• Food Science and Biotechnology
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• v.14 no.3
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• pp.350-354
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• 2005

The effect of the essential oil obtained from Chrysanthemum boreale Makino on the apoptosis of KB cells was investigated. Cytotoxicity and cellular DNA content were analyzed by MTT assay, flow cytometry, agarose gel electrophoresis, and Hoechst 33258 staining. The caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were estimated by Western blotting method. The various cytotoxic effects of the essential oil which are hallmarks of apoptosis, including DNA fragmentation, apoptotic body formation, and sub-G1 DNA content, all progressed in a dose-dependent manner. Treatment with an apoptosis-inducing concentration of the essential oil caused rapid and transient induction of caspase 3 activity. Further, the efficacious induction of PARP cleavage and caspase-3 activation was observed at an essential oil concentration of 0.1 and 0.2 mg/mL for 12 hr.

• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.8-15
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• 2021

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

• Molecules and Cells
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• v.37 no.9
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• pp.644-649
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• 2014

The 3' ends of most eukaryotic messenger RNAs must undergo a maturation step that includes an endonuc-leolytic cleavage followed by addition of a polyadenylate tail. While this reaction is catalyzed by the action of only two enzymes it is supported by an unexpectedly large number of proteins. This complexity reflects the necessity of coordinating this process with other nuclear events, and growing evidence indicates that even more factors than previously thought are necessary to connect 3' processing to additional cellular pathways. In this review we summarize the current understanding of the molecular machinery involved in this step of mRNA maturation, focusing on new core and auxiliary proteins that connect polyadenylation to splicing, DNA damage, transcription and cancer.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.5
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• pp.804-808
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• 1997

The genetic differentiation and characteristics of two oyster populations (Crassostrea gigas) in Korea were assessed based on the restriction fragment length polymorphisms (RFLP) analysis and the restriction patterns of subcloned mtDNA. The restriction fragments of twenty individuals in West Sea revealed an identical pattern, determined by 8 restriction enzymes. On the other hand, two haplotypes having variation at the HindIII site were shown in the specimens from South Sea; minor haplotypes (4 of 20) were similar to the results obtained from individuals in West Sea while major haplotypes were different from those in West Sea. It was suggested that oysters (C. gigas) of West Sea might have been introduced to South Sea. Each mitochondrial DNA from two oyster populations in Korea and from one in Japan was divided to three parts and subcloned into pUC19 to use in genetic studies effectively. Restriction map was constructed based on the cleavage pattern by multiple restriction enzymes.

• BMB Reports
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• v.56 no.3
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• pp.166-171
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• 2023

Monocytes are peripheral leukocytes that function in innate immunity. Excessive triglyceride (TG) accumulation causes monocyte death and thus can compromise innate immunity. However, the mechanisms by which TG mediates monocyte death remain unclear to date. Thus, this study aimed to elucidate the mechanisms by which TG induces monocyte death. Results showed that TG induced monocyte death by activating caspase-3/7 and promoting poly (ADP-ribose) polymerase (PARP) cleavage. In addition, TG induced DNA damage and activated the ataxia telangiectasia mutated (ATM)/checkpoint kinase 2 and ATM-and Rad3-related (ATR)/checkpoint kinase 1 pathways, leading to the cell death. Furthermore, TG-induced DNA damage and monocyte death were mediated by caspase-2 and -8, and caspase-8 acted as an upstream molecule of caspase-2. Taken together, these results suggest that TG-induced monocyte death is mediated via the caspase-8/caspase-2/DNA damage/executioner caspase/PARP pathways.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.43-51
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• 1993

Mitochondrial DNA(mt DNA) of Mammalian is the circular one which the 16.5K base pairs and show the maternal inheritance. Evolutional speed of nucleotide sequence is very fast. So that polymorphic analysis of mt DNA provide the useful informations to investigate the genetic relations of interspecies. Authors trials were focussed to compare with the polymorphic differences of mitochondrial DNA between Jindo and Japanese mongrel dogs. DNA was extracted from bloods of 21 head of Jindo dogs and 20 head of Japanese dogs and isolated using 10 kinds of restriction endonucleases(Apa I, BamH I, Bgl II, EcoR I, EcoR V, Hinc II. Hind III, Pst I, Sty I, Xba I) and then separated by the agarose gel electrophoresis. After sourthern blotting hybridization was completed using the mtDNA of Japanese mongrel dogs as a probe. Autoradiography was used to compare the polymorphism of mtDNA both dogs. The results obtained were as follows; 1. mt DNA of Jindo dog showed polymorphism resulting cleavage with four kinds of restriction endonuclease, Apa I, EcoR V, Hinc II, Sty I. While in the Japanese mongrel dogs observed the polymorphism in the five kinds of restriction endonuclease supplemented with EcoR I. 2. Compared with both dogs the frequency differences of DNA polymorphism were recognized in the specific restriction endonuclease Apa I. Consequently in the restriction endonuclease Apa I both dogs classified with three types as A, B, C however in the Jindo dogs frequency of C type was 71.5 percent but in Japanese mongrel dogs observed 45 percent in the A type. 3. DNA polymorphism obtained from the use of five kinds of restriction endonuclease were classified with seven types. In Jindo dogs frequency was highest in the type 6 as 71.4 percent but in the Japanese mongrel dogs showed 35 percent in the type 5. 4. Genetic distances calculated by NEI method showed 0.0089 in Jindo dogs and was 0.0094 in the Japanese mongrel dogs.

• Archives of Pharmacal Research
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• v.20 no.6
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• pp.550-554
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• 1997

Chlorambucil is known to alkylate primarily N7 of guanine and N3 of adenine to induce DNA monofunctional adducts and interstrand cross-links (ISC). We have investigated the sequence specificity for DNA ISC induced by chlorambucil using duplex oligomers containing a defined cross-linkable sequences $ 5^{I}-A*TT, 5^{I}-G*TTor5^{I}-G*CC$ under bar which asterisk indicates the potential cross-linking site and underlined base indicates the potential cross-linking site on the opposite strand. An analysis of 20% denaturing polyacrylamide gel electrophoresis showed that chlorambucil was albe to induce DNA ISC in the duplex oligomers containing a sequence $5^I-GCC$. The formation of DNA ISC was not observed in the duplex oligomers containing sequences $5^I-ATT$. or $5^I-GTT$. These results indicate that chlorambucil induces guanine-guanine DNA ISC but not guanine-adenine or adenine-adenine DNA ISC. In addition, we have tested the ability of chlorambucil to induce DNA ISC within $5^I-GNNC$ or $5^I-GNNC$sequences using duplex oligomers containing the sequence$5^I-G^4G^3G^2^C$. The result of DNA strand cleavage assay showed that DNA ISC was formed at the $5^I-GGC$ sequence (an 1,3 cross-link, $G^1-G^3$) but not at $5^I-GGGC$ (an 1,4 cross-link, $G^1-G^4$) or $5^I-GC$ sequence (an 1,2 cross-link, $G^1-G^2$).

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.3
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• pp.677-683
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• 2005

The aim of this study was to investigate the apoptotic effect and its mechanism on Radix Aconiti (RA) extract in HL-60 human leukemia cell line. RA extract induced apoptosis as confirmed by discontinuous fragmentation of DNA. To clarify the mechanisms on RA extract-induced apoptosis, we examined the caspase-3, -8 enzyme activity and protein levels including Fas, FasL in HL-60 cells. Treatment with RA extracts resulted in the increase of caspase-3 enzyme activity in a time and dose-dependent manners, which was accompanied by the cleavage of poly-(ADP-ribose) polymerase (PARP). This activation of caspase-3 enzyme resulted from cleavage of procaspase-8, which was followed by increases of FasL, Fas protein expression in RA extracts-treated HL-60 cells. In conclusion, RA extract induced apoptosis of HL-60 human leukemia cell line. This results suggest that the apoptotic mechanisms of RA extract on HL-60 cells involved in FasL, Fas activation, procaspase-8 cleavage, activation of caspase-3 and cleavage of PARP. Collectively, these results suggest that RA may be a valuable agent as a anti-cancer drug.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.3
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• pp.802-810
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• 2005

The tetrahydroisoquinolines included potent cytotoxic agents that showed antitumor activity,antimicrobial activity, and other biological properties. We studied the effect of CDST, 1-Chloromethyl-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-sulfonic acid amide, a newly synthesized anti-cancer agent. The cytotoxic activity of CDST in HL-60 cells was increased in a dose-dependent manner. CDST, tetrahydroisoquinolines derivative, was cytotoxic to HL-60 cells, with IC50 of $80{\mu}g/ml$. Treatment of CDST to HL-60 cells showed the fragmentation of DNA in a dose- and time dependent manner, suggesting that thesecells underwent apoptosis. Treatment of HL-60 cells with CDST was induced in a dose- and time-dependent activation of caspase-3, caspase-8 and proteolytic cleavage of poly(ADP-ribose) polymerase. In caspase activity assay, caspase-3 and -8 was activated after 12 h and 6 h posttreatment, respectively. CDST also caused the release of cytochrome c from mitochondria into the cytosol. CDST-induced cytochrome c release was mediated by caspase-8-dependent cleavage of Bid and Bax translocation. These results suggest that caspase-8 induced Bid cleavage and Bax translocation, caused mitochondrial cytochrome c release, and induce caspase-3 activationduring CDST-induced apoptosis in HL-60 cells.