• Archives of Pharmacal Research
• /
• v.21 no.4
• /
• pp.458-464
• /
• 1998

Search for a new $\alpha$-methylene-$\gamma$-butyrolactone-bearing 6-substituted purine as a potental antitumor agent has led to synthesize seven, hitherto unreported, $5^1$-Methyl-$5^1$-[(6-substituted-9H-purin-9-yl)methyl]-$2^1$-oxo-$3^1$- methylenetetrahydrofurans (H, Cl, l, $CH_3$, $NH_2$, SH, >C=O) (6a-g). These include $5^1$-Methyl-$5^1$-[(9H-purin-9-yl)methyll-$2^1$-oxo-$3^1$ -methylenetetrahydrofurans (6a), $5^1$-Methyl-$5^1$-[(6-chloro-9H-purin-9-yl)methyl]-$2^1$-oxo-$3^1$-methylenetetrahydr ofurans (6b), $5^1$-Methyl-$5^1$-[(6-chloro-9H-purin-9-yl) methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6c), $5^1$-Methyl-$5^1$-[(6-methyl-9H-purin-9-yl) methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6d), $5^1$-Methyl-$5^1$-[(9H-adenin-9-yl)methyll-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6e), $5^1$-Methyl-$5^1$-[(6-mercapto-9H-purin-9-yl) methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6f) and $5^1$-Methyl-$5^1$-[(9H-hypoxanthin-9-yl)methyll-$2^1$-oxo-$3^1$-methylenetetrahydrof urans (6g) which were made by the Reformatsky-type reaction of ethyl $\alpha$-(bromomethyl) acrylate with the corresponding (6-substituted-9H-purin-9-yl)-2-propanone intermediates (5a-g). These ketone intermediates 5a-g, 1-(9H-purin-9-yl)-2-propanone (5a), 1-(6-chloro-9H-purin-9-yl)-2-propanone (5b), 1-(6-iodo-9H-purin-9-yi)-2-propanone (5c), 1-(6-methyl-9H-purin-9-yl)-2-propanone (5d), 1-(9H-adenin-9-yl)-2-propanone (Se), 1-(6-mercapto-9H-purin-9-yl)-2-propanone (5f), and 1-(9H-hypoxanthin-9-yl)-2-propanone (5g) were directly obtained by the alkylation of the 6-substituted purine bases with the chloroacetone in the presence of $K_2$$CO_3$ (or NaH) under DMF (or DMSO). The preliminary in vitro cytotoxcity assay for the synthetic .alpha.-methylene-y-butyro-lactone compounds (6a-g) were determined against three cell lines (PM-3A, P-388, and K-562) and showed the moderate antitumor activity ($IC_50$ ranged from 1.4 to 4.3 $\mu\textrm{g}$/ml) with the compound $5^1$-methyl-$5^1$ -[(9H-hypoxanthin-9-yl)methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofuran (6g) showing the least antitumor activity.

• Radiation Oncology Journal
• /
• v.20 no.3
• /
• pp.253-263
• /
• 2002

Purpose : In order to understand in vivo radiation damage modifying of bFGF on jejunal mucosa, bone marrow and the effect of bFGF on the growth of transplanted mouse sarcoma 180 tumor in mice. Materials and Methods : Mice were treated with $6\;{\mu}g$ of bFGF at 24 hours and 4 hours before exposing to 600 cGy, 800 cGy and 1,000 cGy total body irradiation (TBI), and then exposed to 3,000 cGy local radiation therapy on the tumor bearing thigh. Survival and tumor growth curve were plotted in radiation alone group and combined group of bFGF and irradiation (RT). Histologic examination was performed in another experimental group. Experimental groups consisted of normal control, tumor control, RT (radiation therapy) alone, $6\;{\mu}g$ bFGF alone, combined group of $3\;{\mu}g$ bFGF and irradiation (RT), combined group of $6\;{\mu}g$ bFGF and irradiation (RT). Histologic examination was peformed with H-E staining in marrow, jejunal mucosa, lung and sarcoma 180 bearing tumor. Radiation induced apoptosis was determined in each group with the DNA terminal transferase nick-end labeling method ($ApopTag^{\circledR}$ S7100-kit, Intergen Co.) Results : The results were as follows 1) $6\;{\mu}g$ bFGF given before TBI significantly improved the survival of lethally irradiated mice. bFGF would protect against lethal bone marrow syndrome. 2) $6\;{\mu}g$ bFGF treated group showed a significant higher crypt depth and microvilli length than RT alone group (p<0.05). 3) The bone marrow of bFGF treated group showed less hypocellularity than radiation alone group on day 7 and 14 after TBI (p<0.05), and this protective effect was more evident in $6\;{\mu}g$ bFGF treated group than that of $3\;{\mu}g$ bFGF treated group. 4) bFGF protected against early radiation induced apoptosis in intestinal crypt cell but might have had no antiapoptotic effect in bone marrow stem cell and pulmonary endothelial cells. 5) There was no significant differences in tumor growth rate between tumor control and bFGF alone groups (p>0.05). 6) There were no significant differences in histopathologic findings of lung and mouse sarcoma 180 tumor between radiation alone group and bFGF treated group. Conclusions : Our results suggest that bFGF protects small bowel and bone marrow from acute radiation damage without promoting the inoculated tumor growth in C3H mice. Improved recovery of early responding normal tissue and reduced number of radiation induced apoptosis may be possible mechanism of radioprotective effect of bFGF.

• Journal of Life Science
• /
• v.32 no.2
• /
• pp.135-141
• /
• 2022

Thymosin β-4 (TB4) is a small peptide composed of 43 amino acids. To obtain sufficient biologically active mouse TB4 economically, we cloned and overexpressed this gene in an Escherichia coli system. With the isopropyl β-D-1-thiogalactopyranoside induction of the E. coli transformant, TB4 fusion protein with intein- and chitin-binding domain was successfully expressed in the soluble fraction within the E. coli cell. The TB4-intein - chitin-binding domain fusion protein was purified from the soluble fraction of E. coli cell lysate. The affinity chromatography with chitin beads and dithiothreitol-mediated intein self-cleavage reaction releases the TB4 peptide into the stripping solution. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis and Western blot analyses were used to confirm that the recombinant TB4 peptide was produced with the expected size of 5 kDa. We found that the recombinant TB4 stimulated cell migration in the transwell plate chamber assay. After 18 hr of the treatment of the recombinant TB4 with 1 ng/ml concentration, the migration of the HT1080 cell was increased by 20% compared with that of the chemically synthesized TB4. The recombinant TB4 was also observed to promote the healing of a wound area in C57BL/6 mice by as high as 35% compared with that of the chemically synthesized TB4. These results suggest that the recombinant TB4 has better biological activity for cell migration and wound healing than that of the chemically synthesized TB4 peptide.

• Journal of Life Science
• /
• v.32 no.2
• /
• pp.108-118
• /
• 2022

The subventricular zone (SVZ) in the brain contains neural stem cells (NSCs) that generate new neurons throughout one's lifetime. Many extracellular and intracellular factors that affect cell proliferation and neuronal differentiation of NSCs are already well-known. Recently, L-type calcium channels have been reported to regulate neural development and are present in NSCs, differentiating neuroblasts, and mature neurons in the SVZ. Nifedipine, a blocker of L-type calcium channels, has been long used as a therapeutic drug for hypertension. However, studies on the use of nifedipine to inhibit L-type calcium channels of NSCs are lacking. Herein, we treated NSCs cultured from mouse postnatal SVZ with nifedipine during neuronal differentiation. Nifedipine increased the number of Tuj1-positive neurons but did not significantly change the number of Olig2-positive oligodendrocytes. Nifedipine increased cell division during early differentiation, which was detected using the 5-ethynyl-2'-deoxyuridine incorporation assay and immunocytochemistry assessment by staining the cells with phosphorylated histone H3, a mitosis marker. Nifedipine increased the transcription of Dlx2, a neurogenic transcription factor, and the level of Mash1, a marker for early neurogenesis. In addition to nifedipine, verapamil, which is also an L-type calcium channel blocker, showed a slight increase in neurogenesis, but its statistical significance was very low. In contrast, pimozide, a T-type calcium channel blocker, did not affect neurogenesis, although T-type calcium channel genes Cav3.1, Cav3.2, and Cav3.3 were expressed. In summary, nifedipine might promote the neuronal fate of NSCs during early differentiation and calcium signaling through L-type calcium channels might be involved in neuronal differentiation, especially during the early stages of differentiation.

• Korean Journal of Food Science and Technology
• /
• v.42 no.3
• /
• pp.329-334
• /
• 2010

Escherichia coli O157:H7 causes hemolytic uremic syndrome and hemorrhagic colitis in humans. The objectives of this study were to produce monoclonal antibody(MAb) against E. coli O157:H7 and to develop an enzyme linked immunosorbent assay(ELISA) for the rapid detection of E. coli O157:H7 in agri-stockbreeding. The characterization of MAb produced from hybridoma cell (HKEC 4G8-5) was validated by ELISA and Western blot. The produced MAb was specific to E. coli O157:H7 and showed weak cross-reaction to Staphylococcus aureus. The detection limit of ELISA based on 4G8-5 MAb was $10^5\;cell/mL$. Although the ELISA could not detect E. coli O157:H7 in the meat and sprout samples inoculated with $1{\times}10^1\;cell$/10 g without enrichment, the same samples after enrichment for 6 hr were confirmed to be positive by ELISA. These results indicated that the ELISA combined with short enrichment (6 hr) is useful tool for rapid screening of E. coli O157:H7 in various samples.

• Parasites, Hosts and Diseases
• /
• v.30 no.2
• /
• pp.101-112
• /
• 1992

The natural killer cell activity of splenocytes and TBC, active NK cells, recycling capacity of natural killer cells were observed by means of both the 51Cr-release cytotoxicity assay and single cell cytotoxicity assay against YAC-1. CSH/HeJ mice were infected intranasally with $1{\times}10^4{\;}or{\;}1{\times}10^5$ trophosoites of pathogenic Acanthamoeba culbertsoni. The infected mice showed mortality rate of 34% in $1{\times}10^4$ group and 65% in 1{\times}10^5 group, and mean survival time was $16.40{\pm}3.50$ {\;}and{\;}3.20{\pm}4.09$ days respectively. The cytotoxic activity of natural killer cells of the 2 groups was significantly higher than that of non-infected mice from the 12th hour to the 2nd day after infection, showing the highest on the first day. On the l0th day after infection, the cytotoxic activity of natural killer cells was significantly suppressed as compared with that of the control. There was no significant difference in NK cell cytotoxicity between two infected groups. The targetbinding capacity and active NK cells of natural killer cells in $1{\times}10^5$ trophosoite infected mice was significantly increased on the 12th hour and the first day after infection as compared with the control group. Maximal recycling capacity (MRC) was not changed during the observation period. The present results indicated that the elevation of natural killer cell activity in the mice infected with A. culbertsoni was due to elevation of target.binding capacity and increased active NK cells of natural killer cells, and not due to the maximal recycling capacity of the individual NK cell, and there was no difference between two experimental dose groups.

• Tuberculosis and Respiratory Diseases
• /
• v.47 no.5
• /
• pp.618-628
• /
• 1999

Background: Cell growth is a balance between cell proliferation and cell death. Insulin-like growth factor-I(IGF-I), which binds IGF-I receptor(IGF-IR), mediates cellular proliferation as a potent mitogen. IGF binding protein-3(IGFBP-3) as a circulating major IGFBP can inhibit or enhance the effects of IGF-I on cellular growth by binding IGFs. Methods: We investigated the expressions of mRNA of IGF-I and IGF-IR by northern blot and phosphorylation of IGF-IR with the treatment of IGF-I by western blot in 3T3 fibroblast cells. The cellular proliferations of 3T3 cells with the treatments of IGF-I were evaluated using $^3H$-thymidine incorporation and MTT assay. Also to observe the effect of IGFBP-3 on cellular proliferation, 3T3 cells were treated with anti-IGFBP-3 and ${\alpha}IR_3$(monoclonal antibody to IGF-IR) alone or in combination. Results: Our results demonstrated that 3T3 cells showed mRNA expressions of IGF-I and IGF-IR and the IGF-I increased phosphorylation of IGF-IR. The treatments of 3T3 cells with IGF-I increased cellular proliferation in 5 % and 1 % seruma-containing media, not in serum-free media. The addition of anti-IGFBP-3 to neutralize IGFBP-3 showed 2-fold increase of cellular proliferation, and also co-incubation of anti-IGFBP-3 and ${\alpha}IR_3$ together showed similar increase of cellular proliferation in 3T3 cells. Interestingly, when the cells were pretreated with ${\alpha}IR_3$ for 4 hr, prior to the simultaneous addition of ${\alpha}IR_3$ and anti-IGFBP-3, anti-IGFBP-3-mediated cellular proliferation was decreased to control level. All of these results suggest that free IGF-I released from IGF-I/IGFBP-3 complex would be involved in the cellular proliferation. Conclusion: IGF-I is a mitogen through the activation of IGF-IR in 3T3 cells, and IGFBP-3 could be a potent inhibitor for IGF-I action by binding IGF-I.

• Journal of Life Science
• /
• v.20 no.10
• /
• pp.1519-1524
• /
• 2010

Apigenin (4', 5, 7-trihydroxyflavone), a common dietary flavonoid abundantly present in fruits and vegetables, has shown remarkable anti-proliferative effects against various malignant cell lines. To observe the anti-proliferative effects, oral cavity cancer cell lines, $6{\times}10^3$ cells/well (96 well plate) of KB oral cavity tumor cells were plated and 24 hr later treated with apigenin for one day, after which MTT assay was performed. Apigenin induced cell death in a dose-dependent manner after incubation. Cell viability was significantly decreased in the group treated with 100 ${\mu}M$ apigenin for 24 hr (p<0.05) compared to the control group. To assess apoptosis, the nuclei of KB cells were stained with DAPI. The presence of chromatin condensation in the apigenin treated cells was detected on a fluorescent microscope (${\times}200$). We investigated the in vivo growth inhibitory effects of apigenin on oral cavity cancer KB tumor xenograft subcutaneously implanted in male nude mice. Apigenin was administered to mice by gavage at doses of 25 and 50 mg/kg/day in 0.2ml of PBS. Tumor volume was significantly decreased in 25 and 50 mg/kg apigenin-administration groups compared to the control group. For apoptosis analysis, TUNEL staining was performed. A significant increase in TUNEL positive cells was found in the 25 mg/kg apigenin administration group compared to the non- apigenin administration group. Histopathological changes were not observed. These results indicate that apigenin inhibits oral cavity cancer cell growth through the induction of apoptosis.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.32 no.1 s.55
• /
• pp.59-64
• /
• 2006

Crinum asiaticum Linne var. japonicum has long been used as a rheumatic remedy, an anti-pyretic, an anti-ulcer treatment, and for the alleviation of local pain and fever in Korea and Malaysia. In order to investigate the possibility of Crinum asiaticum Linne var. japonicum extract as a cosmetic ingredient, we measured its anti-inflammatory effect by inhibition of iNOS (inducible nitric oxide synthase), and the release of PGE2, IL-6, and IL-8. HPLC experiment after extraction with 95% ethanol at pH 3.5 showed that Crinum asiaticum Linne var. japonicum was mainly composed of lycorine (up to 1%), a well-known immunosuppressant. The content of lycorine varied depending on the type of tissue analyzed and the extraction method. In anti-inflammatory assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)- activated mouse macrophage RAW 264.7 cells, the ethanolic extract of Crinum asiaticum showed inhibitory activity of NO production in dose-dependent manner ($IC_{50} = 83.5 {\mu}g/mL$). Additional study by RT-PCR demonstrated that the extract of Crinum asiaticum significantly suppressed the expression of the iNOS gene. Moreover, the extract of Crinum asiaticum did not show my cytotoxicity, but did show cell proliferation effect against LPS ($10{\sim}60%$ increase of tell viability). In an assay to determine inhibition of the $H_2O_2$-activated release of PGE2, IL-6, and IL-8 in human normal fibroblast cell lines, the release of PGE2 and IL-6 was almost completely inhibited above concentrations of 0.05% and 1%, respectively. Moreover, the release of IL-8 was completely inhibited over the entire range of concentrations (> 0.0025%). The result showed that the extract of Crinum asiaticum Linne var. japonicum has sufficient anti-inflammatory effect. There-fore, Crinum asiaticum Linne var. japonicum extract may be useful as an ingredient of cosmetic products.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.43 no.2
• /
• pp.224-230
• /
• 2014

Marine algae have long been recognized as a health and beauty food, based on its anti-tumor, anti-inflammatory and anti-obesity activities. In this study, methanol extracts were prepared from 10 different phaeophyta, after which DPPH radical scavenging and cytoprotective activities of HT-22 cells against ${\beta}$-amyloid protein ($A{\beta}$), which has neurotoxic effects, were investigated. In DPPH experiments, Ecklonia cava and Ishige okamurai showed strong ROS scavenging activities, whereas eight other phaeophyta including Petalonia binghamiae (P. bin) showed weak ROS scavenging activities. To validate the cytoprotective effects of 10 different phaeophyta in $A{\beta}$-induced HT-22 cells, protein expression levels of APP, BACE1, iNOS, phosphorylated ERK1/2, phosphorylated p38 and phosphorylated JNK1/2 were determined along with MTT assay. In the MTT assay, P. bin showed the best effective cytoprotective activity at a concentrations of $25{\mu}g/mL$, whereas Sargassum confusum, Colpomenia sinuosa, Myelophycus simplex, and Sargassum hemiphyllum showed potential. Determination of protein expression levels related to $A{\beta}$-induced neurotoxicity in the five selected phaeophyta showed that P. bin inhibited BACE1 and iNOS expression in $A{\beta}$-induced HT-22 cells. These results indicate that the cytoprotective effects of P. bin are mediated by suppressing the pathways involving $A{\beta}$-induced ERK and p38 activation.