• 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.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.113-123
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• 1996

It has been well known that polyamines ensure the stability of chromatin structure and the fidelity of DNA transcription. This study was carried out to evaluate the effect of polyamines on the apoptosis of mouse thymocytes induced by dexamethasone and polyamine synthesis inhibitors. 1) In the histological death findings of thymocytes double-stained with acridine orange and ethidium bromide, the apoptotic and the necrotic fractions (AF; NF) in the control group were $9.4{\pm}4.2%$ and $4.5{\pm}5.3%$, respectively. Dexamethasone $(3\;{\times}\;10^{-8}\;M:\;DX)$ in creased AF upto $52.0{\pm}8.1%$ and did not change NF, but A23187 $(5\;{\times}\;10^{-7}\;M:\;A2)$ increased AF and NF upto $45.0{\pm}8.9%$ and $20.5{\pm}10.6%$, respectively. 2) The thymocyte viability was significantly reduced by DX, DHEA $(1\;{\times}\;10^{-4}\;M)$, A2, DFMO $(1\;{\times}\;10^{-4}\;M)$, and $MGBG\;(1\;{\times}\;10^{-4}\;M)$, respectively. It was, however, little affected by $aminoguanidine\;(1\;{\times}\;10^{-4}\;M:\;AG)$, $putrescine\;(1\;{\times}\;10^{-5}\;M:\;PT)$, $spermidine\;(1\;{\times}\;10^{-5}\;M:\;SD)$, and $spermine\;(1\;{\times}\;10^{-5}\;M:\;SM)$. 3) The genomic DNA of mouse thymocyte was markedly fragmented by DX and A2, respectively, and to a lesser extent, by DHEA, but was little affected by MGBG, DFMO, AG, and each of polyamines. 4) The DX induced reduction of thymocyte viability was moderately attenuated by DHEA, but little affected by DFMO, MGBC, and AG. However, SM significantly attenuated the viability reduction induced by A2 as well as DX. 5) The thymocyte viability reduction by MGBG and DFMO was significantly attenuated by only SM among three polyamines applied in this study. 6) The thymocyte viability redution by combined treatments of DX with DFMO and MGBG, respectively, was significantly attenuated by SM, and moderately by PT. But the viability reduction by combined treatment of DX with AG or DHEA was not affected by polyamines. These results suggest that polyamines, particularly spermine, might play the inhibitory role in thymocyte apoptosis and the inhibitory effect can be ascribed in part to the increase of polyamine uptake by thymocytes pretreated with DFMO and MGBG.

• The Korean Journal of Nuclear Medicine
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• v.28 no.2
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• pp.220-226
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• 1994

This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR ) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patient's serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was defected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows ; 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79%) of the same group with conventional method. 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possiblity of false positivity and false negativity.

• Journal of Animal Science and Technology
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• v.48 no.5
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• pp.637-650
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• 2006

Genomic DNA isolated from two crucian carp species obtained from Yesan (Carassius auratus) and Dangjin (Carassius cuvieri) in Korea were amplified at several times by polymerase chain reaction (PCR). The amplified products were separated by agarose gel electrophoresis (AGE) with oligonucleotides decamer primer and stained with ethidium bromide. The seven arbitrarily selected primers OPC-11, OPC-14, OPC-18, OPD- 02, OPD-11, OPD-15 and OPD-20 generated the shared loci by each species, the polymorphic and specific loci. The seven primers generated the total 458 loci that can be scored from the crucian carp obtained in C. auratus species. 358 fragments were generated from the species obtained in C. cuvieri species. The size of DNA fragments varies from 150 to 1,600bp. The complexity of the banding patterns varies dramatically between the primers and two locations. In this study, 458 loci were identified in the crucian carp species from Yesan and 358 in the crucian carp species from Dangjin: 84 polymorphic loci (18.3%) in the C. auratus species and 48 (13.4%) in the C. cuvieri species. 154 shared loci by each species, the average 22 per primer, were observed in the C. auratus species and 187 loci, the average 26.7 per primer, in the Dangjin species. Based on the average bandsharing (BS) values of all samples, the similarity matrix ranged from 0.434 to 0.868 in the C. auratus species and from 0.449 to 0.924 in the C. cuvieri species. The average BS value was 0.641±0.013 within the C. auratus species and 0.684±0.013 within the C. cuvieri species. The average BS value between two crucian carp species 0.484 ± 0.007, ranged from 0.307 to 0.682. The BS value between the individual No. 09 and No. 16 was 0.682, which was the highest between two crucian carp species. Compared separately, the BS value of individuals within the C. cuvieri species was higher than the C. auratus species. The dendrogram obtained by the seven primers, indicates three genetic clusters: cluster 1 (AURATUS No. 01, 02, 03, 04, 05, 06, 07, 08, 09, 10 and 11), cluster 2 (CUVIERI No. 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21) and cluster 3 (CUVIERI no. 22). The shortest genetic distance displaying significant molecular difference was between the individual AURATUS No. 09 and AURATUS No. 08 from Yesan (genetic distance=0.064). The longest genetic distance displaying significant molecular differences was between the individual CUVIERI No. 17 and AURATUS No. 11 between two crucian carp species (0.477). RAPD-PCR analysis has revealed the significant genetic distance between two crucian carp species pairs.(Key words: Carassius auratus, Carassius cuvieri, Crucian Carp, DNA Polymorphism, Genetic Distance)

• Tuberculosis and Respiratory Diseases
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• v.39 no.6
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• pp.517-525
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• 1992

Background: Since its development by Saiki et al, polymerase chain reaction (PCR) has been very useful in various fields of molecular biology. PCR can be used for the detection of a very small amount of microbial agent, and is especially useful in those patients who are difficult to diagnose microbiologically or serologically. Mycobacterium tuberculosis is a very slowly growing organism and AFB staining frequently shows false negative results, and therefore PCR would be a very rapid, easy, and sensitive diagnostic method for the diagnosis of Mycobacterium tuberculosis. Method: To compare PCR with conventional methods in diagnosing Mycobacterium tuberculosis in sputum, we used sputa of patients who visited or were admitted to Seoul National University Hospital. The amplification targets were 383 base pair DNA, a part of 2520 base pair DNA encoding 65 kD Mycobacterium tuberculosis specific protein (the primers are TB-1, -2), and 123 base pair DNA, a part of IS6110 fragment, which multiple copies are known to exsist PCR one genome (the primers are Sal I-1, -2). We also requested AFB staing and culture to the lab of Seoul National University Hospital with the same sample and compared the results. Results: 1) Using TB-1, -2 primers, PCR was positive in 73.1% (19/26) of culture positive sputa, in 12.5% (1/8) of culture negative. but clinically diagnosed tuberculous sputa, and was negative in all sputa of patients who were clinically diagnosed as non-tuberculous etiology. 2) Using Sal I-I, -2 primers, PCR was positive in 94.1% (32/34) of culture positive sputa, in 23.1% (6/26) of culture negative, but clinically diagnosed tuberculous sputa, and was negative in 87.5% (14/16) of sputa from patients who were clinically diagnosed as non-tuberculous etiology. Conclusion: PCR could be a very rapid, sensitive and specific method for the diagnosis of Mycobacterium tuberculosis in sputa, and further studies should be followed for the development of easier method.

• Korean Journal of Medicinal Crop Science
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• v.11 no.1
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• pp.5-12
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• 2003

The biological activities of extracts from Rubus coreanus Miq. were compared. About 70% of the growth of human hepatocarcinoma and 79% of human gastric cancer cell was inhibited in adding 1.0 mg/ml of the extracts of Rubus coreanus Miq. respectively. The growth of human breast cancer cells was also inhibited in adding 1.0 mg/ml of the extracts as well as 78% of the human cancer cells. It was proved that the growth of human normal lung cell, scored as 15% for the extracts. Overall selectivity of the extracts on several human cancer cell line was over 5, which is higher than those from the Rubus coreanus Miq. The growth of both human immune B and T cells was enhanced up to 1.4 to 1.8 times by adding the extracts, compared to the controls. The secretion of tumor necrosis $factor-alpha(TNF-{\alpha})$ from T cell was also increased up to 78.8 pg/ml in adding the ethanol extract (0.5 mg/ml). Ethanol extract also increased up to about 70 pg/ml of interleukin-6(IL-6) from B cell. For screening regulate function of blood pressure, angiotensin converting enzyme(ACE) activity was inhibited up to 25% by adding the ethanol extract (1.0 mg/ml). In testing the hypoglycemic activity, 20% of ${\alpha}-glucosidase$ activity was inhibited for the extracts (0.5 mg/ml). GST activity was increased in the range of 1.2 to 1.6 times by adding extracts.

• Korean Journal of Medicinal Crop Science
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• v.11 no.1
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• pp.13-18
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• 2003

The biological activities of extracts from Rosa rugosae Radix were compared. About 78% of the growth of human hepato- carcinoma and 68% of human gastric cancer cell was inhibited in adding 0.5 mg/ml of the extracts of Rosa rugosae Radix respectively. The growth of human breast cancer cells was also inhibited in adding 0.5 mg/ml of the extracts as well as 66% of the human cancer cells. It was proved that the growth of human normal lung cell, scored as 20% for the extracts. Overall selectivity of the extracts on several human cancer cell line was over 4, which is higher than those from the Rosa rugosae Radix. The growth of both human immune B and T cells was enhanced up to 1.2 to 1.5 times by adding the extracts, compared to the controls. The secretion of tumor necrosis factor-alpha$(TNF-{\alpha})$ from T cell was also increased up to 61.9 pg/ml in adding the ethanol extract (0.5 mg/ml). Ethanol extract also increased up to about 61.3 pg/ml of interleukin-6(IL-6) from B cell.

• Development and Reproduction
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• v.10 no.4
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• pp.227-238
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• 2006

Genomic DNAs(gDNAs) were isolated from the venus clam(Gomphina aequilatera) from Samcheok(venus clam from Samcheok; VCS) and Wonsan(venus clam from Wonsan; VCW) located in the East Sea of the Korean Peninsula. The amplified products were generated by agarose gel electrophoresis(AGE) with oligonucleotides primer, detected by staining with ethidium bromide and viewed by ultraviolet ray. The seven arbitrarily selected primers BION-21, BION-23, BION-25, BION-27, BION-29, BION-31 and BION-33 generated the shared loci, polymorphic, and specific loci, with the molecular sizes ranging from 150 bp to 2,400 bp. In this study, 147 polymorphic loci(147/954 loci, 15.41%) in VCS population and 274(274/996 loci, 27.51%) in VCW population were generated with seven primers. These results suggest the genetic variation in VCW population is higher than in VCS population. Especially, the 700 bp bands generated by the primer BION-21 were identified commonly in two Gomphina populations, which identified populations and/or species. This specific primer was found to be useful in the identification of individuals and/or population, resulting from the different DNA polymorphism among individuals/species/population. Two Gomphina populations between the individual SAMCHEOK no. 03 and WONSAN no. 22 showed the longest genetic distance(0.696) in comparison with other individuals used. The complete linkage cluster analysis indicating three genetic groupings and dendrogram revealed close relationships among individual identities within two geographical populations of venus clam(G. aequilatera) from the Samcheok and Wonsan. The intra-species classification and clustering analyses inferred from molecular markers supported the traditional taxonomy of the species based on morphological characters such as shell size, shape and color. Accordingly, as mentioned above, RAPD analysis showed that VCS population was more or less separated from VCW population.