Growth of Campanula punctata 'Rubriflora' plantlets, as affected by three levels of photosynthetic photon flux (PPF), 70, 110, and $220{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, two levels of $CO_2$ concentration, 500 and $1,500{\mu}mol{\cdot}m^{-1}$, and two levels of number of air exchanges per hour (NAEH), 0.1 and $2.8 h^{-l}$, was studied. Explants were obtained from photomixotrophically-micropropagated plantlets. Four explants were planted in each $3.7{\times}10^{-4}m^3$ polycarbonate box containing MS basal medium and no added sucrose. Explants were cultured under cool-white fluorescent lamps for $16h{\cdot}d^{-1},\;at\;25\pm1^{\circ}C$ temperature, and $70\~80\%$ relative humidity In treatments of $2.8h^{-1}$ NAEH, a 10mm round hole made on the vessel cap was sealed with a microporous filter. For higher $CO_2$ concentrations in the culture room, $CO_2$ gas was provided from a tank of liquefied $CO_2$. Fresh and dry weights, length of the longest root, and number of leaves significantly increased with increasing PPF and especially $CO_2$ concentration. Length of the longest root, number of leaves, fresh and dry weights, and chlorophyll concentration were enhanced with increased NAEH. However, leaf area was the smallest in the $220{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}\;PPF\;2.8h^{-1}$ NAEH and especially, $1,500{\mu}mol{\cdot}mol^{-1}\;CO_2$ concentration treatment. Treatment effect became more produced with time. Overall, treatment with $220{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}\;PPF\;and\;1,500{\mu}mol{\cdot}mol^{-1}\;CO_2$ gave the most vigorous growth.
Liver regeneration is a result of highly coordinated proliferation of hepatocytes and nonparenchymal liver cells. Partial hepatectomy (PH) is the most often used stimulus to study liver regeneration because, compared with other methods that use hepatic toxins, it is not associated with the tissue injury and inflammation, and the initiation of the regenerative stimulus is precisely defined. Granulocyte macrophage-colony stimulating factor (GM-CSF), which is a cytokine able to regulate the proliferation and differentiation of epithelial cells, was first identified as the most potent mitogen for bone marrow. Particularly, rrhGM-CSF, which is highly glycosylated and sustained longer than any other types of GM-CSF in the blood circulation, was specifically produced from rice cell culture. In this experiment, effects of rrhGM-CSF administration were evaluated in the regenerating liver after 78% PH of rats. Morphological changes induced by PH were characterized by destroyed hepatocyte plate around the central vein and enlarged nuclear cytoplasmic ratio and increased hepatocytes with two nuclei. And then, proliferation of liver cells (parenchymal and nonparenchymal) and rearrangement of plates and lobules seemed to be carried out during liver regeneration. These alterations in the experimental group preceded those of the control. Since proliferating cell nuclear antigen (PCNA) is known to be a nuclear protein maximally elevated in the S phase of proliferating cells, the protein was used as a marker of liver regeneration after PH in rats. PCNA levels by western blot analysis and immunohistology were compared between the two groups. PCNA protein expression of two groups at 12 hr and 24 hr after injury showed similar pattern. The protein expression showed the peak at 3 days in both groups, however, the protein level of the experimental group was higher than that of the control. On immunohistochemical observations, the reaction product of PCNA was localized at the nuclei of proliferating cells and the positive reaction in experimental group at 3 days was clearly stronger than that in control group. The results by Western blotting and immunohistology for PCNA showed similar pattern in terms of the protein levels. In conclusion, rrhGM-CSF administration during liver regeneration after 78% PH accelerated breakdown and restoration of the hepatic plate and expression of PCNA. These results suggest that rrhGM-CSF might play an important role during liver regeneration in rats.
Park H. S.;Kim T. S.;Jung S. Y.;Lee Y. H.;Jung J. Y.
Journal of Embryo Transfer
/
v.20
no.2
/
pp.105-112
/
2005
The present study was conducted to examine some factors affecting in vitro development of oocytes from somatic cell nuclear transfer (SCNT) in Korean native goats. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean Native goats. For nuclear transfer, the fibroblasts from caprine ear cells and fetal fibroblasts were surgically harvested and were cultured in vitro until cell confluency in serum-starvation condition (TCM-199 + $0.5\%$ FBS) for 3 to 5 days. The zona pellucidae of matured oocytes were partially drilled by laser irradiation. A single somatic cell was individually transferred into each enucleated oocyte. The reconstructed oocytes were then electrically fused and activated. Activated NT embryos were cultured in mSOF medium supplemented with $0.8\%\;BSA\;6\~7\;day\;at\;39^{\circ}C,\;5\%\;CO_2,\;5\%\;O_2,\;90\%\;N_2$ in air. There were no significant difference in the number of embryos cleaved and 4-cell development between the fibroblast nuclei from mature ear cells and fetal cells, but the rate of 8-cell development was higher (P<0.05) in ear cells $(40.5\%)$ than in fetal cells $(55.5\%)$. However, the embryo development to morula or blastocyst was not significantly different between both the groups$(6.7\%\;vs\;16.0\%)$, respectively. The number of embryo cleaved $(79.0\%)$ were higher (P<0.05) in the oocytes activated with ionomycin+6-DMAP than in the oocytes activated electrically $(9.5\%)$. The development of fused embryos to morula or blastocyst was found $15.6\%$ in ionomycin+6-DMAP, but no morula or blastocysts were developed in electrical stimulation. The development rate of SCNT embryos to morula or blastocyst was love. (P<0.05) in SCNT embryos $(19.0\%\;vs\;0.0\%)$ than that in parthenotes $(66.1\%\;vs\;59.1\%)$. In the parthenotes, the cleavage rate and development to morula or blastocyst were significantly higher (P<0.05) as $86.8\%\;and\;50.0\%$ in ovulated oocytes than in follicular oocytes $(69.0\%\;vs\;23.6\%)$, respectively. These results suggest that some factors Including superovulation treatment, oocyte source, maturation of follicular oocytes, activation method and culture condition may affect in vitro developmental capability of embryos produced by somatic cell nuclear transfer in Korean Native goats, and the fusion rate be greatly low compared with other species.
Cho S. R.;Choi S. H.;Kim H. J.;Han M. H.;Choe C. Y.;Chung Y. G.;Son D. S.
Journal of Embryo Transfer
/
v.20
no.2
/
pp.169-176
/
2005
Loop-mediated isothermal amplification (LAMP) is novel DNA amplification methods that amplifies a target sequence specifically under isothemal condition. The present study was to assess the in vitro viability afier biopsy and sexing rate of different types of embryo biopsied. In vivo compact morulae and blastocyst embryos were obtained from Korean Native Cow (KNC) superovulated with FSH (Antorin, R-10) on 7 Day after artificial insemination. in vitro compact morulae and blastocyst embryos were obtained with KNC or Holsteins that were gained on 6, 7 or 8 day after in vitro fertilization(IVF) with frozen semen. Biopsy of bovine embryo was carried out in a $80{\mu}l$ drop with $Ca^{2+}-Mg^{2+}$ free D-PBS and the viability of biopsied embryos were evaluated in IVMD (IFP, Japan) medium at 12 hrs culture time. The sex ratio of biopsied Hanwoo embryos were male vs. female of $43.5\%\;vs.\;56.5\%$ in vivo and $33.9\%\;vs.\;49.2\%$ in vitro respectively, and male rate of biopsied Holstein embryos were significantly higher than female $(70.8\;vs.\;29.2\%)$. and indefinite rate of in vitro embryos was $16.9\%$ and in vivo was not. The degeneration rate of biopsied embryo, in vitro embryos were significantly higher than in vivo $(13.2\%\;vs,\;0.0\%,\;p<0.05)$. The survivability of in vivo embryo were between biopsied following punching method was significantly (P<0.05) higher than bisection method produced embryos $(100\%\;vs.\;83.3\%)$ and in vitro had no difference. However, the degeneration rate of biopsied embryo by bisection method was significantly higher than punching methods between in vivo and in vitro $(16.7\;vs.\;22.6\%,\;respectively,\;p<0.05)$. In conclusion, these results indicate that punching method was optimal and survivability after embryo biopsy was useful for reducing the damage caused by the embryo biopsy procedure for LAMP-based embryo sexing.
It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs (cumulus oocytes complexes) in vitro. Attempts had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of FGF (fibroblast growth factor) on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in HPM 199 (Inst. of Functional peptide, Japan) containing 0.1, 1 and 10 ng/ml FGF for 24 hr, maturation rates to metaphase II ($70.0{\sim}75.0%$) were significantly higher (p<0.05) than that of control group (0 ng/ml FGF, 37.5%). When matured COCs with FGF were cultured in maturation medium after in vitro fertilization, developmental rates to blastocysts were 9.5, 0 and 2.9%, respectively, compared to 25.0% of the control group (p<0.05). When the matured COCs with FGF were cultured in HPM 199 (IFP971, Inst. of Functional peptide, Japan) containing 10% FBS, 0.8% BSA or 0.1% PVA (polyvinyl alcohol), the blastocyst formation rates were 12.4, 12.8 and 8.5%, respectively, while the rates of matured COCs with FGF and cultured with IVMD and IVD (Inst. of Functional peptide, Japan) without serum were 38.4% and 34.8%, respectively (p<0.05). These results suggested that FGF is available for in vitro maturation of bovine COCs and is not suitable for in vitro development, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine embryo development.
The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.
This study was conducted to find out the optimum container for increasing acclimatization rate of in vitro mass propagated plantlets of Ever-bearing strawberry (Fragaria ${\times}$ ananassa Duch.) via bioreactor. Four types of containers were used such as transparent plastic container (TPC), plug tray (PT), I-pot (IP), and black vinyl pot (BVP). Number of date maintaining soil water content above 10% was five days in TPC, three to four days in BVP, two days in PT, and one day in IP. Survival rate of plantlets was 80% in BVP, 70% in TPC, 55% in IP, and 15% in PT. In TPC, growth increment of plantlets was the greatest among all the tested containers and the lowest in IP. Numbers of runner per plant were 3.3 in BVP, 2.9 in TPC, 1.6 in PT, and 1.2 in IP. Total cost was 44,405,300 won/10 a in BVP, resulting in reducing more 6,659,400 won/10 a than IP's (51,064,700 won/10 a). Around 102,718 plants/10 a were produced by using BVP, suggesting that 30,265.1 plants/10 a more could be produced than IP (72,452.9 plants/10 a). Production cost per plant was 432.3 won in BVP, resulting in reducing 272.5 won than IP's (704.8 won). As a result, BVP was appropriate for acclimatization of in vitro plantlets through bioreactor system.
This study was performed to evaluate the bactericidal efficacy of Fumagari OPP$^{(R)}$, fumigation disinfectant, containing 20% ortho-phenylphenol against Staphylococcus aureus (S. aureus). In this research, efficacy test of fumigant against S. aureus was carried out according to French standard NF T 72-281. S. aureus working culture suspension number (N value), all of the colony numbers on the carriers exposed with the fumigant (n1, n2, and n3), the number of bacterial test suspentions by pour plate method (N1), the number of bacterial test suspentions by filter membrane method (N2) and the mean number of bacteria recovered on the control-carriers (T value) were obtained from the preliminary test. In addition, the reduction number of S. aureus exposed with the fumigant (d value) was calculated using T value, the mean number of bacteria in recovery solution (n'1) and the mean number of bacteria on carriers plated in agar (n'2). N value was $4.0{\times}10^8$ CFU/mL, and n1, n2, and n3 were higher than 0.5N1, 0.5N2 and 0.5N1, respectively. Additionally, T value was $3.4{\times}10^6$ CFU/mL. In the bactericidal effect of the fumigant, the d value was 6.43 logCFU/mL. According to the French standard for the fumigant, the d value for the effective bactericidal fumigant should be over than 5 logCFU/mL. The results indicated that Fumagari OPP$^{(R)}$ had an effective bactericidal activity against S. aureus, then the fumigant can be applied to disinfect food materials and kitchen appliances contaminated with pathogenic bacteria.
This study aimed to develop polyclonal antibodies to regional inedible adipocytes of Korean native cattle (Hanwoo) and investigate cross-reactivity of the antibodies. Patterns in plasma membrane proteins (PMPs) from abdominal and subcutaneous adipocytes of Hanwoo isolated by collagenase digestion were investigated using SDS-PAGE. As antigens, abdominal and subcutaneous adipocyte PMPs of Hanwoo were injected to sheep 3 times at 3 wk intervals for passive immunization, and non-immunized serum and antisera were collected before and after the injections. Titers of the antisera obtained from sheep and their cross-reactivities with heart, kidney, liver, lung, muscle, and spleen of Hanwoo were determined by ELISA. Isolation and culture of abdominal and subcutaneous adipocytes of Hanwoo were performed for analysing LDH concentration. Based on the SDS-PAGE analysis, specific proteins of PMPs in abdominal and subcutaneous adipocytes appeared despite rather similar patterns between both adipocytes. At the level of 1:1,000 dilution, little antibody reactivity appeared in non-immunized serum whereas the antisera had relatively strong reactivity up to the level of 1:128,000 and 1:64,000 dilution. These findings may indicate that strong antibodies against adipocyte PMPs can be developed using an immunological approach. Extremely low reactivities of abdominal and subcutaneous adipocyte antisera were detected with PMPs of the organs. Both antisera strongly reacted with each adipocyte PMPs and showed statistically (p<0.01) higher cross-reactivities compared with non-immunized serum. In conclusion, these results may indicate that the present polyclonal antibodies against regional inedible adipocyte PMPs are well developed and have safety in cross-reactivities with body organs. Further studies on in vivo cross-reactivity and fat reduction of the antibodies against abdominal and subcutaneous adipocytes PMPs of Hanwoo should be required for inedible fat-reduced high quality beef production.
With the introduction of digital technology since the late 1970s, we have been shifting from the industrial era into the information age. New communication systems have changed our concept of reality. Korea has a wealthy communication heritage with its own language and alphabet. However, Korean typography today has struggled to keep its originality, and does not hold up well to international standards. Korean contemporary typography is not efficient for communicating. It is not orderly, organised and simple. It has currently become complicated and decorative. Moreover, many young designers are attracted by imitations of western trends. It is now time for Korea to reveal its own identity. How can it develop a new typographic language that is more sympathetic to Korean tradition\ulcorner How will Korean information design produce a contemporary style with international relevance that contributes to world culture\ulcorner This thesis will be developed a new Korean typographic language that relies more on Korean traditions. Simultaneously, in this thesis will be examined Swiss typography as a relevant style for a new Korean typography to incorporate. Swiss typography maintained a similar philosophy to Korean tradition with its emphasis on clarity and simplicity. This study will be explored the potential of creating a contemporary Korean typographic solution which combines the traditions of Korea with the clarity of Swiss typography. The first attitude of the new typographic language should focus on legibility. The second condition of the new typographic philosophy based on the ability of designers is interpretation of context. The third concept of the new language is founded on an objective, rational design attitude. The final mental attitude of the new typographic language should feel a deep obligation to traditions. It is crucial time for Korean alphabet to establish a relevant standard rather than goes uncritically with the international current. Korea has had worthy traditions. It is suggested that the answers to 'good design' lie in the study of Korea's own history. Simultaneously, the research of Swiss design which is a
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