• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.109-109
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• 1997

Inhibitory effect on DNA topoisomerase-I, rate of glutathione conjugation and cytotoxicity of naphthoquinone derivatives were correlated. During 5 min exposure of the derivatives to glutathione (GSH), it was found that 14% of 5,8-dimethoxy-1,4-naphthoquinone(DMNQ) was converted into a GSH-conjugate, whereas 5,8-dihydroxy-1,4-naphthoquinone(DHNQ) did not interact with GSH, implying that DMNQ exerted higher electrophilicity than DHNQ. However, DHNQ (IC$\_$50/, 0.15 ${\mu}$M) showed stronger cytotoxicity in L1210 cells than DMNQ(IC$\_$50/, 0.45 ${\mu}$M). The stronger cytotoxicity of DHNQ, compared to DMNQ, could be ascribed to more rapid redox cycling. Both naphthoquinones (IC$\_$50/, 60-65 ${\mu}$M) exhibiting about the same inhibitory effect on DNA topoisomerase-I were more potent than 1,4-naphthoquinone(1,4-NQ, IC$\_$50/, 134 ${\mu}$M). Thus, 5,8-oxy groups in the structure seem to be important for the inhibition of the enzyme. DMNQ showed a broader dose range while maintaining a good antitumor activity against S-180 fluid tumor. For these reasons, DMNQ was taken as useful pharmacophore for structural modification. Introduction of 1-hydroxyalkyl groups at C-2 of DMNQ lowered all of the activities mentioned above, while acetylation of 1-hydroxyalkyl moiety enhanced the activities by 4-5 times. Introduction of the same side chains at C-6 exhibited stronger activities than 2-substituted ones. Based on these results it was suggested that the quinonoid moiety in 6-substituted DMNQ was more exposed to cellular nucleophiles such as DNA, thiols of enzymes and so on. The synthesis of DHNQ or DMNQ derivatives are going on, and the corelationship between structure-activity will be discussed.

• Journal of Genetic Medicine
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• v.12 no.2
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• pp.79-84
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• 2015

Purpose: We developed and validated a fetal trisomy detection method for use as a noninvasive prenatal test (NIPT) including a Clinical Laboratory Improvement Amendments (CLIA)-certified bioinformatics pipeline on a cloud-based computing system using both Illumina and Life Technology sequencing platforms for 221 Korean clinical samples. We determined the necessary proportions of the fetal fraction in the cell-free DNA (cfDNA) sample for NIPT of trisomies 13, 18, and 21 through a limit of quantification (LOQ) test. Materials and Methods: Next-generation sequencing libraries from 221 clinical samples and three positive controls were generated using Illumina and Life Technology chemistries. Sequencing results were uploaded to a cloud and mapped on the human reference genome (GRCh37/hg19) using bioinformatics tools. Based on Z-scores calculated by normalization of the mapped read counts, final aneuploidy reports were automatically generated for fetal aneuploidy determination. Results: We identified in total 29 aneuploid samples, and additional analytical methods performed to confirm the results showed that one of these was a false-positive. The LOQ test showed that the proportion of fetal fraction in the cfDNA sample would affect the interpretation of the aneuploidy results. Conclusion: Noninvasive chromosome examination (NICE), a CLIA-certified NIPT with a cloud-based bioinformatics platform, showed unambiguous success in fetus aneuploidy detection.

• Journal of Sasang Constitutional Medicine
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• v.18 no.3
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• pp.131-144
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• 2006

1. Objectives This research uses the DNA chip, which includes 16,383 gene code, and various statistic prediction way that shows objectification index for the objectification of constitution diagnosis. 2. Methods Drawing blood whose constitution is confirmed, and analyze its gene information by using 1.7k DNA chip to find the gene correlation through multivariate statistical method. 3. Results and Conclusions Distinctive genes such as AK001919, U09384, NM_001805, X99962, NM_004796, AK026738, AL050148, BC002538, AK027074, AK026219, AF087962, AL390142, NM_015372, AL157466, NM_002446, AK024523, NM_014706, NM_014746 and AL137544 were related to Taeumin; AL157448, NM_005957, NM_005656, NM_017548, AK027246, NM_003025, NM_012302 and NM_005905 were represented in Soeumin, while AK026503, AF147325, NM_002076, AF147307, AK001375, NM_003740, NM_005114, AB007890, NM_005505, NM_015900, NM_014936, Z70694, AB023154, U52076, NM_004360, NM_005835, NM_017528, AF087987, NM_014897, AK021720, NM_006420, AJ277915, AK002118 and AK021918 were for Soyangin. This study figured out the possibility to develop the prediction system by sorting each constitution's gene, and research each constitution's distinctive character of manifestation pattern.

• ALGAE
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• v.38 no.2
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• pp.127-139
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• 2023

Cryptic stages of diverse macroalgae present in natural substrata, "the bank of microscopic forms", were isolated into clonal cultures and identified based on both morphological characteristics and DNA barcoding. Approximately 120 clonal isolates from 308 natural substratum samples were collected from the entire coastline of Kuwait. Amongst these isolates, 77 (64%) were identified through DNA barcoding using the nuclear ribosomal small subunit, RuBisCO spacer (ITS2, tufa, rbcL, psaA, and psbA) and sequencing. Twenty-six isolates (34%) were identified in the division Chlorophyta, 18 (23%) as Phaeophyceae, and 33 (43%) as Rhodophyta. For all DNA sequences in this study, species-level cut off applied was ≥98% homology which depend entirely on the markers used. Three putative new records of Chlorophyta new for the Arabian Gulf were made: Cladophora laetevirens (Dillwyn) Kützing, Ulva torta (Mertens) Trevisan and Ulvella leptochaete (Huber) R. Nielsen, C. J. O'Kelly & B. Wysor in Nielsen, while Cladophora gracilis Kützing and Ulva ohnoi M. Hiraoka & S. Shimada are new records for Kuwait. For Phaeophyceae, Ectocarpus subulatus Kützing and Elachista stellaris Areschoug were new records for the Gulf and Kuwait. In the Rhodophyta, Acrochaetium secundatum (Lyngbye) Nägeli in Nägeli & Cramer, Ceramium affine Setchell & N. L. Gardner, Gelidium pusillum var. pakistanicum Afaq-Husain & Shameel and Dasya caraibica Børgesen are new records for the Gulf and Kuwait, while the red alga Stylonema alsidii (Zanardini) K. Drew is a new record for Kuwait. Several isolates identified corresponded to genera not previously reported in Kuwait and / or the Arabian Gulf, such as Porphyrostromium Trevisan, a new genus from the Bangiales, and two unidentified species for the Planophilaceae Škaloud & Leliaert. The isolates cultivated from substrata enhance understanding of the marine macroalgal diversity in the region and confirmed that the Germling Emergence Method is suitable for determining the actual diversity of a given study area through isolation from cryptic life-history phases.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.113-119
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• 2012

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

• Journal of Microbiology
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• v.44 no.6
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• pp.671-673
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• 2006

A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminogly-coside phosphotransferase gene (aph) from Tn903, a lacZ' gene for $\alpha$-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DHS${\alpha}$ and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.6
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• pp.776-783
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• 2008

In this study, we investigated the genetic diversity and phylogenetic relationship of six duck populations by employing the genetic polymorphisms of 20 microsatellites. The parameters used in this study included number of alleles, average effective numbers of alleles (E) and average rates of heterozygosity of each population. The results showed that all the microsatellite loci were highly polymorphic except that the locus AJ515896 in Muscovy duck was 0. The average PIC (0.762), average h (0.7843) and average E (5.261) of the six duck populations were all high, indicating that the gene polymorphisms and genetic diversity were high. The test of Hardy-Weinberg equilibrium showed that the six populations in this study were all in Hardy-Weinberg disequilibrium. The F-statistic analysis results showed the range of FST was from 0.0205 (AJ515895) to 0.2558 (AJ515896). The mean FST was 0.0936. Phylogenetic study revealed that Peking duck (Z1 and Z4), Shaoxing duck, Cherry Valley duck and Aobaixing duck were clustered in one group, while the Muscovy duck was clustered in one group alone. The phylogenetic relationships among different populations were in accordance with their breeding history and distribution. Our data suggested that the 20 microsatellite loci were effective markers for analysis of genetic relationships among duck populations.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.18-24
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• 2001

Expression of the baculovirus major envelope glycoprotein gene(gp64) is regulated by transcription from botha early and late promoters. To develop a transient expression vector under the control of gp64 gene promoter, the gp64 gene of Bombyx mori nucleopolyhedrovirus-K1(BmNPV-K1) was characterized. The gp64 gene was local-ized at EcoR I-Pst I 7.38-kb fragment of the BmNPV-K1 genome. The EcorR 1-Pst I 7.38-kb fragment was cloned and the nucleotide sequence of 2,277 bases including the coding region of gp64 gene was determined. Based on these results, transient expression vector using gp64 gene promoter was constructed and named as pBm64. E.coli lacZ gene was introduced onto pBm64 as a reporter gene and expressed transiently in B. mori 5(Bm 5) cells. The expression vector transfected into the cells was maintained stably for 1 to 5 days. In order to confirm the expression of the reporter gene by gp64 promoter, recombinant virus was constructed. The recombinant virus has two independent transcription units in opposite orientations with two promoters; gp64 and polyhedrin gene promoters each initiating transcription of $\beta$-galactosidase and polyhedrin, respectively. Polyhedra formation and expression of $\beta$-galactosidase in Bm5 cells infected with the recombinant virus were observed with phase contrast microscope and in situ staining.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.167-176
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• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

• Molecules and Cells
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• v.27 no.5
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• pp.583-589
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• 2009

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5'-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between -98 and +31, and between -73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between -98 and -56 and between -73 and -41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilrs GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.