• Journal of Life Science
• /
• v.28 no.12
• /
• pp.1448-1454
• /
• 2018

Insects have been investigated as a novel source of food and biomaterial in several recent studies. However, their osteoblastogenic cell activity has not been sufficiently researched and so, to investigate the potential of this natural material for promoting osteoblastogenesis, we studied the activity of Locusta migratoria ethanol extract (LME) on MG-63 pre-osteoblast cells. The cytotoxicity and proliferation effects of LME on MG-63 cells were measured by MTS assay, and there was no cytotoxicity up to $1,000{\mu}g/ml$. With LME treatment of 500 and $1,000{\mu}g/ml$ for 48 hr, cell proliferation increased to 105% and 116% versus control, respectively. The osteoblastogenic activity of the LME was measured through alkaline phosphatase (ALP) staining at three and five days. As a result, both 500 and $1,000{\mu}g/ml$ LME concentrations were seen to increase ALP activity by more than three times compared with control at three and five days. In addition, the expression level of the osteogenic markers ALP and RUNX2 was markedly increased after LME treatment. These results demonstrate that Locusta migratoria ethanol extract promotes osteoblastogenesis as evidenced by the increased osteogenic markers and suggest that LME may be a potential agent for bone formation and osteoporosis prevention.

• Journal of Life Science
• /
• v.29 no.9
• /
• pp.1027-1033
• /
• 2019

Recently, Korea has seen a rapid increase in the elderly population. As a result, osteoporosis, a geriatric disease, has become a social problem. To investigate the novel and natural materials for promoting osteoblastogenesis, we investigated the osteoblastogenic activity of Tenebrio molitor larvae oil (TMO) on the MG-63 preosteoblast cells. The cytotoxicity and proliferation effects of TMO on MG-63 cells were measured by MTS assay. There was no cytotoxicity up to $80{\mu}g/ml$. At 40 and $80{\mu}g/ml$ of TMO (treated for 48 hr), cell proliferation was elevated about 120% compared to the control. The osteoblastogenic activity of TMO was measured with alkaline phosphatase (ALP) activity at 5 days. Doses of 5 to $80{\mu}g/ml$ of TMO increased ALP activities significantly compared with the control. In addition, expression of ALP and Runx2 (osteoblastogenic markers) were markedly increased after treatment of TMO for 5 days. These results provide evidence that TMO promotes osteoblastogenesis by increasing the gene and protein expression of ALP and Runx2, and they suggest that TMO may be a potential agent for bone formation and preventing osteoporosis.

• The Journal of Korean Obstetrics and Gynecology
• /
• v.30 no.2
• /
• pp.49-70
• /
• 2017

Objectives: The present study is to observe the skin-regeneration, anti-wrinkle, whitening and skin moisturizing effects of Cheongsangbangpung-tang (CSBPT) with cytotoxicity. Methods: In the present study, cytotoxicity of CSBPT lyophilized aqueous extracts (yield=18.71%) was experimented against human normal fibroblast cells and B16F10 murine melanoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay, and skin regeneration and anti-wrinkle effects were also showed through the assay of collagen type I synthesis by an enzyme immunoassay (EIA) kit as comparing with transforming growth factor (TGF)-${\beta}1$, hyaluronidase, collagenase and matrix metalloproteinase (MMP)-1 inhibitory assays as comparing with oleanolic acid (OA), and elastase inhibitory effects as comparing with phosphoramidon disodium salt (PP). In addition, whitening effects of CSBPT were observed by tyrosinase inhibitory assay and melanin formation test in B16/F10 melanoma cells as comparing with arbutin, and skin moisturizing effects were measured through mouse skin water contents test, respectively. Results: No CSBPT treatment related cytotoxic effects were demonstrated against human normal fibroblast cells and B16/F10 murine melanoma cells. CSBPT concentration-dependent increased collagen type I synthesis at human normal fibroblast cells. It also effectively suspreessed hyaluronidase, collagenase, elastase and MMP-1 activities, which were enzymes that related to declining of ECM and formation of wrinkle. CSBPT supressed B16/F10 melanoma cells's melanin productions with tyrosinase activity, which was an enzyme connected with melanin formation, and dose-dependent and significant increases of skin water contents were detected in CSBPT treated mouse skin as compared with vehicle control skins. Conclusions: CSBPT showed favorable and enough skin regeneration, anti-wrinkle, whitening and skin moisturizing effects at least in a condition of this experiment. However, more detail mechanism and in vivo skin protective efficacy studies should be conducted in future with the screening of the biological active compounds in individual herbs of Cheongsangbangpung-tang.

• Tuberculosis and Respiratory Diseases
• /
• v.47 no.2
• /
• pp.172-183
• /
• 1999

Background: Nitric oxide is a short-lived effector molecule derived from L-arginine by the nitric oxide synthase(NOS). Nitric oxide plays a role in a number of physiologic and pathophysiologic functions including host defense, edema formation, and regulation of smooth muscle tone. Some kinds of cells including macrophage are known to produce large quantities of nitric oxide in response to inflammatory stimuli such as interleukin-$1\beta$(IL-$1\beta$), tumor necrosis factor-$\alpha$(TNF-$\alpha$), interferon-$\gamma$(IFN-$\gamma$) and lipopolysaccharide(LPS). Reactive oxygen species are also known to be important in the pathogenesis of acute cell and tissue injury such as acute lung injury model Methods: Using the RA W264.7 cells, we have examined the ability of oxidant hydrogen peroxide($H_2O_2$) to stimulate nitric oxide production and inducible NOS mRNA expression. Also, we have examined the effects of NOS inhibitors and antioxidants on $H_2O_2$ induced nitric oxide production. Results: Stimulation of RAW264.7 cells with combinations of 100 ng/ml IL-$1\beta$, 100 ng/ml TNF-$\alpha$, and 100 U/ml IFN-$\gamma$ or 100 U/ml IFN-$\gamma$ and $1{\mu}g/ml$ LPS induced the synthesis of nitric oxide as measured by the oxidation products nitrite($NO_2^-$) and nitrate($NO_3^-$). Addition of $250 {\mu}M-2$ mM $H_2O_2$ to the cytokines significantly augmented the synthesis of $NO_2^-$ and $NO_3^-$(p<0.05). When cells were incubated with increasing concentrations of $H_2O_2$ in the presence of IL-$1\beta$, TNF-$\alpha$ and IFN-$\gamma$ at constant level, the synthesis of $NO_2^-$ and $NO_3^-$ was dose-dependently increased(p<0.05). $N^G$-nitro-L-arginine methyl ester(L-NAME), dose dependently, significantly inhibited the formation of $NO_2^-$ and $NO_3^-$ in cells stimulated with LPS, IFN-$\gamma$ and $H_2O_2$ at constant level(p<0.05). Catalase significantly inhibited the $H_2O_2$-induced augmentation of cytokine-induced $NO_2^-$ and $NO_3^-$ formation(p<0.05). But, boiled catalase did not produce a significant inhibition in comparison with the native enzyme. Another antioxidant 2-mercaptoethanol and orthophenanthroline dose-dependently suppressed $NO_2^-$ and $NO_3^-$ synthesis(p<0.05). Northern blotting demonstrated that H:02 synergistically stimulated the cytokine-induced iNOS mRNA expression in RA W264.7. Conclusion: These results suggest that $H_2O_2$ contributes to inflammatory process by augmenting the iNOS expression and nitric oxide synthesis induced by cytokines.

• Korean Journal of Poultry Science
• /
• v.44 no.1
• /
• pp.19-28
• /
• 2017

The eggshell is an intricate and highly ordered structure composed of multiple layers and a calcified matrix. The eggshell is formed at the uterine segment of the chicken oviduct. In this study, histological changes in the uterine endometrium and the expression of the eggshell-related genes were investigated according to hen age. We analyzed the expression of eggshell protein-related genes, such as OCX-32, OCX-36, OC-17, OC-116, and eggshell-ion-related genes, such as CABL-1, SPP1, SCNN1G, ATP2A2, CA2, and CALM1. In chicken uterine endometrium, histological deformation, fibrosis, atrophy and elimination of micro-villi were found with increasing hen age. The concentration of blood-ion components did not significantly change with age. The amount of telomeric DNA in uterine endometrial cells decreased with increasing hen age. The expression of most of the eggshell-related genes changed significantly with increasing hen age. The expression of some ovo-proteins, which play a role in eggshell formation, increased with increasing hen age; however, there were no significant correlations among eggshell protein genes. Eggshell ion-related genes, such as ATP2A2, SCNN1G, CA2, and CALM1, were closely related to each other. The OCX-32 and OCX-36 genes were closely related to some of the eggshell ion genes. Eggshell protein-related genes, such as the OCX-32, OCX-36 genes and ion-related genes such as CALB-1, ATP2A2, SCNN1G, CA2, CALM1, affected eggshell formation, mutually or independently. This study shows that, uterine although endometrial cell damage occurs with increasing hen age, normal eggshells can be formed in old hens. This suggests that eggshell protein-and eggshell ion-related genes also control the homeostasis of eggshell formation.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.35 no.3
• /
• pp.203-208
• /
• 2009

Doenjang (Korean fermented soybean paste) is a unique fermented food in Korea. It has been traditionally manufactured from soybeans, by Jang Yang process. We focused on the newly formed compound in highly aged Doenjang and its biological activity. One new o-dihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone and two known o-dihydroxyisoflavone derivatives were isolated from 5-year-old Doenjang and evaluated as potent antioxidant and whitening effect by comparing with other known isoflavone. 7,8,4'-Trihydroxyisoflavone (compound 1), 7,3',4'-trihydroxyisoflavone (compound 2) and 6,7,4'-trihydroxyisoflavone (compound 3) inhibited DPPH (diphenyl-1-picrylhydrazyl) formation by 50 % at a concentration of $21.5{\pm}0.2$, $28.7{\pm}0.4$ and $32.6{\pm}0.6$ ($IC_{50}$) respectively, whereas daidzein showed weak DPPH radical scavenging activity. In superoxide scavenging effect were measured in one assay. Compound 1 ($IC_{50}=18.10{\pm}0.2{\mu}M$) and 2 ($IC_{50}=10.54{\pm}0.4{\mu}M$) show significant inhibitory activity and greater effect than L-ascorbic acid. But compound 3 and daidzein showed lower inhibition activity. Also, o-dihydroxyisoflavone derivatives evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. Compound 1 ($IC_{50}=11.21{\pm}0.2{\mu}M$), compound 2 ($IC_{50}=5.23{\pm}0.6{\mu}M$) exhibited significant inhibitory effect on tyrosinase activity. Furthermore, those compounds are significantly suppressed the cellular melanin formation by 50 % at a concentration of $12.23{\pm}0.7{\mu}M$ (1) and $7.83{\pm}0.7{\mu}M$ (2). This result suggests that 7,3',4'-trihydroxyisoflavone from highly aged Doenjang could be used as an active ingredient for cosmetics.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.27 no.2
• /
• pp.318-332
• /
• 2000

The purpose of this study was to investigate the effects of demineralized freeze-dried bone (DFDB) on mechanically exposed pulp of dog by evaluating the pulpal inflammation and healing process, formation of dental hard tissue, and structural changes of fibroblasts of the remaining pulp tissue. Teeth of 4 dogs, weighing 10kg, were used in this study. Class V cavities were prepared followed by exposed the pulp tissue mechanically by sterilized round bur. In control group, exposed pulps were capped with calcium hydroxide paste followed by sealed with IRM. In experimental groups, the exposed pulps of one group were capped with the collagen and those of the other group were capped with DFDB. All cavities were sealed with same manor as control group. The animals were sacrificed at the intervals of 3, 7, 14, and 28 days for histopathlogic evaluation. The specimens were observed by the light microscope and trans-electron microscope. The results were as follows: 1. Pulp necrosis was not observed in all groups. Inflammatory response was disappeared from 1 week in control group and group 2. But it was not disappeared until 2 weeks and also irregular arrangement of odontoblasts was showed at the lateral walls of root canal just beneath the amputated site of the pulp in group 1. 2. Dentinal bridge was formed incompletely at 2 weeks but it was formed completely at 4 weeks in control group. Odontoid tissue was also found in control group at 4 weeks from treatment. Amputated site of pulp was encapsulated with fibrous tissue and odontoblast and dentinal bridge was not found in group 1. Preodontoid tissue and reparative dentin which were formed by odontoblast differentiated around DFDB were found, but dentinal bridge was not found in group 2. 3. Cell with large basophillic-stained nuclei infiltrated to amputated site and DFDB at 1 week from treatment in control group and group 2. They were found more in group 2 than in control group. Odontoblasts arranged more regularly and reparative dentin was found more as time elapsed. 4. Dentin-formative odontoblasts which showed ultramicrostructure of cytoplasm with polarized nucleus, rEM, Golgi complex, secretory granules, secretion of organic matrix in control of group and group 2. In regards to above results, the demineralized freeze-dried bone(DFDB) induce odontoblastic differentiation and further come up to the dentin formation in amputated pulp.

• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.1-7
• /
• 2002

The present study was carried out to investigate the effects of maturation media on penetrability of pig oocytes by liquid boar sperm coincubated with different sperm concentrations in a modified Tris­buffered medium (mTBM). Follicular oocytes collected from ovaries of prepubertal gilts were matured in a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. The sperm­ich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ far 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes in 5% $CO_2$in air at 38.5$^{\circ}C$, cumulus cells were removed and oocytes (30~40) were coincubated for 6 h in 0.5 $m\ell$ mTBM fertilization medium with five different (1$\times$10$^{6}$ , 2$\times$10$^{6}$ , 4$\times$10$^{6}$ , 6$\times$10$^{6}$, 10$\times$10$^{6}$ $m\ell$) sperm concentrations. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ NCSU-23 culture medium fur further culture of 6 h. At 12 h after IVF, sperm penetration, polyspermy and male pronuclear formation of oocytes were evaluated. Oocytes of NCSU-23 maturation medium decreased polyspermy and increased male pronuclear formation compared to those of mTCM­199 and mWaymouth MB 752/1 maturation media. Of oocytes matured in NCSU-23 medium and inseminated in mTBM medium with 2~4$\times$10$^{6}$ $m\ell$ sperm concentrations, 50.8~50.9% showed sperm penetration, 13.3~19.5% polyspermy and 43.9~45.4% male pronuclear formation. In conclusion, we found out that oocytes matured in NCSU­23 medium and inseminated in mTBM medium showed superior in­vitro fertilization compared to those matured in mTCM­199 and mWaymouth MB 752/1 maturation media and inseminated in mTBM medium. The optimum sperm concentrations for in-vitro fertilization of oocytes matured in NCSU-23 medium by liquid boar semen stored at 17$^{\circ}C$ for 5 days were 2~4$\times$10$^{6}$ $m\ell$.

• Korean Journal of Veterinary Research
• /
• v.15 no.1
• /
• pp.57-67
• /
• 1975

The poxvirus group is considered to be a typical cytoplasmic inclusion forming virus. Every poxvirus has been reported to produce only one kind of inclusion in the infected tissues. A vague concept that inclusions of poxviruses are eosinophilic or acidophilic has prevailed. Although many papers and theories about the nature of the inclusion have been presented, most of them are not quite convincing on the point of the relations with virus multiplication, and an analysis of papers published showed that there seem to be many discrepancies in the descriptions of the nature of the poxvirus inclusions. Comparative studies on host-virus interaction with cowpox, orf, swinepox and fowlpox viruses which selected from each Group (I-IV) of poxviruses were performed from the morphological and virological standpoints. At first, in cowpox virus-FL cell system, as a comparative model, cytoplasmic inclusion, nucleic acid metabolism by autoradiography and detection of viral antigen by immunofluorescence were studied and obtained the results as follows: 1. The focus-like cytopathic effect (CPE) at early stage developed to entire culture at terminal stage of infection, and also the developing status of CPE was correlated to viral doses for inoculation. Two kinds of cytoplasmic inclusions which named A and B type were easily observed by Giemsa, hematoxylin-eosin (H & E) and May-Greenwald Giemsa (MGG) stainings in the infected cells. The B type inclusions were formed at early stage of infection and the A type inclusions were produced subsequently the B type formation. The B type which common type inclusion in poxviruses was a small compact or aggregate at early stage and developed to a large diffuse body at terminal stage of infection. On the other hand, the A type inclusion which depend upon the kind of virus was appeared as round and discrete shape, and its size and number was increased gradually during the culture period. It was characteristic to form distinct halos around the both types of inclusions in acid fixed, H & E stained preparations of infected cultures. The B type inclusion was always positive in Feulgen reaction and showed as DNA containing body but the A type inclusion was not. 2. In the relationship between inclusion and DNA metabolism of infected cells by the qualitative autoradiography using 3H-thymidine, the appearance of silver grains was coincided with B type inclusion but not with A type inclusion. This showed that the DNA synthesis was proceeded in all B type inclusions except those in the terminal stage with a diffuse form. This suggested that the B type inclusions are only sites of DNA synthesis and this was proceeded after the cell infection independently. The activity of DNA synthesis of the inclusions was nearly the same as that of the nucleic of normal cells and non-inclusion bearing cells. and non-inclusion bearing cells. Regardless of the size of the degree of DNA synthesis of the B type inclusion, inclusion bearing cells all showed remarkable suppression of nuclear DNA synthesis. 3. By the direct fluorescent antibody technique viral antigen in infected cells was detected. The B type inclusions have been proved to contain a great deal of viral antigen, whereas the basic substance of A type inclusion did not show antigenicity except the round edge. It was suggested that the round edge fluorescence might be caused by the glare of cytoplasmic viral antigen which pushed out and concentrated by the A type inclusion development. 4. Hemorrhagic red pock formations on chorioallantoic membrane of embryonated chicken egg had proved the characteristic of used viral strain. 5. By the above studies on the nature of two types of inclusions and the role they play in virus multiplication, it was concluded that the B type inclusion must be the site of the synthesis of viral DNA and protein as well as the site of the virus.

• Journal of Embryo Transfer
• /
• v.19 no.2
• /
• pp.133-145
• /
• 2004

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.