• The Korean Journal of Pesticide Science
• /
• v.7 no.3
• /
• pp.159-168
• /
• 2003

With microtiter plate, the assay method was developed for detecting the fungicidal activity of new compounds against spore germination, spore adhesion and mycelial growth of Colletotrichum sp. JC24 cal1Sing red pepper anthracnose. Also, the effects of some commercialized fungicides on fungal development like above mentioned were investigated by measuring the optical density of mycelia grown into wells of microtiter plate. For the standardization of assay method, some factors, such as the treatment of MTT and/or propanol, inodulum density and incubation period, affecting on mycelial optical density were investigated. For obtaining precise and consistent mycelial optical density, it was necessary the treatment of MTT for 12 hrs and propanol for 1 hr. inoculum density adjusted to $1\times10^5$ spores/mL and incubation period for 36 hrs at $25^{\circ}C$. For fungicidal activities, 6 protective fungicides, 6 ones inhibiting sterol biosynthesis, and one inhibiting respiration were used in this study. While mancozeb, chlorothalonil and dithianon among 6 protective fungicides inhibited strongly spore germination, adhesion, and mycelial growth at $6.25{\mu}g/mL$, propineb, iminoctadine and fluazinam inhibited intermediately spore germination and mycelial growth at $100{\mu}g/mL$. Washing above 3 fungicides with new PD broth, their activity against spore adhesion decreased. With hexaconazole, tebuconazole and myclobutanil, the tendency of the activity against fungal differentiation of the early infection stage was similar to the latter group of protective fungicides, showing the decrease of the inhibitory activity against spore adhesion by washing 2 hrs after incubation. However, kresoxim-methyl inhibited spore adhesion distinctly, depending on the applied concentrations. Based on these results, it might be able to assess the fungicidal activity of many compounds against spore germination, adhesion and mycelial growth by the use of microtiter plate in vitro. Using the assay developed in this report, it was possible to investigate the inhibitory activity of some commercialized fungicides, too.

• The Journal of Korean Academy of Prosthodontics
• /
• v.41 no.2
• /
• pp.182-206
• /
• 2003

Statement of problem : Successful osseointegration of endosseous threaded implants is dependent on many factors. These may include the surface characteristics and gross geometry of implants, the quality and quantity of bone where implants are placed, and the magnitude and direction of stress in functional occlusion. Therefore clinical quantitative measurement of primary stability at placement and functional state of implant may play a role in prediction of possible clinical symptoms and the renovation of implant geometry, types and surface characteristic according to each patients conditions. Ultimately, it may increase success rate of implants. Purpose : Many available non-invasive techniques used for the clinical measurement of implant stability and osseointegration include percussion, radiography, the $Periotest^{(R)}$, Dental Fine $Tester^{(R)}$ and so on. There is, however, relatively little research undertaken to standardize quantitative measurement of stability of implant and osseointegration due to the various clinical applications performed by each individual operator. Therefore, in order to develop non-invasive experimental method to measure stability of implant quantitatively, the resonance frequency analyzer to measure the natural frequency of specific substance was developed in the procedure of this study. Material & method : To test the stability of the resonance frequency analyzer developed in this study, following methods and materials were used : 1) In-vitro study: the implant was placed in both epoxy resin of which physical properties are similar to the bone stiffness of human and fresh cow rib bone specimen. Then the resonance frequency values of them were measured and analyzed. In an attempt to test the reliability of the data gathered with the resonance frequency analyzer, comparative analysis with the data from the Periotest was conducted. 2) In-vivo study: the implants were inserted into the tibiae of 10 New Zealand rabbits and the resonance frequency value of them with connected abutments at healing time are measured immediately after insertion and gauged every 4 weeks for 16 weeks. Results : Results from these studies were such as follows : The same length implants placed in Hot Melt showed the repetitive resonance frequency values. As the length of abutment increased, the resonance frequency value changed significantly (p<0.01). As the thickness of transducer increased in order of 0.5, 1.0 and 2.0 mm, the resonance frequency value significantly increased (p<0.05). The implants placed in PL-2 and epoxy resin with different exposure degree resulted in the increase of resonance frequency value as the exposure degree of implants and the length of abutment decreased. In comparative experiment based on physical properties, as the thickness of transducer increased, the resonance frequency value increased significantly(p<0.01). As the stiffness of substances where implants were placed increased, and the effective length of implants decreased, the resonance frequencies value increased significantly (p<0.05). In the experiment with cow rib bone specimen, the increase of the length of abutment resulted in significant difference between the results from resonance frequency analyzer and the $Periotest^{(R)}$. There was no difference with significant meaning in the comparison based on the direction of measurement between the resonance frequency value and the $Periotest^{(R)}$ value (p<0.05). In-vivo experiment resulted in repetitive patternes of resonance frequency. As the time elapsed, the resonance frequency value increased significantly with the exception of 4th and 8th week (p<0.05). Conclusion : The development of resonance frequency analyzer is an attempt to standardize the quantitative measurement of stability of implant and osseointegration and compensate for the reliability of data from other non-invasive measuring devices It is considered that further research is needed to improve the efficiency of clinical application of resonance frequency analyzer. In addition, further investigation is warranted on the standardized quantitative analysis of the stability of implant.

• 한국유가공학회:학술대회논문집
• /
• 2007.09a
• /
• pp.49-58
• /
• 2007

Inflammatory process leads to the well-known mucosal damage and therefore a further disturbance of the epithelial barrier function, resulting abnormal intestinal wall function, even further accelerating the inflammatory process[1]. Despite of the records, etiology and pathogenesis of IBD remain rather unclear. There are many studies over the past couple of years have led to great advanced in understanding the inflammatory bowel disease(IBD) and their underlying pathophysiologic mechanisms. From the current understanding, it is likely that chronic inflammation in IBD is due to aggressive cellular immune responses including increased serum concentrations of different cytokines. Therefore, targeted molecules can be specifically eliminated in their expression directly on the transcriptional level. Interesting therapeutic trials are expected against adhesion molecules and pro-inflammatory cytokines such as TNF-${\alpha}$. The future development of immune therapies in IBD therefore holds great promises for better treatment modalities of IBD but will also open important new insights into a further understanding of inflammation pathophysiology. Treatment of cytokine inhibitors such as Immunex(Enbrel) and J&J/Centocor(Remicade) which are mouse-derived monoclonal antibodies have been shown in several studies to modulate the symptoms of patients, however, theses TNF inhibitors also have an adverse effect immune-related problems and also are costly and must be administered by injection. Because of the eventual development of unwanted side effects, these two products are used in only a select patient population. The present study was performed to elucidate the ability of TNF-${\alpha}$ antibodies produced in sheep colostrums to neutralize TNF-${\alpha}$ action in a cell-based bioassay and in a small animal model of intestinal inflammation. In vitro study, inhibitory effect of anti-TNF-${\alpha}$ antibody from the sheep was determined by cell bioassay. The antibody from the sheep at 1 in 10,000 dilution was able to completely inhibit TNF-${\alpha}$ activity in the cell bioassay. The antibodies from the same sheep, but different milkings, exhibited some variability in inhibition of TNF-${\alpha}$ activity, but were all greater than the control sample. In vivo study, the degree of inflammation was severe to experiment, despite of the initial pilot trial, main trial 1 was unable to figure out of any effect of antibody to reduce the impact of PAF and LPS. Main rat trial 2 resulted no significant symptoms like characteristic acute diarrhea and weight loss of colitis. This study suggested that colostrums from sheep immunized against TNF-${\alpha}$ significantly inhibited TNF-${\alpha}$ bioactivity in the cell based assay. And the higher than anticipated variability in the two animal models precluded assessment of the ability of antibody to prevent TNF-${\alpha}$ induced intestinal damage in the intact animal. Further study will require to find out an alternative animal model, which is more acceptable to test anti-TNF-${\alpha}$ IgA therapy for reducing the impact of inflammation on gut dysfunction. And subsequent pre-clinical and clinical testing also need generation of more antibody as current supplies are low.

• Tuberculosis and Respiratory Diseases
• /
• v.63 no.3
• /
• pp.242-250
• /
• 2007

Background: Abnormal angiogenesis can induce hypoxia within a highly proliferating tumor mass, and these hypoxic conditions can in turn create clinical problems, such as resistance to chemotherapy. However, the mechanism by which hypoxia induces these changes has not yet been determined. Therefore, this study was conducted to determine how hypoxia induces changes in cell viability and extracellular microenvironments in an in vitro culture system using non-small cell lung cancer cells. Methods: The non-small cell lung cancer cell line, A549 was cultured in DMEM or RPMI-1640 media that contained fetal bovine serum. A decrease in the oxygen tension of the media that contained the culture was then induced in a hypoxia microchamber using a $CO_2-N_2$ gas mixture. A gas analysis and an MTT assay were then conducted. Results: (1) The decrease in oxygen tension was checked the anaerobic gas mixture for 30 min and then reoxygenation was induced by adding a 5% $CO_2-room$ air gas mixture to the chamber. (2) Purging with the anaerobic gas mixture was found to decrease the further oxygen tension of cell culture media. (3) The low oxygen tension resulted in a low pH, lactic acidosis and a decreased glucose concentration in the media. (4) The decrease in glucose concentration that was observed as a result of hypoxia was markedly different when different types of media were evaluated. (5) The decrease in oxygen tension inhibited proliferation of A549 cells. Conclusion: These data suggests that tumor hypoxia is associated with acidosis and hypoglycemia, which have been implicated in the development of resistance to chemotherapy and radiotherapy.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2007.11a
• /
• pp.79-92
• /
• 2007

Oxidative stress have known to be a risk factor for the degenerative processes and closely related to a lot of diseases. It is well established that antioxidants are good in protection and therapeutic means against oxidative damage. There is increasing interest in natural antioxidants and many natural antioxidants have been found and utilized as the possible protection for various diseases and skin aging. We have screened natural antioxidant agents for cosmeceuticals, nutraceuticals, and drugs as therapeutic and preventive means against oxidative stress, and have developed a number of novel antioxidants from various natural sources. A novel melanin synthesis inhibitor, Melanocin A, isolated from the metabolite of a fungal strain Eupenicillium shearii F80695 inhibited mushroom tyrosinase and melanin biosynthesis of B16 melanoma cells with $IC_{50}$ value of 9.0 nM and MIC value of $0.9\;{\mu}M$, respectively. Melanocin A also exhibited potent antioxidant activity by scavenging of DPPH and superoxide anion radicals. UV was found to increase the level of hydrogen peroxides and other reactive oxygen species (ROS) in skin tissues. This increase in ROS may not only alter the structure and function of many genes and proteins directly but may also modulate their expressions through signal transduction pathways and, ultimately, lead to skin damage. We investigated the effect of Melanocin A on UV-induced premature skin aging. Firstly, the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT in vitro was investigated. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated UV-induced skin changes in hairless mice in vivo by Melanocin A. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. These results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging. Terrein is a bioactive fungal metabolite isolated from Penicillium species. Terrein has a relatively simple structure and can be easily synthesized. However, the biologic effects of terrein are comparatively unknown. We found for the first time that terrein potently inhibit melanin production in melanocytes and has a strong hypopigmentary effect in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment of Mel-Ab cells with terrein (10-100 mM) for 4 days significantly reduced melanin levels in a dose-dependent manner. In addition, terrein at the same concentration also reduced tyrosinase activity. We then investigated whether terrein influences the extracellular signal-regulated protein kinase (ERK) pathway and the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. Terrein was found to induce sustained ERK activation and MITF down-regulation, and luciferase assays showed that terrein inhibits MITF promoter activity in a dose-dependent manner. To elucidate the correlation between ERK pathway activation and a decreased MITF transcriptional level, PD98059, a specific inhibitor of the ERK pathway, was applied before terrain treatment and found to abrogate the terrein-induced MITF attenuation. Terrein also reduced the tyrosinase protein level for at least 72 h. These results suggest that terrain reduces melanin synthesis by reducing tyrosinase production via ERK activation, and that this is followed by MITF down-regulation.

• Journal of Embryo Transfer
• /
• v.22 no.2
• /
• pp.89-95
• /
• 2007

The present study was conducted to examine some factors affecting in vitro development and fecundity of embryos recloned with somatic cell nuclear transfer (SCNT). Fibroblast cells retrieved from the ear of a 3-week-old, cloned Korean goat (Jinsoonny) were used as karyoplast donors and serum-starvation was conducted in tissue culture medium (TCM)-199 supplemented with 0.5% FBS. Recipient oocytes were surgically collected by flushing the oviducts 35 h after hCG injection following FSH priming. The zonae pellucidae of the oocytes were partially perforated with a laser drill and a donor cell was transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $%O_2$, 90% $N_2$ for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients and pregnancy was subsequently diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion rate following 1st fusion pulse was higher (p<0.05) in 2nd cloning (65.9%) compared to 1st cloning (51.0%), but it was not different in the other groups. The rate of cleavage after fusion was significantly higher (p<0.05) in 1st (77.7%) than in 2nd cloning (56.0%). A total of 175 re-cloned embryos were transferred into 28 recipients. On day 21 and 60 after transfer, 11 (39.3%) and 4 recipients (17.4%) were pregnancy, respectively. In comparison of pregnancy rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were pregnant on days 21 and 63 while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. These results show that recloning of goat can be achieved by SCNT and estrous synchronization between donor and recipient animals may be one of the major factors affecting success rate.

• Clinical and Experimental Reproductive Medicine
• /
• v.33 no.4
• /
• pp.237-243
• /
• 2006

Objective: The aim of this study was to evaluate the efficacy of frozen-thawed ET in poor prognosis patients such as the old age (38~44 years; OA group) and the patients who did not achieve clinical pregnancy with the first fresh ET cycle (non-pregnant patients; NP group). Methods: Laboratory and clinical data were collected from fresh and frozen-thawed ET cycles of OA and NP group. Controlled ovarian hyperstimulation (COH) and conventional insemination or ICSI, in vitro culture and ET were performed by routine procedures. Supernumerary embryos were frozen by the slow freezing method, and frozen embryos were thawed by the rapid thawing method. Embryo development, pregnancy and implantation rates were statistically analyzed by Student t-test and chi square test Results: Mean ages were similar between fresh ET ($40.0{\pm}1.8$ years, n=206) and frozen-thawed ET ($39.9{\pm}1.9$ years, n=69) cycles in OA group. However, the clinical pregnancy and implantation rate of subsequent frozen-thawed ET significantly higher than those of fresh ET cycles (29.0% and 11.2% vs. 16.5% and 7.0%, p<0.05). In NP group, there was no difference in the mean age between fresh ET ($31.2{\pm}2.3$ years, n=40) and frozen-thawed ET ($31.9{\pm}3.1$ years, n=119) in subsequent cycles. The clinical pregnancy and implantation rates were similar between the subsequent fresh ET (42.5% and 22.6%) and the frozen-thawed ET (40.3% and 18.8%). Conclusion: In old age patients, higher pregnancy rate of frozen-thawed ET compared to fresh ET cycles in this study. It may be related that better uterine environments for implantation in frozen-thawed ET cycles than that of non-physiological hormonal condition in uterus of fresh COH cycles.

• Korean Journal of Microbiology
• /
• v.43 no.4
• /
• pp.243-249
• /
• 2007

The centromere is a highly differentiated structure of the chromosome that fulfills a multitude of essential mitotic and meiotic functions. Alphoid DNA (${\alpha}$-satellite) is the most abundant family of repeated DNA found at the centromere of all human chromosomes, and chromosomes of primates in general. The most important parts in the development of Human Artificial Chromosomes (HACs), are the isolation and maintenance of stability of centromeric region. For isolation of this region, we could use the targeting hook with alphoid DNA repeat and cloned by Transformation-Associated Recombination (TAR) cloning technique in yeast Saccharomyces cerevisiae. The method includes rolling-circle amplification (RCA) of repeats in vitro to 5 kb-length and elongation of the RCA products by homologous recombination in yeast. Four types of $35\;kb{\sim}50\;kb$ of centromeric DNA repeat arrays (2, 4, 5, 6 mer) are used to examine the stability of repeats in homologous recombination mutant strains (rad51, rad52, and rad54). Following the transformation into wild type, rad51 and rad54 mutant strains, there were frequent changes in inserted size. A rad52 mutant strain showed extremely low transformation frequency, but increased stability of centromeric DNA repeat arrays at least 3 times higher than other strains. Based on these results, the incidence of large mutations could be reduced using a rad52 mutant strain in maintenance of centromeric DNA repeat arrays. This genetic method may use more general application in the maintenance of tandem repeats in construction of HAC.

• Reproductive and Developmental Biology
• /
• v.31 no.1
• /
• pp.35-41
• /
• 2007

This study was conducted to examine the effects of energy substrates in different conoentration of carbohydrates in the human oviduct and uterus on the in vitro development of mouse 2-cell embryos. Two-cell embryos were collected from ICR female mice at $46{\sim}50\;hr$ after 5 IU hCG injection and cultured in three different media [control: 0 mM, Guoup A: glucose (G) 0.5 mM + pyruvate (P) 0.32 mM + lactate (L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM] for 72 hr. Rates of morula formation of group A (72.3%) and B (56.6%) were significantly higher higher (p<0.05) than that of control (34.9%) at 24 hr. However, blastocyst rate was significantly higher (p<0.05) in control (51.8%) than group A (39.8%) and B (28.9%) at hr. At 72 hr, no differences were found in the number of zona-intact, zona-escape and total blastocysts among groups. Mean and ICM cell numbers were significantly higher (p<0.05) in group A (78.0, 13.4) and B (64.4, 11.8) than control (53.1, 5.7), respectively, The percent of ICM were significantly higher (p<0.05) in group A (22.9%) and B (23.7%) than control (14.2%). No differences were round in the TE cell numbers ($34.1{\sim}45.1$). The ICM:TE ratio was significantly higher $34.1{\sim}45.1$ in control (1:6.0) than group A (1:3.4) and B (1:3.4). This study shows that energy substrates added to culture media especially, the oviductal level of carbohydrates increase the developmental capacity of 2-cell mouse embryos.

• Journal of Embryo Transfer
• /
• v.26 no.3
• /
• pp.147-152
• /
• 2011

This study was conducted to examine whether efficiency of oocyte production from superovulated prepubertal goats. Fifteen prepubertal and twenty adult goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen implanted CIDR for 10 days. Superovulation treatment of the goats received twice daily intramuscular injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotrophin treated goats were injected with 10 mg $PGF_2{\alpha}$ on Day 8 and 400~600 IU hCG in the afternoon on Day 10. Oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 h after hCG injection through mid-ventral incision. The in vivo matured oocytes was activated by ionomycin (5 min) and 6-DMAP (3.5~4 h). The activated oocytes were cultured in mSOF medium containing 0.8% BSA at 38.5$^{\circ}C$ in an atmosphere of 5% CO$_2$, 5% O$_2$, 90% N$_2$ for 7~8 days. There was no significant difference in the mean number of CL and in vivo matured and follicular oocytes recovered. But, quality of I + II grade follicular oocytes was lower (p<0.05) in the prepubertal goat (25.0%) than the adults (52.4%). The same results were also observed in the cleavage and blastocyst rate of activated oocytcs. The cleavage and blastocyst rate from prepubertal derived oocytes were lower (p<0.05) in the prepubertal goat (54.5%, 23.3%) than the adult goat (86.8%, 46.6%). Considering overall these results, we suggest that maturation of donor goats is a major factor affecting recovered oocytes quality and in vitro development of activated goat oocytes.