• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.6 no.1
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

• Toxicological Research
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• v.24 no.3
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• pp.175-182
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• 2008

DNA-dependent protein kinase(DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be $2^{nd}$ upstream kinase for protein kinase B(PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells(MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK(M059J) and a wild-type of DNA-PK(M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.423-428
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• 2000

In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

• Tuberculosis and Respiratory Diseases
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• v.49 no.4
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• pp.453-465
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• 2000

Background : Lung carcinogenesis is a multistage process involving alterations in multiple genes and diverse pathway. Mutational activation of oncogenes and inactivation of tumor suppressor genes, and subsequent increased genetic instability are the major genetic events. The p53 gene and FHIT gene as tumor suppressor genes contribute to the pathogenesis of lung cancer, evidenced by mutation, microsatellite instability(MI) and loss of heterozygosity(LOH). Methods : We analysed genetic mutations of p53 and FHIT gene in 29 surgical specimens of nonsmall cell lung cancer using PCR-single strand conformation polymorphism, DNA sequencing and RT-PCR. MI and LOH were analyzed in loci of D3S1285, D9S171, and TP53. Results : In 2 cases, point mutation of p53 gene was observed on exon 5. MI of 3 times and LOH of 14 times were observed in at least one locus. In terms of the location of microsatellite, D3S1285 as a marker of FH1T was observed in 5 cases out of 26 specimens; D9S171 as a marker of p16 in 5 out of 17; and TP53 as a marker of p53 in 7 out of 27. In view of histologic type, squamous cell carcinoma presented higher frequency of microsatellite alteration, compared to others. Mutation of FHIT gene was observed in 11 cases and 6 cases of those were point mutation as a silent substitution on exon 8. FHIT mRNA expression exhibited deletion on exon 6 to 9 in 4 cases among 15 specimens, presenting beta-actin normally. Conclusion : Our results show comparable frequency of genetic alteration in nonsmall cell lung cancer to previous studies of Western countries. Microsatellite analysis might have a role as a tumor marker especially in squamous cell carcinoma. Understanding molecular abnormalities involved in the pathogenesis could potentially lead to prevention, earlier diagnosis and the development of novel investigational approaches to the treatment of lung cancer.

• Journal of the Korean Arthroscopy Society
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• v.9 no.1
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• pp.51-55
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• 2005

Purpose: To compare the stability and clinical result after anterior cruciate ligament reconstructed knee after graft fixation using Intrafix in tibial tunnel with or without additional tibial post fixation. Materials and Methods: We analyzed 37 cases which were treated with four-strand hamstring tendon autograft during the period from May 2002 to January 2003. The grafts were fixed with Rigidfix system (Mitek Product, Johnson and Johnson, USA) in femur tunnel and Intrafix system (Mitek Product, Johnson and Johnson, USA) in tibial tunnel. After tibial fixation, additional tibial post fixation was done, which was determined by the serial case number prospectively. Patients were followed for average of fourteen months(range, thirteen to twenty-five months) At the time of final follow-up, patients were evaluated in terms of Lachman test, pivot shift test, Lysholm scores, IKDC (International Knee Documentation Committee) assessment, side-to-side KT-1000 maximum-manual arthrometer differences. Results: At last follow-up, Lysholm score was average 93.1(range: 65 to 98), IKDC assessment revealed that 26 cases had score of A, 10 cases had score of B and 1 case had score of C. The average maximum-manual KT-1000 arthrometer side ?to-side difference was 2.5 mm$(0{\sim}6mm)$. There was one case in which the Lachman test was graded as 2+ and four cases in which the Lachman test was graded as 1+ and the remaining thirty-two cases were normal by Lachman test. One case had a 2+ pivot-shift, and 2 cases had a 11 pivot-shift. The remaining 34 knees were normal on pivot -shift testing. The average maximum-manual KT-1000 arthrometer side-to-side difference was average 2.8 mm$(0{\sim}6mm)$ in Intrafix only group and average 2.2 mm$(0{\sim}4mm)$ in additional fixation group (P>0.05). Conclusion: Without additional tibial fixation, the stability of the anterior cruciate reconstructed knee with hamstring graft which was fixed with Intrafix was restored.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.289-295
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• 2005

This study was performed to investigate the relationship between Heat shock protein 70 (HSP70) gene polymorphism and in vitro fertilization(IVF) embryo development in the pigs. The single strand conformation polymorphism(SSCP) genotypes from HSP70 K1, K3 and K4 PCR products were detected different patterns. In cleavage rate of oocyte fertilized in vitro, HSP70 K1-AA genotype($73.1\%$) and K1-AB genotype($62.3\%$) showed significantly higher oocyte cleavage rate than HSP70 K1-BB genotype($49.3\%$)(p<0.05). And HSP70 K3-AA genotype ($72.4\%$) and K3-AB($62.2\%$) also showed significantly higher oocyte cleavage rate than HSP70 K3-BB genotype($49.1\%$)(p<0.05). The IVF embryo development of 2-cell stage according to HSP70 genotypes of sperm and pig breeds also showed a significant difference. The number of embryos developed to 2-cell stage in Landrace(28.8) and Duroc(29.8) were significantly higher than in Yorkshire(10.9)(p<0.05). And also HSP70 K4-AB genotype group(29.6) higher than HSP70 K4-AA genotype group(10.6)(p<0.05). However, the number of embryos developed to blastocyst stage did not showed significant differences among breeds as well as HSP70 genotypes. These resrults suggest that in vitro development in porcine early embryos may be affected by HSP70 genotypes and breeds.

• Radiation Oncology Journal
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• v.22 no.3
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• pp.208-216
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• 2004

Purpose: DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase consisting of a 470 kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex, called Ku, which is composed of 70 kDa(Ku 70) and 86 kDa (Ku 80) proteins. The DNA-PK has been shown to play a pivotal role in rejoining DNA double-strand-breaks (dsb) in mammalian cells. The purpose of this study is to examine the relationship between the level of Ku expression and radiation sensitivity. Methods and Materials: Nine head and neck, cancer cell lines showed various intrinsic radiation sensitivities. Among the nine, AMC-HN-3 cell was the most sensitive for X-ray irradiation and AMC-HN-9 cell was the most resistance. The most sensitive and resistant cell lines were selected and the test sensitivity of radiation and expression of Ku were measured. Radiation sensitivity was obtained by colony forming assay and Ku protein expression using Western blot analysis. Results: Ku80 increased expression by radiation, wheres Ku70 did not. Overexpression of Ku80 protein increased radiation resistance in AMC-HN9 cell line. There was a correlation between Ku8O expression and radiation resistance. Ku80 was shown to play an important role in radiation damage response. Conclusion: Induction of Ku80 expression had an important role in DNA damage repair by radiation. Ku80 expression may be an effective predictive assay of radiosensitivity on head and neck cancer.

• Journal of the Korean Wood Science and Technology
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• v.38 no.1
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• pp.1-10
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• 2010

In this study, to study an effective use and improve strength performances of woods and wood-based materials, three-ply hybrid laminated woods which are composed of spruce in the face and three kinds of wood-based boards (MDF, PB, OSB) in the core were manufactured, and the effect of constitution elements used for the core laminae on bending creep performances was investigated. The shape of creep curves showed exponential function plots which the upper right side was increased, and differed among the kinds of wood-based boards used for the core laminae of hybrid laminated wood. The creep deformation perpendicular to the grain of faces of hybrid laminated woods was in order $C_{\perp}$(P) > $C_{\perp}$(M) > $C_{\perp}$(O) with PB, MDF and OSB in the core, respectively. It was found that the creep deformation arranged with OSB in the core had 2 times smaller than those arranged with MDF and PB in the core. By hybrid laminating, the creep deformation of spruce perpendicular to the grain was markedly decreased. On the other hand, the creep deformation parallel to the grain of the faces ($C_{\parallel}$ type) of hybrid laminated woods was in order $C_{\parallel}$(P) > $C_{\parallel}$(O) > $C_{\parallel}$(M) with PB, OSB and MDF in the core. The ratios among three hybrid laminated woods were considerably decreased, especially the difference between $C_{\parallel}$(P) and $C_{\parallel}$(O) hybrid laminated woods arranged with PB and OSB in the core was very small. These values showed 0.108~0.464 times smaller than creep deformation of three wood-based boards and it was found that creep deformation of three wood-based boards was considerably decreased by hybrid laminating. Creep anisotropy of hybrid laminated woods was greater in creep deformation than in initial deformation, whereas it was found that the values was much smaller than that of spruce parallel laminated woods.

• Food Science of Animal Resources
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• v.24 no.4
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• pp.349-354
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• 2004

Stress-susceptible pigs have been known as the porcine stress syndrome (PSS), swine PSS, also known as malignant hyperthermia (MH), is characterized as sudden death and production of poor meat quality such as PSE (pale, soft and exudative) meat after slaughtering. PSS and PSE meat cause major economic losses in the pig industry. A point mutation in the gene coding for the ryanodine receptor (RYR1) in porcine skeletal muscle, also known calcium (Ca$^{2+}$) release channel, has been associated with swine PSS and halothane sensitivity. We used the PCR-RFLP(restriction fragment length polymorphism) and PCR-SSCP (single strand conformation polymorphism) methods to detect the PSS gene mutation (C1843T) in the RYR1 gene and to estimate genotype frequencies of PSS gene in Korean pig breed populations. In PCR-RFLP and SSCP analyses, three genotypes of homozygous normal (N/M), heterozygous carrier (N/n) and homozygous recessive mutant (n/n) were detected using agarose or polyacrylamide gel electrophoresis, respectively. The proportions of normal, carrier and PSS pigs were 57.1, 35.7 and 7.1％ for Landrace, 82.5, 15.8 and 1.7％ far L. Yorkshire, 95.2, 4.8 and 0.0％ for Duroc and 72.0, 22.7 and 5.3％ for Crossbreed. Consequently, DNA-based diagnosis for the identification of stress-susceptible pigs of PSS and pigs producing PSE meat is a powerful technique. Especially, PCR-SSCP method may be useful as a rapid, sensitive and inexpensive test for the large-scale screening of PSS genotypes and pigs with PSE meat in the pork industry.y.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.10
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• pp.1271-1278
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• 2007

In this study, the ability of Styela clava or Styela plicata to reduce ethanol-induced hepatotoxicity and hepatic and leucocytic DNA damages was evaluated. Twenty four male SD rats were given 25% ethanol containing water (ad lib, p.o.) and divided into 3 groups; ethanol treated control group (EtOH), ethano1+3% S. clava (EtOH+SC), and ethano1+3% S. plicata (EtOH+SP). After 6 weeks, the supplementation of S. clava reduced the plasma ALT, ALP and LDH activities significantly (p<0.05), while S. plicata induced significant decrease in the plasma LDH activity only. The comet assay was employed to quantify the alcohol-induced DNA damage in rat hepatocytes and leucocytes. A significant protective effect on hepatic and leucocytic DNA damages was observed in S. clava or S. plicata supplemented groups compared to the EtOH control group. The hepatic DNA damage was correlated positively with plasma ALP and LDH activities. These results demonstrated that S. clava or S. plicata supplementation protected alcohol-induced hepatic and leucocytic DNA damage.