $70{\mu}sec$ and that processing of a transplanted nucleus in IVM oocytes may be affected by maturation age of recipient oocytes.

• 한국수정란이식학회지
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• 제26권4호
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• pp.251-256
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• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.